Physiology and chemistry of parathyroid hormone

Physiology and Chemistry of Parathyroid Hormone J. A. PARSONS J. T. POTTS, Jr

Physiological history The first great landmark in understanding parathyroid function was the work of MacCallum and Voegtlin (1909), showing that the removal of all four glands from dogs lowered their plasma calcium concentration and led to tetany, which could be temporarily relieved by injecting calcium chloride. The second was the success of Collip and his collaborators in preparing parathyroid extracts which could restore the calcium level in parathyroidectomised dogs or raise it in normal dogs (Collip, 1925). In the 60 years since the work of MacCallum and Voegtlin, parathyroid hormone has been shown to produce its effects by means of four separate major actions. These are as follows, in the order in which they were shown to exist. 1. An increase in urinary excretion of phosphate. This was an early observation, both in dogs (Greenwald, 1911, 1926) and in man (Albright and Ellsworth, 1929). 2. Acceleration of the metabolic destruction of bone. It remained in doubt whether this was a direct action until (a) parathyroids were transplanted to lie in contact with intracerebral bone grafts or against the cranial vault, which became eroded (Barnicot, 1948; Chang, 1951); (b) parathyroid extract (PTE) was shown to stimulate resorption of bone in tissue culture (Gaillard, 1955); and (c) PTE was shown to cause hypercalcaemia in nephrectomised rats. Such rats are abnormal in many ways, but respond reproducibly to PTE if their plasma levels of phosphate and other ions have been maintained within physiological limits by a technique such as peritoneal lavage (Talmage and Elliott, 1956, 1958a). 3. A decrease in urinary excretion of calcium preceding any change in plasma calcium concentration. Although hypercalcaemia may increase the filtered load and thus the net calcium loss in the urine, it can be shown that parathyroid hormone always raises the proportion of filtered calcium which is reabsorbed in the tubules (Talbot et aI, 1952; Talmage and Kraintz, 1954; Kleeman et aI, 1961).

33

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4. An increase in absorption of calcium from the intestine, demonstrated by measuring the uptake of radiocalcium from ligated gut loops (Talmage and Elliott, 1958b) and isolated, everted gut sacs (Rasmussen, 1959). With the advantage of hindsight, we can now identify the prime function of parathyroid hormone (PTH) as raising the calcium concentration of the plasma and understand how these four major actions contribute to that end. The second of them provides calcium rapidly by mobilising a small fraction of the great stores in the skeleton, while the third and fourth operate in the long term to increase the total calcium content of the body. However, this logical simplicity was obscured for many years by the attachment of incorrect significance to the first action discovered-the phosphaturia. Phosphaturia is among the earliest detectable responses to PTH, and in the 1920s it was the most obvious experimentally. Albright and his colleagues (1929,1948) put forward the hypothesis that it represented the primary action, and that parathyroid hormone raised the plasma calcium concentration only indirectly by an undefined physicochemical process whose effect was to keep constant the product of plasma concentrations of calcium and phosphate ions. The value of [Ca2 +] X [HP042-] was the most nearly constant of the various products calculated. Unfortunately, convincing experiments to put this intellectually stimulating hypothesis into proper perspective were not devised for a long while and the debate with those who maintained that parathyroid hormone had a direct action on bone continued for 20 years (Thomson and Collip, 1932; McLean and Bloom, 1937; Greep, 1948). Toward its end, Albright himself wrote 'It is amusing that, just as the authors have had to admit that there may be a direct action on bone tissue, the chief proponent of the other school, Dr J. B. Collip, has swung around somewhat to the authors' original point of view' (Albright and Reifenstein, 1948). Since the work of Barnicott, Chang, Gaillard and Talmage referred to earlier, there has been no doubt that parathyroid hormone has a direct action on bone, stimulating its breakdown. The fact that PTH also causes phosphaturia is nonetheless of great physiological significance. Its importance appears to lie in promoting disposal of the phosphate load from resorbing bone. Without it, the plasma phosphate level would tend to rise, interfering peripherally with the hypercalcaemic response (Neufeld and Collip, 1942; see also the section on bone) and permitting the ion concentration product of calcium and phosphate in the plasma to reach a dangerous level. Indeed, grossly excessive doses of parathyroid hormone do cause generalised calcification of soft tissues (Hueper, 1927). This probably follows damage to the kidneys by precipitation of calcium and phosphate in the distal tubules. The remaining step to establish the parathyroids as organs for maintaining the plasma calcium concentration was to demonstrate that PTH secretion is controlled in such a way as to defend against hypocalcaemia. In 1942, Patt and Luckhardt showed that the perfusate from dog parathyroids, perfused with low-calcium blood, raised the plasma calcium concentration when injected into normal dogs, whereas after perfusion with normal blood the perfusate lacked this property. This made it clear that the parathyroids were part of a negative feedback control system, but the operation of this

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

35

system could not be studied in detail without measurements of the circulating hormone level. Such measurements became possible when a radioimmunoassay was developed for parathyroid hormone in the blood (Berson et aI, 1963). The assay has shown that plasma PTH concentration has a linear inverse relationship to plasma calcium over a wide range, and is not directly affected by the plasma level of phosphate (Potts et aI, 1968a).

Chemical history Understanding of the physiology of the parathyroids, like that of other glands, has depended at many points on advances in chemical knowledge. The chemistry of PTH has presented an unusual number of difficulties and 45 years elapsed between preparation of the first active extracts and the establishment of its aminoacid sequence. This contrasts strikingly with a corresponding interval of four years in the case of calcitonin, the other peptide hormone concerned with calcium regulation (Copp, 1969; Hirsch and Munson. 1969). The delay seems to have been partly due to lack of a convenient and sensitive bioassay and partly to the chemical nature of parathyroid hormone, which is readily inactivated by atmospheric oxidation or by peptidases. Also, starting material for isolation is difficult to obtain, since the parathyroids are characterised by a high rate of synthesis and store very little hormone-typically 0·004 per cent of their fresh weight, whereas the pituitary contains up' to 5 per cent of its weight of growth hormone. PTH appears to be strongly associated with other proteins in the gland, and, as described in the section on chemistry, reasonable yields can only be obtained by using a dissociating solvent for initial extraction. Progress toward isolation was impossible until it was recognised that hot hydrochloric acid (introduced for initial extraction by Collip in 1925) causes cleavage of the native hormone, producing an extract containing multiple bioactive fragments of the molecule. Hot acid extracts of the parathyroids will be referred to throughout this review as PTE. Less than one per cent of their solid content consists of biologically active peptide and they have proved unsuitable for chronic administration (Melick et aI, 1967). After so many years, it is regrettable that no other form of parathyroid hormone is commercially available for diagnostic tests* or for clinical trials of the type discussed in the section on PTH and the small intestine. Now that a synthetic fragment of the bovine hormone sequence has been shown to be biologically active (Potts et aI, 1971a) one may hope that the need will be met by a commercial source of synthetic material. *e.g. the Ellsworth-Howard test to distinguish hypoparathyroidism from pseudo-hypoparathyroidism. PTE (200 units) is administered intravenously to the fasting subject and the phosphorus content of urine specimens is determined hourly for three hours before and three to five hours after medication. Absence of a brisk phosphaturic response is diagnostic of the peripheral insensitivity to parathyroid hormone which is now known to be the distinguishing feature of pseudo-hypoparathyrodism (Ellsworth and Howard, 1934; Albright and Reifenstein, 1948; Chase, Melson and Aurbach, 1969b).

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PARATHYROID HORMONE AND BONE The 8 kg of bone in an average adult represent one of the largest masses of target tissue directly affected by any hormone, whereas the parathyroids are the smallest endocrine glands, weighing in total 20 to 40 mg. The association of the two tissues has been clear since the studies on osteitis fibrosa cystica by Askanazy (1904) and Mandl (l926,a,b). It is borne out by a study of comparative endocrinology. The parathyroids appear to have evolved in vertebrates as they left the calcium-rich ocean and adapted to life on land. They are found in all terrestrial vertebrates, but not in fish (Greep, 1963).

Calcium mobilisation It has been accepted since the work of Collip and his collaborators that injections of parathyroid hormone cause hypercalcaemia in fasting animals and that the calcium which floods into the blood and urine must come from bone. Our understanding of the mechanism of this response is still incomplete Some of the reasons for this obscurity and slow progress are now clear and are given below, providing a convenient framework for a summary of our present knowledge. 1. The magnitude and time-course of PTH-induced hypercalcaemia depend principally on the effectiveness of an animal'sfeedback calcium regulation, and vary greatly between species. For instance, PTH causes virtually no acute hypercalcaemia in intact adult rats. Any tendency of the calcium level to rise is opposed by suppression of endogenous PTH output and increase in secretion of the hypocalcaemic hormone, calcitonin. The principal action of calcitonin is to inhibit calcium mobilisation from bone (Milhaud, Perault and Moukhtar, 1965) and in some species it provides a very effective defence against acute hypercalcaemia (Copp, 1969; Hirsch and Munson, 1969). In the dog, on the other hand (as shown in Figure I), intravenous PTH consistently causes hypercalcaemia within two hours (Parsons, Neer and Potts, 1971) and it does so even more rapidly in the laying hen (Candlish and Taylor, 1970) and in lO-day-old chicks (Parsons and Robinson, I972b). The rat is not refractory to parathyroid hormone, as was once believed. At the age of three days, baby rats show a brisk response (Garel, 1969). Removal of the parathyroids, or the whole thyroparathyroid mass from adult rats also unmasks an acute hypercalcaemic response, maximal within a few hours (Tweedy and Chandler, 1929; Munson, 1955; Causton, Chorlton and Rose, 1965). Even in intact adults, PTH rapidly affects bone histology and Figure l(a). Chart of plasma calcium concentration against time in 8 successive experiments using 3 dogs. Each line of connected dots represents 1 experiment and refers to a vertical line on the left whose height represents a change in plasma calcium concentration of 1 mg/ 100 mI. The position of the 10 mg/IOO mllevel is shown in each case and the animal used is indicated by a code letter on the tight. Parathyroid hormone (20 USP U/kg) was injected intravenously as shown by the arrows and dotted line. (From Parsons, Neer and Potts, 1971, by courtesy of the publishers.) Figure I(b). Mean changes in plasma calcium (. ) and inorganic phosphate (0 ) at selected time intervals after parathyroid hormone injection, calculated from the calcium data of Figure l(a) and corresponding phosphate analyses. Vertical bars show standard errors of the means. (From Parsons, Neer and Potts, 1971, by courtesy of the publishers.)

37

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

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38

PARSONS AND

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POTTS, JR.

the plasma calcium level rises slowly if hormone injections are repeated day after day (Puglsey, 1932; Pugsley and Selye, 1933). This is consistent with other observations suggesting that calcitonin cannot provide long-term protection of bone against calcium mobilisation, either in tissue culture (Raisz et al, 1968) or in vivo (see references in Robinson, Rafferty and Parsons, 1972). 2. Calcium appears to serve as a second messenger in the skeletal action of PTH, as well as being the substance whose transport is controlled. One of the earliest responses to parathyroid hormone is a small uptake of calcium into the skeleton, apparently due to a specific increase in permeability of bone cell membranes. Evidence for this was obtained by injecting PTH intravenously, a procedure which causes the calcium shift to begin suddenly throughout the skeleton and reveals it by (a) transient hypocalcaemia (Figure 1) and (b) alteration in the initial distribution of a simultaneous dose of radiocalcium (Figure 2) (Parsons et al, 1971; Parsons and Robinson, 1971).

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Figure 2. Distribution of a dose of radiocalcium 10 minutes after intravenous injection to rats, and the changes produced by adding PTH (30 USP units per rat) to the isotope solution. By making reasonable assumptions more than 90 per cent of the dose was accounted for. Calculations are based on mean values, control and hormone-treated groups each containing nine rats. The width of each column (shown thus ~ 0·19 -) represents the fraction of the dose attributable in control animals to the tissues analysed. Where only a sample of tissue was taken, these dose fractions were calculated using the figures for total tissue mass given in the methods section. The figure for skin refers to the whole pelt. The height of each column represents the percentage change of tissue uptake in PTH-treated rats compared with controls. Note that in the controls half the dose of isotope had entered the skeleton at the time of sampling. The dotted line approximately indicates the mean increase in skeletal uptake caused by the hormone. All differences between control and PTH-treated groups were significant (0'001 < P < 0'01) except those for the scapulae, which were particularly difficultto dissect without loss. (From Parsons and Robinson, 1971 by courtesy of the publishers.)

39

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

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Figure 3. Results of adding calcium chloride (15 urnol per bird) to intravenous injections of parathyroid hormone. Values shown are mean plasma calcium concentrations from groups of five chicks (10 days old), bled one hour after injection. The control groups received respectively vehicle alone or vehicle plus calcium. (From Parsons and Robinson, 1972b, by courtesy of the publishers.)

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PTH /CALCIUM INTERVAL (rnins) Figure 4. Time-dependence of calcium chloride enhancement of the hypercalcaemic response of chicks to intravenous PTH (6 units/bird). Calcium was either added to the PTH solution (interval = 0) or given by the other wingvein at varying intervals after the hormone. Birds were bled one hour after receiving the hormone and other details are as in Figure 3. The absence of enhancement at an interval of 16 minutes is based on separate experiments with appropriate controls, because this late injection of calcium had not completely disappeared from plasma by the time of bleeding (Parsons and Robinson, unpublished data).

40

J. A.

PARSONS AND

J. T.

POTTS, JR.

The calcium entering cells under the influence of PTH may serve as a 'second messenger', to use the phrase of Sutherland (1970). This was first suggested on the grounds that the characteristic responses of bone to PTH can be imitated in vitro by raising the calcium concentration of the medium (Talmage, Cooper and Park, 1970). Such a mechanism is strongly supported by the finding that a small intravenous calcium injection given within four minutes of an intravenous dose of PTH greatly enhances calcium mobilisation from the skeleton an hour or two later. There is no enhancement if the interval between the PTH and calcium injections exceeds eight minutes (see Figures 3 and 4) (Parsons, Reit and Robinson, 1972b). It is already known that the adenyl cyclase mechanism of bone cell membranes is activated by PTH (Chase, Fedak and Aurbach, 1969a). Activation of all the components of the parathyroid response probably requires simultaneous increases in the intracellular concentrations both of calcium and cyclic AMP (Rasmussen, 1970). 3. The magnitude of the response to parathyroid hormone depends on the preexisting level ofplasma calcium. Evanson (1966) gave a series of infusions of PTE to patients with various disorders of calcium metabolism, and noted a strong correlation between the calcium rise and the initial calcium level. Au and Raisz (1967) showed that D-deficient parathyroidectomised rats had a mean plasma calcium of 4 mg/IOO ml and failed to respond to injection of PTE. Responsiveness could be restored either by feeding large amounts of lactose, which increased calcium absorption from the diet, or by repeated parenteral injections of calcium. We have found that thyroparathyroidectomised rats (plasma calcium 6 mg/IOO ml) were unresponsive to intravenously injected PTH, whereas similar animals fed a high-calcium, low-phosphate diet for two weeks (plasma calcium 9 mg/IOO ml) showed striking hypercalcaemia two hours after PTH injection (Parsons, Rafferty and Robinson, unpublished data). Defective mobilisation of calcium from bone in animals with low plasma calcium could also contribute to the pathogenesis of 'milk fever' in cattle. Even marked hypertrophy of the parathyroids and high circulating levels of PTH are unable to correct the profound hypocalcaemia of this condition (Mayer et ai, 1969). One possible explanation for this dependence of the skeletal response is that in hypocalcaemia less calcium enters bone cells as a 'second messenger'. However, as suggested by Evanson (1966), it is also possible that in chronic hypocalcaemia the skeleton contains a significant proportion of nonmineralised osteoid (osteomalacia) and that calcium mobilised by PTH is trapped by osteoid and fails to reach the circulation. The nature of the dependence cannot be simple, because it is undisputed that PTH causes mobilisation of bone calcium in recently parathyroidectomised rats, despite marked initial hypocalcaemia (Munson, 1955; Causton et al, 1965). The matter clearly requires further investigation, taking into account both the duration of the hypocalcaemia and the rate of calcium mobilisation in response to the hormone. The action of calcitonin (CT) on bone may involve lowering of the intra-

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

41

cellular calcium concentration (Rasmussen and Tenenhouse, 1970). If some constituents of the skeletal response to PTH were mediated by calcium while others result from the activation of adenyl cyclase, this hypothesis could account for the partial nature of PTH-CT antagonism (Robinson et ai, 1972b). Such a possibility requires testing in a bone tissue culture system. 4. Hypercalcaemia induced by the action of PTH on bone is almost certainly complex, containing separable fast and slow components. The proliferation of osteoclasts and increase in enzyme synthesis which are induced by parathyroid hormone (see later section) undoubtedly account for much of its action in accelerating bone resorption. However, three types of evidence suggest that the hypercalcaemic response contains a fast component with a separate mechanism. (a) The very speed of the response in some systems is enough to make it improbable that mobilisation of calcium from bone can depend entirely on enzyme synthesis. The plasma calcium level rises within 10 minutes in the laying hen (Candlish and Taylor, 1970), a fact confirmed in our laboratories using 10-day-old chicks. Parsons and Robinson (1968) saw PTH-induced calcium mobilisation within 10 minutes in isolated blood-perfused dog bone. (b) As shown in Figure 5, inhibition of protein synthesis by pretreating rats with actinomycin D does not prevent PTH from causing an initial rise in plasma calcium (Rasmussen, Arnaud and Hawker, 1964).This observation is perhaps more significant than the fact that the delayed component of the response was abolished, since actinomycin D is a general poison as well as an inhibitor of RNA synthesis (Brazell and Owen, 1971). B

A

-ACTINOMYCIN

+ACTINOMYCIN

Figure 5. Changes in plasma calcium (open circles) and plasma phosphate (closed circles) as a function of time after intraperitoneal injection of 200 ug of purified bovine parathyroid hormone to parathyroidectomised rats weighing 150 g. The values on the left are for control animals, and those on the right for animals given 1 ug of actinomycin D per gram of body weight two hours before the injection of hormone. Each point represents the mean of 12 rats. (From Rasmussen, Arnaud and Hawker, 1964, by courtesy of the authors and the publishers.)

(c) A recent short communication, with few experimental details, makes the important statement that parathyroid hormone begins to raise plasma calcium by inhibiting bone formation. This was based on the changes in

42

J. A.

PARSONS AND

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blood specific activity three hours after injecting PTE to rats labelled earlier in the same day with calcium-45 (Milhaud, Le Du and Perault-Staub, 1971).

5. Unknown variables probably also modify PTH-induced mobilisation of calcium. Responses vary unpredictably even in the same animal when subcutaneous or intravenous doses of parathyroid hormone are repeated (Allardyce, 1931; Parsons et al, 1971; Parsons and Robinson, unpublished observations). In addition to circulating levels of calcitonin, already discussed, this variability may depend on acid-base balance (Cuisinier-Gleizes, Mathieu and Royer, 1967) and on circulating levels of phosphate (Neufeld and Collip, 1942), both of which are known to modify the response to PTH. It is not clear whether the inhibitory action of phosphate is a direct one, or mediated by a reduction in plasma calcium. Changes in blood flow might also introduce variability by changing the partial pressure of oxygen in bone cells (Goldhaber, 1963) and by altering the proportion of a dose of PTH which could reach the skeleton before being destroyed. Destruction of bone matrix Mineral constituents account for only half the fresh weight of the skeleton, and parathyroid hormone has an equally striking effect in accelerating destruction of bone matrix. The controversy over whether the resorptive process first attacks bone crystals or the organic component has led to little advance in understanding; it was discussed by Rasmussen and De Luca (1963, page 136) and more recently by Raisz et al (1968). Two separate chemical processes are needed to dissolve the two components, and both must clearly occur together for the whole tissue to be removed. Matrix and mineral are so intimately related that any process which blocks removal of one seems likely to interfere with access to the other. There are several types of biochemical measurement which give a direct indication of matrix destruction. Injections of PTH have been shown to increase plasma and urinary levels of hydroxyproline (Bates, McGowen and Talmage, 1962; Keiser et al, 1964) and hydroxylysine (Pinnell and Krane, 1972). They also mobilise 35S injected as sulphate 24 hours previously (Bronner, 1960) and deplete the hexosamine content of bone (Johnston, Deiss and Holmes, 1961). Measurements of hydroxyproline (Harris and Sjoerdsma, 1966) and hydroxylysine (Pinnell and Krane, 1972) appear to provide reliable indices of collagen destruction but do not differentiate between skin and bone collagen. The observation of Pinnell and Krane that the ratio of galactosyl-hydroxylysine to glucosyl-galactosyl-hydroxylysine differs in collagen from these two tissues may make it possible to develop a highly specific indicator of bone collagen destruction. The mobilisation of sulphate and hexosamine, referred to above, provide some index of resorption of the non-collagenous fraction of bone matrix. Cellular and enzymic mechanisms of resorption The histological changes seen in bone in hyperparathyroidism are variable and complex. A single section may show evidence of excessive resorption (osteoporosis, osteitis fibrosa) at the same time as excessive and disordered formation (osteosclerosis, 'woven bone') and incomplete calcification (osteo-

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

43

malacia, see Chapter by Bordier). Correspondingly, there is in vivo or in vitro evidence for a direct action of parathyroid hormone on all the main cell types recognised in bone. A direct effect in stimulating resorption of osteoclasts was first demonstrated by Barnicot (1948) in the important paper in which he described the grafting of parathyroid tissue and bone chips into the cranium of mice. Later evidence on the mechanism of the response is well reviewed by Vaughan (1970). Bingham, Brazell and Owen (1969) in a careful, quantitative study, reported an increase in the total number of osteoclasts, beginning 22 to 26 hours after injection of PTE to young rabbits. They concluded that earlier changes in enzyme action, described below, were a consequence of increased metabolic activity of pre-existing osteoclasts. There is a body of evidence to suggest that parathyroid hormone also mobilises calcium from bone by an action on osteocytes (Belanger et ai, 1963; Talmage et ai, 1965; Baud, 1966; Belanger, 1969). The suggestion is based on the change in appearance of osteocytes and the bone around them after exposure to parathyroid hormone in vitro or in vivo. There is no indication of how such an effect might be produced, no convincing evidence having yet been reported of PTH-induced biochemical changes in osteocytes. Osteoclasts, however, rapidly show major biochemical changes in response to parathyroid hormone. The synthesis of cytoplasmic RNA increases three or fourfold within a few hours (Figure 6), and uptake of 3H-glucosamine and 3H-Ieucine is also increased (Owen and Shetlar, 1968; Bingham, 1968). Subsequently, an increase is observed in secretion of collagenase (Woods and Nichols, 1965) and in both secretion and synthesis of at least eight other enzymes which are associated with Iysosomes and may be concerned with matrix destruction (Vaes, 1967, 1968). The latter are all hydroxylases with optimal activity in the acid range and include a hyaluronidase, a cathepsin and an acid phosphatase. The latter is especially interesting in connection with the old observation that acid phosphatase is associated with sites of bone resorption, whereas at sites of formation an alkaline phosphatase is found (Schajowicz and Cabrini, 1954, 1958). All these changes in enzyme activity are probably preceded both by admission of calcium to the cell (already discussed) and by activation of adenyl cyclase. Something is already known of the intervening mechanism. Many of the enzymes in bone are released or activated by a rise in calcium concentration (Talmage et ai, 1970). Others may be activated by phosphorylation via a protein kinase, on the model of phosphorylase b kinase kinase (Krebs et ai, 1966). Many tissues whose hormonal responses depend on activation of adenyl cyclase have been found to contain a cyclic AMPdependent protein kinase (Kuo and Greengard, 1969). Cyclic AMP has been shown to activate two of these by dissociating them from an inhibitor protein (Tao, Salas and Lipman, 1970; Erlichman, Hirsch and Rosen, 1971). It is usually assumed that PTH-induced mobilisation of calcium requires formation of acid, though it is not clear whether this would be necessary for the fast component, which might be accounted for by transfer of calcium already in solution. The earlier theory of Neuman and his colleagues that calcium mobilisation is due to accumulation of citrate appears to have been

44

J. A. PTE

PARSONS AND

J. T.

POTTS, JR.

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Figure 6. Effects of PTE on incorporation of 3H-uridine in different bone cell types in the mid-shaft of young rabbits. The amount of 3H-uridine incorporated into nuclear and cytoplasmic RNA over a short period of time in different cell types was measured at various times after injection of PTE. Results were compared between PTE-treated and paired control animals in the same litter. Each bar or pair of bars in the histogram represents the ratio of the results from each pair of animals. Osteoblasts and preosteoblasts were studied on the periosteal surface and osteoclasts and mesenchyme cells on the endosteal surface of the mid-shaft of the femur of each animal. Results for different cell types from the same animals appear vertically above each other in the histograms. (From Bingham, Brazell and Owen, 1969, by courtesy of the authors and the publishers.)

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

45

abandoned, as the citrate-forming capacity of bone is inadequate to account for the rate at which calcium is set free. Furthermore, accumulation of citrate induced by fluoracetate (Raisz, Au and Tepperman, 1961) or oestradiol (Vaes and Nicols, 1961) is not accompanied by calcium mobilisation. The question of acid formation is well reviewed by Vaes (1967, pages 44-47 and 56-60), who calculates that enough lactate is formed after addition of PTH to calvaria in vitro to account for the mobilisation of calcium. This suggested mechanism clearly requires further study. Biphasic action on osteoblasts; anabolic effect There is evidence for two other effects of parathyroid hormone on the skeleton, one running parallel to its obvious catabolic action on bone tissue, the other anabolic. (a) There is a rapid depression of the metabolic activity of osteoblasts. This has been detected in several ways: (i) by Gaillard (1961, 1965) in tissue culture, as a loss of basophilia, accompanying morphological changes; (ii) by Flanagan and Nichols (1964) as a depression of 14C-glycine incorporation into collagen, measured by isolating proline and hydroxyproline from explanted bone chips incubated in vitro; and (iii) by Owen and Bingham (1968) as a depression of RNA synthesis in vivo, in the bone of young rabbits, affecting preosteoblasts as well as osteoblasts. (b) There is evidence that parathyroid hormone has an anabolic action, although this is contrary to the general view of PTH as an agent of bone destruction. Selye (1932) and Pugsley and Selye (1933) found that the hypercalcaemia caused by daily injection of 20 units of PTE to young rats was not sustained. The plasma calcium level reached its maximum at four days, as did the proliferation of bone cells, both osteoclasts and osteoblasts. By the twelfth day, the calcium and the osteoclast count had returned to normal but numerous osteoblasts persisted. If treatment was continued, huge amounts of bone tissue were formed, leading to a picture which was described as that of 'marbled bone'. Treatment with lower doses of PTE (5 units on alternate days) led directly to the osteoblastic stage, apparently without proliferation of osteoclasts. Similar effects of PTE on bone were observed independently by Jaffe (1933) and were confirmed by Burrows (1938). These results might have been due to compensatory secretion of calcitonin, but Kalu et al (1970) have recently repeated the experiment, using thyroparathyroidectomised rats and highly purified PTH. Bone mineralisation was assessed by radiographic densitometry, and a clear increase was seen after 21 days' treatment with 50 units of PTE or PTH per day. Not only deposition of mineral but also formation of matrix was stimulated, as shown by the increased incorporation of 3H-proline in treated animals. These interesting observations are hard to explain. There is no evidence whether the anabolic effect is exerted directly on osteoblasts, which would imply that PTH first depresses and then stimulates them. The matter is discussed, in connection with the finding that calcitonin does not block the PTH-induced initial shift of calcium into bone, by Robinson et al (1972b). They conclude that chronic administration of PTH together with calcitonin would be worth testing in osteoporosis. The time-course and dose-dependence of the anabolic effect clearly require further study.

46

J. A.

PARSONS AND

J. T.

POTTS, JR.

PARATHYROID HORMONE AND THE KIDNEY Parathyroid hormone has two renal actions, which appear to be separate. As described in the historical section, phosphaturia was the first of the hormone's many physiological effects to be recognised. The action of decreasing urinary calcium excretion was discovered more recently, since it can be studied only by correcting for, or controlling, changes in plasma calcium level.

Phosphaturia It was undecided for many years whether phosphaturia was a response to the hypercalcaemic hormone itself or due to a contaminant-either a separate 'phosphaturic parathyroid hormone' or a non-hormonal component of parathyroid extracts. Both may be true, since it is now recognised that renal phosphaturia is a non-specific response and can have more than one mechanism. For instance, treatment with formaldehyde abolishes the hypercalcaemic activity of PTE Lilly, but not its phosphaturic potency (Stewart and Bowen, 1952). This would bedevil any attempt to use a phosphaturic assay to monitor purification of a separate phosphaturic principle; there are probably several and, so far as we know, isolation has never been attempted. It is established that very highly purified PTH causes phosphaturia (Aurbach, 1959a; Pullman et ai, 1960) and that a synthetic fragment with the N-terminal sequence of bovine PTH causes all the characteristic responses to the intact molecule, phosphaturia included (Potts et al, 1971a). Studies are in progress in our laboratories to determine whether the ratio of phosphaturic to hypercalcaemic potency varies significantly among a series of such bioactive fragments. Phosphaturia could be caused by an increase in glomerular filtration, a fall in tubular reabsorption of phosphate, or tubular phosphate secretion. In the studies of Pullman et al (1960), highly purified PTH was infused into one renal artery of a dog. It caused phosphaturia which was largely or entirely unilateral and could be acccounted for by an inhibition of tubular reabsorption of phosphate, Bartter (1961), using carefully controlled clearance studies in dogs and man, also found that infusion of PTE diminished phosphate reabsorption, with no consistent effect on filtration rate and no evidence for secretion. Samiy, Hirsch and Ramsay (1965) confirmed these findings in intact and thyroparathyroidectomised dogs and used the stop-flow technique to show that the PTH-inhibitable phosphate reabsorption took place largely in the proximal tubule. Injection of 32p during the period of stopped flow provided strong evidence against phosphate secretion, because none appeared in collected samples until new filtrate began to arrive. Micropuncture, one of the most sophisticated techniques for investigating renal function, was first applied to the study of phosphaturia by Strickler et al (1964). Their work confirmed that, in the rat, phosphate reabsorption takes place almost entirely in the proximal tubule. A partiularly elegant study of PTH-induced phosphaturia by the micropuncture method has recently been carried out in the dog by Goldberg et al (1972). They were able to confirm earlier findings regarding the nature and location of the effect, and to establish

47

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

the fact, previously hinted at by indirect evidence, that PTH inhibits reabsorption of sodium in the proximal tubule even more strikingly than reabsorption of phosphate. In fact, in molar terms, PTH causes the tubule to reject approximately 100 times more sodium than phosphate. Goldberg and his colleagues advance the hypothesis that PTH acts primarily on sodium reabsorption in the proximal tubule, the inhibition of proximal phosphate reabsorption being in some way a consequence of this event. The sodium rejected by the proximal tubule is all reabsorbed distally whereas phosphate is not, so that only the phosphate reaches the urine in increased amounts after PTH injection. Another point shown by this important study was that the same mechanism appeared to operate when dibutyryl cyclic AMP was infused systemically or directly into one renal artery. The effect was not produced by infusion of another adenine nucleotide (5'-AMP) and gave strong support to the concept that the phosphaturic action of PTH is mediated through the adenyl cyclase mechanism, as reported by Aurbach and his colleagues (Figure 7.) PTH 7.5 ,.,.9 . . . - . Phosphate

!

2.0

~--.()

0.5

Cyclic AMP

0.4

1.6

o ~ r

c

"E

.....

." (;

III

-a

0.3 »

1.2

E

~

~ ILl

~ :z: 0.8 a..

I I I

a

I

1Il

:z: a..

::>

3

0.2 ~ III

..... 3 s:

I

0.4 /

P

~

/1> - -0..,

/A...

b/

. . "(f/

0.1

'b

,/

d

o OL----.L.----.L.----..1.-----'--' 2

0

TIME (hours)

Figure 7. The effect of parathyroid hormone on the excretion of phosphate and cyclic 3',5'-AMP by a parathyroidectomised rat. Parathyroid hormone (7'5 ug) was injected intravenously over a 2-minute period at the point shown by the arrow. (From Chase and Aurbach, 1968, by courtesy of the authors and the"publishers).

Calcium retention The other established renal action of parathyroid hormone, that of decreasing calcium excretion, apparently involves a different mechanism and a different site. It was initially difficult to isolate the effect of PTH on renal calcium

J. A.

48

J. T.

PARSONS AND

POTTS, JR.

clearance in the intact animal or man, since mobilisation of calcium from the bone tends to raise blood calcium concentration and provide a greater filtered load. Talmage, Kraintz and Buchanan (1955), using rats, demonstrated that parathyroidectomy increased calcium excretion by the kidney at the same time as lowering the plasma calcium level, while hourly injections of PTE restored both these variables toward normal. Kleeman and his colleagues made corresponding observations in man, confirming that PTE specifically decreased the renal clearance of calcium (Kleeman et aI, 1961; Bernstein, Kleeman and Maxwell, 1963). Eisenberg (1968) showed that this effect was independent of changes in plasma calcium level by giving PTH to hypoparathyroid patients continuously infused with calcium. Widrow and Levinsky (1962), using a stop-flow technique in the dog, showed that the effect was due to enhancement of calcium reabsorption at a distal tubular site. This localisation was confirmed in rats by Frick et al (1965), who showed further that the rate of calcium reabsorption in the proximal tubule was unaffected by PTH. There does not yet appear to be any direct evidence whether this calcium-retaining action of PTH is mediated by the adenyl cyclase mechanism. The role of PTH-induced calcium retention in homeostasis has been discussed by Kleeman and his colleagues and by Nordin and Peacock (1969). Figure 8 illustrates the striking observation of the latter authors that the regression of urinary calcium excretion on plasma calcium was consistently displaced to the left in hypoparathyroid subjects and to the right in hyperparathyroidism. I

6

1'4

I

I I I I

~~ 1'2

I

~lli

6 6

'" ~ 1'0

~: ~~ ~~

0'8

~ ~ 0'6 \oj'

..... ~

~'- 0'4

0'2 0

6

I

6

I I I

,,

~6At~6 • 6 7 8 5

,,

9

10

,, ,, , ,

I

I

• •• " •••••. • •

M~j~~r ....11

SERUM- CALCIUM

,,

I

,

,

, ,,

,

I

6

1f6~/ 6 6 , 6 6 6 66 6, 6iJ1e. 6 6 6 6 ... 6 66 666 6 6A

...

,

I

6

, ,, , ,,

•••

•

II 12 13 14 (mg. per 100 mI.)

15

Figure 8. Relation between urinary calcium excretion (as mg/l00 ml glomerular filtrate) and serum calcium during calcium loading in clinical hypoparathyroidism (~) and hyperparathyroidism ( .). The solid and broken lines show the mean values (± 2 s.d.) obtained in normal subjects. and the shaded area represents the normal basal range. Methods used are described by Peacock, Robertson and Nordin (1969). (From Nordin and Peacock, 1969, by courtesy of the authors and the publishers).

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

49

It seems to us that a prima facie case has been made for a major renal role in calcium homeostasis. The relative importance of renal and osseous regulation under different circumstances has become controversial, but the matter cannot be much clarified by assertion and counter-assertion. Quantitative studies of the whole problem are required and must take account of the different types of regulation needed to counteract acute and chronic disturbances of the plasma calcium level. The fastest regulation probably depends on simple buffering by the large exchangeable calcium pool in bone, and the slowest must involve the long-term balance between absorption from the intestine and excretion in urine and faeces. It is in the intermediate range of speeds that renal and osseous regulation compete for dominance. The mean rate of bone formation and resorption estimated in adults by Neer et al (1967) was 600 mg/day, close to the upper limit of urinary calcium excretion which has even been observed in normal subjects (Nordin, Hodgkinson and Peacock, 1967). A renal mechanism could compensate rapidly for hypercalcaemia, but its effectiveness against hypocalcaemic challenge must depend on the pre-existing rate of urinary loss and thus on the dietary status.

PARATHYROID HORMONE AND THE SMALL INTESTINE Calcium absorption The effect of PTH on intestinal calcium absorption was the last to be established and is still the least thoroughly studied of the major actions of the hormone. The first decisive evidence for an action on the intestine came from experiments on the rate of transfer of radiocalcium out of ligated loops of the rat small intestine (Talmage and Elliott, 1958b) and into isolated everted intestinal sacs from the same species (Rasmussen, 1959). In both cases, parathyroidectomy of the rats four hours before setting up the experiment greatly decreased the calcium-transporting activity of the gut. However, not all experimenters were able to repeat such findings in the rat (e.g. Gran, 1960; Wasserman and Comar, 1961). Important early observations by Biilbring (1931), which might have indicated the reason for such disagreement, appear to have been overlooked. Techniques were not available in 1931 for a direct study of intestinal calcium absorption, but Biilbring showed that injecting PTE to rats influenced the retention of ingested calcium in a way which varied with the dietary calcium level. On a highcalcium diet, PTE reduced retention; on a normal-calcium diet it had little effect; but on a low-calcium diet, it produced a clear increase in the percentage retained. This dependence of PTH action on diet was rediscovered in 1966 by Shah and Draper, whose illuminating study showed it to have an intestinal mechanism. On an adequate calcium intake (calcium 1·2 per cent by weight of diet), calcium absorption fell only 13 per cent after parathyroidectomy, an effect which was not significant. However, it fell 26 per cent after parathyroidectomy on a 0·6 per cent calcium diet and 55 per cent on 0·3 per cent calcium, both these results being highly significant. Comparable experiments by Kimberg, Schachter and Schenker (1961), directed to the general question

50

J. A.

PARSONS AND

J. T.

POTTS, JR.

of adaptation to a low calcium intake, are difficult to evaluate because the P :Ca ratio in the low-calcium diet was as high as 25: 1 (parathyroidectomised rats became extremely hypocalcaemic on this diet and the survival rate was only 5/9). Parathyroid hormone has also been shown to affect calcium absorption in man. It is an old clinical observation that hyperparathyroid patients often have a low faecal calcium excretion, in spite of their hypercalcaemia (e.g. Albright et aI, 1932). It only became possible to analyse the mechanisms which might account for such a finding when isotopic tracers were introduced for the clinical study of calcium metabolism. Early techniques gave figures for all the major calcium fluxes of the body, but were laborious, involving analysis of faecal and urine collections for two weeks, plus the drawing of about 20 blood samples on the day of isotope injection and daily samples for the next five days. A physiological variable such as the absorption coefficient a (calcium absorbed/calcium ingested) could be estimated with considerable precision, but the figure obtained was a two-week integrated value, and many precautions were needed to make it reasonable to assume steady-state conditions. More practical limited procedures have therefore been introduced which estimate a over a short period, without permitting calculation of all calcium fluxes. Such estimates contain uncorrected errors, but correlate reasonably with each other and with clinical observations (Avioli et aI, 1965; DeGrazia et aI, 1965; Birge et aI, 1969). By the use of such a method, it has been shown that injection of parathyroid extract (200 units twice daily for three to four days) increased the calcium absorption coefficient in every case in a series of six normal subjects and one hypoparathyroid patient receiving vitamin D. The tests were performed on a 'low normal' calcium intake, the diet containing 400 mg of calcium per day, and the average increase in calcium absorption coefficient was 20 per cent (Wills et al, 1970). The mechanism of this PTH-induced increase in calcium absorption is unknown. It has been seen after injecting PTE to dogs, first operated on to create Thiry-Vella fistulae of the jejunum and then parathyroidectomised (Cramer, 1963), and in parathyroidectomised rats bearing ligated intestinal loops, after injection of PTE or highly purified PTH (MacIntyre and Robinson, 1969-see Figure 9). In these rat experiments, parathyroid hormone enhanced calcium absorption injejunum and ileum as well as duodenum. This accords well with the finding of Kimberg et al (1961) that under conditions of calcium deprivation, almost the entire small intestine of young rats can transport calcium against a concentration gradient. An enhancement of calcium absorption by PTE was also seen in an earlier rat study, using thyroparathyroidectomised animals and depending on an indirect kinetic estimate of intestinal absorption (Aubert et aI, 1964). In both the dog and rat experiments, the effect of injected parathyroid hormone began only after 24 hours and one might suppose that it depended on interaction in some way with induction of the>vitamin D-dependent calcium-transporting protein described by Wasserman and Taylor (1969). But such a mechanism is far from being established. Attempts to-demonstrate an action of PTH on intestinal calcium transport in vitro have been largely unsuccessful. This may be due to the unphysiological nature of isolated,

51

PHYSIOLOGY AND CHEMISTRY OF PARA TH YROID HORMONE

partly anoxic gut sacs, in which transported substances must diffuse across both mucosa and serosa. In the onl y study which has so far emplo yed a gut preparation perfused in vitro via the vascular bed , acti ve calcium transport began to increase 30 minutes after highly purified PTH was added to the perfusion fluid (Olson , De Luca and Potts, 1962). Ind uction of protein synthesis seems an unlikely explanation for th is more rapid response. One might think rather of a possible effect of PTH on membrane permeabi lity to calcium at some rate-limiting point in a pre-existing calcium transport system . Further work is clearly required on the intestinal response to PTH which , although undramatic , may play an important role in long-term adaptation to a low calcium intake. Nothing is known of its relationship to the postulated regulating mechanism involving dihydrox ylated cholecalciferols, discussed by Boyle, Gray and De Luca (1972) a nd Kodicek (1972).

o Control • P. lE. treatedanimals

•• 0 • e0

100 80 60

.,

~

...

..ca CI

40

..ca '"

10~T "' E

•

0

o•

0

0

0

•

Duodenum

0

lh io 3b

I

I

45 60

I

!

90

120

•

•

-~

v

"' 80

~

~

60 40 20 0

•

••

•• 0 00 0

0

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Jejunum

120

Figure 9. Effect of parathyroid extract on calcium absorption from ligated intestinal loops in parathyroidectomised rat s. Eight subcutaneous injections of extract (75 units) were given during the 48 hours preceeding the experiment. Radiocalcium solution containing 20 mEq /l stable calcium was injected into the loops at time zero and animals were sacrificed at varying intervals. Each point represents the mean of the values from two rat s. (From MacInt yre and Robinson, 1969, by courtesy of the authors and the publishers).

P hosphate absorption In the only study of which we are aware, parathyroid extract was shown to increase active uptake of phosphate by the rat duodenum, studied in isolated everted sacs perfused via th e lumen (BorIe, Keutmann and Neuman, 1963).

52

J. A.

PARSONS AND

J. T.

POTTS, JR.

Magnesium absorption

It has been claimed that parathyroid hormone is a major factor in magnesium homeostasis (MacIntyre, Boss and Troughton, 1963; MacIntyre and Robinson, 1969). The rat experiments of MacIntyre and his colleagues showed that highly purified PTH causes magnesium retention by the kidney and increases intestinal absorption of magnesium in much the same way as that of calcium. Some evidence to complete the postulated negative feedback control loop was provided by the results of varying the magnesium concentration in blood perfusing the parathyroid glands of sheep (Buckle et al, 1968), although unphysiological magnesium concentrations were used. Raising the magnesium lowered PTH secretion and vice versa. The ratio of calcium to magnesium in the diet can vary widely and the problem of how a single control channel could simultaneously account for homeostasis of two apparently independent variables has not been studied.

OTHER ACTIONS OF PARATHYROID HORMONE Hypoparathyroidism

It may be that parathyroid hormone acts on other tissues in addition to the bone, kidney and gut. Fourman and Royer's illuminating discussion of hypoparathyroidism refers to many symptoms and signs which are not corrected by treatment with a high calcium intake and vitamin D (Fourman and Royer, 1968, pages 319-339). For instance, unrecognised actions on calcium distribution in the nervous system might account for the high incidence of severe mental depression and the frequent occurrence of tetany, in spite of normal plasma levels of calcium and phosphate. Nearly 50 per cent of hypoparathyroid patients have cataracts. Although lens opacities have been produced experimentally by simple hypocalcaemia (Swan and Salit, 1941), the cataracts often progress in spite of treatment and it has been reported that PTE directly affects the calcium permeability of the lens capsule in vitro (Clarr, 1939). It has long been recognised that teeth which erupt during parathyroid deficiency remain permanently abnormal (acalcified). This was established by the experiments of Erdheim (1911), later extended in collaboration with Albright (Albright and Reifenstein, 1948). It is not clear whether this effect results from hypocalcaemia. Evidence for other sites of action There is some experimental evidence to hint at a direct action of PTH at three other sites. Toverud and Munson (1956) showed that parathyroidectomy of lactating rats increased the calcium concentration in the milk in spite of causing hypocalcaemia. Andreyer and Pugsley (1933), using dogs, reported that an increased serum calcium brought about by PTE was accompanied by a greater increase in the salivary calcium. Correspondingly, Kraintz, Kraintz and Talmage (1965) found that parathyroidectomy increased the calcium content of the salivary gland of rats, which may reflect diminished calcium secretion. These observations require extension.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

53

The evidence which we have left until last is in some ways the most intriguing. It is an old observation that intravenous injection of parathyroid hormone causes a transient fall in blood pressure. When PTE was used, this could have been an effect of histamine or some other non-hormonal constituent, but we have seen it consistently when giving highly purified PTH to dogs. By the use of electromagnetic flow transducers, Charbon (1968,a,b) showed that the hypotension was due to selecti ve increase of t he blood flow in the hepatic and renal arteries, developing within a minute of injection. This is the only evidence of which we are aware for a hepatic action of PTH in vivo, despite the extensive work showing an effect (of doubtful specificity) of PTH on liver mitochondria in vitro. The liver is the only major viscus not known to be affected by parathyroid hormone, and the results of further investigation are eagerly awaited.

CHEMISTRY

Isolation Significant progress toward the isolation of parathyroid hormone did not begin until rat bioassays were developed which were precise and more convenient than the original dog assay (Davies, Gordon and Mussett, 1954; Munson, 1955). Attempts to purify hot hydrochloric acid extracts prepared by the method of Collip (Collip, 1925; Collip and Clark, 1925) then led to the realisation that they contained several bioactive peptides. Yields were low and there was little improvement in specific biological activity (Handler, Cohn and Dratz, 1954; Aurbach, Beck and Astwood, 1958; Aurbach, 1959a). Having correctly concluded that the difficulty resulted from cleavage of the hormonal polypeptide during exposure to acid at 100 °C, Aurbach (1959a) introduced a method using phenol for initial extraction which eliminated this problem and gave high yields of hormonal peptide. Rasmussen, Sze and Young (1964) developed initial extraction with 8-molar urea and cysteine in cold hydrochloric acid as an alternative method for the same purpose. Further treatment of these unfragmented initial extracts by solvent and salt fractionation , followed by countercurrent distribution , yielded a preparation which was very pure, though still not homogeneous (A urbach, 1959a; Rasmussen and Craig, 1969). Gel filtration on Sephadex proved more suitable than countercurrent distribution for preparation of such material on a large scale (Rasmussen and Craig, 1962; Aurbach and Potts, 1964). When the Sephadex eluates were subjected to ion-exchange chromatography on carboxymethylcellulose (CMC) a further small degree of purification was obtained, permitting preliminary determination of the amino acid composition of bovine PTH. It was also possible to prepare and isolate subfragments of the hormone resulting from cleavage by trypsin and specific chemical reagents (Potts, Aurbach and Sherwood, 1966) and some information was obtained about the sequence of the amino-terminal region (Potts et aI, 1968b) . However, the CMC preparation still showed several bands on disc gel electrophoresis, and end-group analysis showed 10 to 20 per cent contamination by two peptides which were not fully characterised, but could be recognised by possession of amino-terminal leucine and valine. Determination of the amino acid sequence was therefore impracticable.

J. A.

54

PARSONS AND

J. T.

POTTS, JR.

Structure of the bovine and porcine hormones The last two years have seen a great acceleration of progress in parathyroid hormone chemistry. This largely reflects improvement in methods for peptide isolation, for sequence analysis by the automated Edman technique, and for solid-state synthesis with appropriate precautions to ensure homogeneity (Niall, 1971; Tregear, 1972; Potts et aI, I972a). In the case of bovine PTH, the problem of isolation was eventually overcome by adding 8-molar urea to the ammonium acetate buffers used to develop the gradient elution on carboxymethylcellulose. This allowed the isolation of three distinct but closely related forms of bovine parathyroid hormone, completely free from non-hormonal contaminants (Keutmann et aI, 1971). The amino acid composition of two of these isohormones, termed bovine parathyroid hormone I and II, is shown in Table I. BPTH I is devoid of threonine, whereas BPTH II contains one residue of threonine and one fewer valine residue. The precise chemical nature and significance of a third form of the hormone, the most basic in elution from carboxymethylcellulose, is still unclear. Table 1. Amino Acid Composition of Parathyroid Hormones" Amino acid Aspartic Asparagine Threonine Serine Glutamic Glutamine Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Total

Bovine I

Bovine II

Porcine

6 3

6 3 I 8 6 5 2 4 7 7 2 3 8 I 2 I 9 4 5 84

5 3

0 8 6 5 2 4 7 8 2 3 8 I 2 I 9 4 5 84

0 8 6 5 2 5 6 9 I 3 10

0 I 1 9 5 5 84

aCombined data from acid and enzymatic hydrolyses shown as residues per mole.

Porcine parathyroid hormone was purified by similar methods (Woodhead et aI, 1971), except that the use of urea in the final carboxymethylcellulose step was not found to be necessary. A small amount of human parathyroid hormone has also been isolated (O'Riordan, Potts and Aurbach, 1971) but the quantities obtained after fractionation in solvents, gel filtration and ionexchange chromatography were not sufficient to provide definitive information on purity. Porcine and human PTH are closely similar to the bovine isohormones. All five of these molecules are single-chain polypeptides, devoid of cysteine and hence containing no intrachain disulphide bonds, and have a free a-amino group on the amino-terminal residue.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

55

Complete amino acid sequences of bovine PTH I and porcine PTH have now been determined (Brewer and Ronan, 1970; Niall et ai, 1970; Potts et al, 1971a) and are illustrated in Figure 10. There are 10 differences between these hormones in overall amino acid composition, reflecting a reduction in the content of five residues and a corresponding increase in the content of others. This would lead to a prediction of five sequence differences, but in fact seven are found, accounted for by internal rearrangement of serine and alanine residues. It has not yet been established at which site within the sequence of bovine PTH II the replacement of valine or the addition of threonine has occurred, nor is it known whether there are internal rearrangements between the sequences of bovine PTH I and II. 1

Figure 10. Comparison of amino acid sequences of bovine PTH-I and porcine PTH. The bovine sequence is shown as a continuous chain and the seven positions at which the porcine differs from the bovine are shown by shaded circles, with the substituted residue shown alongside.

Human parathyroid hormone Our knowledge of the chemistry of human parathyroid hormone is still very limited. Existing information from amino acid analysis (O'Riordan, Potts and Aurbach, 1971) shows that it is similar in overall composition to bovine

J. A.

56

PARSONS AND

J. T.

POTTS, JR.

and porcine PTH, but definite differences were found, as expected from immunological evidence (O'Riordan et al, 1969). Structure-activity relationships Much can now be said about the relationship between structure and biological activity in parathyroid hormones. The earlier studies have been confirmed which indicated that amino-terminal fragments of the molecule, prepared by cleavage in acid at elevated temperatures, are biologically active. The polypeptide fragment consisting of the amino-terminal 29 residues of bovine PTH was isolated after hydrolysis in 0·03 normal HCL at 110 °C for four hours (Keutmann et al, 1972) and shown to act on both bone and kidney, resulting in an increase in serum calcium, phosphaturia, and specific stimulation of renal adenyl cyclase. Further information on the requirements for biological activity was provided by synthesis of a number of peptides representing various regions of the amino-terminal sequence of either bovine or porcine PTH, and also analogues of those sequences. Those peptides so far studied are illustrated in Figures 11 and 12. Six of them have proved active (Potts et al, 1972b) but, in others, specific structural deletions resulted in loss of biological activity. The inactive sequences synthesised include 1-13,2-34 and 14-34. In addition, a peptide corresponding to the sequence 1-20 and peptides from the middle and carboxyl-terminal portions of the molecule were isolated from tryptic 1

20

13

29 30

34

44

84

52

olo-Iys- org-gln osp-phe-org-org----- gin

Activity

+

~

1

olo--------phe 1

+

29

ala-----gln 1

+

20

olo----arg 1

13

olo-Iys 2

34

yol-------phe 14

34

his - - - - - phe 26

44

I y s - - - - - org 53

84

Iys----gln Figure 11. Biological activity of various natural and synthetic fragments of parathyroid hormone. Minimum length essential for activity lies between residues 1-20 and 1-29. Elimination of the amino-terminal alanine results in complete loss of biological potency.

57

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

digests of bovine parathyroid hormone and shown to be inactive. The overall results lead to the following conclusions which, presumably, apply by analogy to the human peptide (Potts et aI, 1971a, 1972b). I. The amino-terminal position, represented by alanine in the bovine and serine in the porcine molecule, is critical for biological activity. Alanine or serine can be replaced by tyrosine with retention of activity, but deletion of an amino acid at this position results in complete loss of activity in the renal adenyl cyclase assay. 2. The importance of the amino-terminal position is further emphasised by the finding that addition of tyrosine at the 'minus l' position results in marked lowering of activity, while the derivative in which the chain has been lengthened by three residues is inactive (Figure 12). 3. The fact that bovine and porcine PTH are both inactivated by oxidation of methionine must be due to introduction of the highly charged suiphoxide or sulphone residues, since methionine is not essential for biological activity. This is shown by two observations: (a) porcine PTH, containing only one methionine (position 8) is no less active than bovine PTH, with two (positions 8 and 18); (b) a synthetic derivative of the porcine sequence, in which norleucine replaces the methionine at position 8, is highly active in the renal adenyl cyclase assay. 4. No peptides shorter than 1-29 have shown activity in the in vitro assay, even when assayed in combination. It can therefore be concluded that the structural requirements for biological activity involve a continuous peptide sequence beginning with residue 1, extending at least as far as position 20 (arginine), but not necessarily as far as position 29 (glutamine). Accordingly, one can predict with considerable confidence that any circulaing fragment of endogenous parathyroid hormone which does not contain this amino-terminal sequence in an intact form will be biologically inactive. , - - - - 2 T 8 '8 ~.,

f

olo-Yol--phe-mel--mel----phe Iyr - Yol-- phe- mel - - mel - - - - p h e

BPTH Iyr-olo-Yol--phe-mel--mel - - - - p h e Iyr-gly-gly-olo-Yol--phe-mel--mel----phe

PPTH

C

30

ser-Yol--Ieu-mel---Ieu--osp ser-Yol--Ieu-norleu--)eu--osp

Figure 12. Synthetic analogues of the active amino-terminal portion of bovine and porcine parathyroid hormone. Aia LPhe34 represents the active synthetic fragment of bovine PTH 1 (see Figure 11). Extension of the sequence by a single tyrosine at the amino terminus, or substitution of tyrosine for alanine at the amino terminus, results in retention of some biological activity. Extension of the amino terminus by three residues (Tyr-Gly-Gly) results in loss of all biological activity. The sequence of I-3D of porcine PTH, and substitution of norleucine for the single methionine at position 8, both showed retention of significant biological activity.

J. A.

58

PARSONS AND

J. T.

POTTS, JR.

ASSAY OF PARATHYROID HORMONE

Bioassay Parathyroid hormone has such a complex action that it is particularly difficult to find a response suitable for precise measurement of activity. Hypercalcaemia, for example, depends on many factors, principally the following: the rate of absorption of the dose (generally subcutaneous); the rate of inactivation of hormone (at the injection site as well as after absorption); the bone blood flow; the sensitivity of PTH receptors; levels of and homeostatic changes in endogenous calcium-regulating hormones; mobilisation of calcium and its redistribution among exchangeable pools; and renal calcium losses. Phosphaturia is a somewhat simpler response, but as discussed in the kidney section, it is not specific for PTH. Phosphaturic assays are not more precise than those depending on calcium mobilisation, and are best reserved for situations where they are physiologically appropriate. References are given by Ziegler et al (1967) and we will not discuss them further here. The specific assays which have been widely used are compared in Table 2. Others are included which are either new Or illustrate interesting points, such as the method of Biering (1950), using intact rats. A more complete list of early assay methods is given by Greep and Kenny (1955). Where great sensitivity is needed and large numbers of pure materials are to be compared, there are clear advantages in using one of the technically complex in vitro methods. But it will always be necessary to use animal assays to settle questions relating to activity in vivo. They are often easier and have special advantages, such as the ability to accept crude tissue extracts or column effluents containing acid, ammonia or urea. Historically, the greatest contributions to progress in the chemistry of PTH were made by using dogs (Collip, 1925) and parathyroidectomised rats, the method of Munson (1955, 1961) having been the most widely used of all. We recently developed a hypercalcaemic assay using lO-day-old chicks which has the advantages of sensitivity, ease and speed. No surgery is required and samples are drawn one hour after injection (Parsons et aI, 1972). It has a good index of precision, due partly to the injection of calcium with the samples to increase the slope of the log dose-response line, and partly to use of the intravenous route, minimising some of the time-dependent variables in the response. Table 3 summarises repeated comparisons of two ampouled preparations of Sephadex PTH, in the chick assay and the rat assay of Munson.

Biological standards The U.S.P. units in which parathyroid potency is usually stated are not units of a biological standard. By definition, they are 'animal units', with the possibility which this implies of variation between animals of different strains, in different laboratories, on different diets and at different seasons. One unit is 'one-hundreth of the amount required to raise the calcium content of 100 ml of the blood serum of normal dogs 1 mg within 16 to 18 hours after administration' (U.S. Pharmacopoeia XI (1935) to XVIII (1970)).

'"

:I: -< en

s

8

Table 2. Comparison of Specific Bioassays For Parathyr oid Hormone Response measured

Hypercalcaemia

Route of administration

Tes t an imal or system

Z

0 ·20 a

Subcutaneous Subcutaneous

150-1000

0 -16°

10- 100

0 ,23-0-49 0 '23-0 -27

T PT X rat

Intrapcri toncal Subcutaneou s Sub cutaneous Sub cutaneou s Subcutaneou s

In vitro

PTX -parathyroidectomised

0-22

5-20 5-20 0-5-20 0-25- 1'0 50-200 2'5-20 1- 10 0 -04- 1'0

0 ·20-0 ·30 0 ·14 0 -15

0-2- J'0

0 ·08

TPTX-thyroparathyroidectomi sed

() :I:

m ~

Rat PTX rat

Ra t kidney a denyl cyclase

Reference

~ ;;0::

100-300

Intravenous In vitr o

o

Reported index of precision

Subcutaneous

Chicken Mo use ca lvaria

-c

>

Dog

T PTX mo use Neonata l rat Chicken Calcium concentration in medium Formation of cyclic 3',5 '-AMP

Approximate worki ng do se range (USP unit s per animal or per tube)

0 -15 0-27 0-28 -

Collip and C lark ( 1925), Miller (1938), Bliss and Ros e (1940) Biering (1950) Davies, Gordon and Mu ssell (1954) Munson (1955,1 961) Causton, Chorlt on and Rose ( 1965) A mer (1968) Bethu ne, Inoue a nd Turpin (1967) Garel (1969) Polin, Sturkie and Hunsake r (1957) Ra oul, Marnay-Gulat a nd Aldbais (971) Parsons et a1 (1972) Za nelli, Lea and Ni sbet (969)

-<

o-n -e

>

::c

~

:I:

-<

~

o :I:

o

::c

~

o

Z m

Marcus and Aurbach (1969)

aCalculated from pu blished data

VI.

'-0

J. A.

60

PARSONS AND

J. T.

POTTS, JR.

In practice, however, the situation has not been so unsatisfactory as this might suggest. Most manufacturers and laboratories who have prepared material for research purposes have assayed it in the rat against successive batches of Parathyroid Extract Lilly. These batches have in turn been calibrated by the U.S.P. dog assay, which, as Munson said in 1961, 'is remarkably dependable, at least in the hands of pharmacologists at the Eli Lilly Company'. Table 3. Example of Chick Bioassay of Bovine Parathyroid Hormone

Potency a (95 % confidence (U/mg) limits) Chick hypercalcaemia assay 2050 (1320-2880) 2070 (1440-3080) 2460 (1890-3270) Mean 2190 Rat hypercalcaemia assay (Munson) 2430 (1630-3850) 1800 (1040-3080) Mean 2120

A

0'14 0·19 0·15 0·24 0·27

aUnknown in terms of 70/312 treated as standard (assumed potency 500 Ujampoule)

The first ampouled PTH standard for general distribution was issued in 1970 (M.R.C. Research Standard A for Parathyroid Hormone, Bovine; obtainable from Division of Biological Standards, National Institute for Medical Research, Hampstead, London, N.W.3), using a donated sample of the bovine hormone purified to the stage of trichloracetic acid precipitation (Aurbach, 1959a). Following a collaborative study (Robinson, Berryman and Parsons, 1972a), it was assigned a potency of 200 units/ampoule on the basis that this figure most nearly preserves continuity with the U.S.P. unit. This research standard appears satisfactory for use in most bioassay systems, but may not be suitable in vitro or in the intravenous chick assay (Parsons et al, 1972). Table 4 summarises three assays of the purest available bovine parathyroid hormone (Urea-CMC PTH) in terms of the standard, using the rat method of Munson (Keutmann, Rafferty, Potts and Parsons, unpublished data). Table 4. Bioassay of Very Highly Purified Bovine Parathyroid Hormone (Urea-CMC BPTH) against Research Standard A. Rat hypercalcaemia assay (Munson, 1961)

Potency a (95 % confidence (Ujmg) limits) 2380 (1460-4070) 2860 (1930-4520) 2470 (1880-3240) Weighted mean 2540 (2090-4000)

A 0·26 0,20 0·14

aUnknown in terms of M.R.C. Research Standard A, whose assigned potency is 200 U/ampou!e.

It is expected that a separate standard will be necessary for radioimmunoassay of PTH, which presents particular pro blems, discussed in a later section.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

61

Solvents

When handling parathyroid hormone in dilute solution, precautions against inactivation are essential. Like most peptides , it is readily adsorbed on glass or plastic. Experience with calcitonin makes it unsafe to assume that this tendency will be lessened by treatment with silicones (Parsons , 1968). PTH also readily loses biological activity by atmospheric oxidation. Immunological activity is more stable, since most antisera fail to distinguish between oxidised and reduced methionine residues. In our laboratories, solut ions for bioassay are therefore made in a vehicle containing one per cent sodium acetate , 0·5 per cent L-cysteine hydrochloride and 0·1 per cent crystalline bovine serum albumin, adjusted to pH 4. Many albumin preparations contain peptidases, most active near neutrality. These enzymes are virtually inactive at pH 4, but if acidity must be avoided, or solutions are to be kept, all enzymic activity can be destroyed by heating the vehicle at 56°C for one hour. Aurbach (l959b) recommends the addition of 30 per cent gelatin to PTH solutions for bioassay. This considerably delays absorption of hormone from a subcutaneous injection site. We have not found it necessary in carrying out the Munson assay and believe that it complicates the interpretation of results. Another function of the gelatin is probably to prevent adsorption of hormone, but this is adequately fulfilled by the 0·1 per cent albumin solution , which is not viscous. Assay in the blood: radioimmunoassay

Normal circulating levels of parathyroid hormone are undetectable by known specific bioassays; even the sensitive in vitro methods cannot accept sufficient serum. Attempts to concentrate the hormone chemically before assay gave unconvincing results, because the non-specific phosphaturic assay was used. The development of a specific radioimmunoasssy for bovine PTH (crossreacting well with human PTH) made it possible to measure circulating levels in some normal subjects for the first time. Raised levels were found in hyperparathyroid patients (Berson et al, 1963). It is clear why bioassay methods were unsuccessful, since it is now recognised that the normal level in the cow and man is less than 1 ng/ml. As discussed in the next section, only 10 to 15 per cent of this is likely to be biologically active, so ,that there is probably less than one unit of biological activity in the whole circulation. Independent radioimmunoassays have now been developed in a number of other laboratories (e.g. Tashjian, Frantz and Lee, 1966; Berson and Yalow, 1968; Reiss and Canterbury, 1968; Arnaud, Tsao and Littledike, 1971; Addison et al, 1971). The assay of Addison et a1 (1971) depends on allowing the hormone being measured to bind purified 125I-labelled antibodies. The other assays, reviewed by Deftos and Potts (1969), depend on allowing the hormone being measured to compete with purified iodinated PTH (the tracer) for binding to antibody. They differ in the species from which the antibody was derived and the type of hormone used as a tracer. All are basically assays for bovine or porcine PTH, which have been shown to detect the human hormone.

62

J. A.

PARSONS AND

J. T.

POTTS, JR.

Immunoassays are being used to investigate the nature of the defect in parathyroid hormone production in a variety of diseases. These include primary and secondary hyperparathyroidism (the latter accompanying such conditions as chronic renal failure and steatorrhoea with osteomalacia) and the ectopic PTH syndrome. The latter is clinically identical with primary hyperparathyroidism, except that the hormone is produced by a non-parathyroid tumour. Dent and his colleagues have been interested in the possibility that a parathyroid adenoma may develop in a small proportion of patients with secondary hyperparathyroidism. This is what is meant by 'tertiary hyperparathyroidism', a non-descriptive name adopted from St Goar (Davies, Dent and Watson, 1968). It seems impossible to decide on retrospective evidence whether such adenomatous change occurs frequently enough to be regarded as a clinical entity, because adenoma may have been the original pathology. If criteria can be found to distinguish by radioimmunoassay between adenoma and chief-cell hyperplasia, a prospective investigation may become possible.

The nature of the circulating hormone lmmunoassays using different antisera (like bioassays carried out in different animals) vary strikingly in the characteristics of a complex molecule which they recognise. It is therefore essential to establish in each assay that the hormone being measured in plasma is immunologically indistinguishable from a dilution of the hormone in the standard employed. Any differences will lead to serious errors in quantitative estimations. Initial studies suggested that PTH in human and bovine plasma was immunologically identical to standards extracted from glands or adenomas (Berson et al, 1963; Sherwood et ai, 1966). However, more recent studies have shown that circulating immunoreactive PTH is heterogeneous, consisting of a family of related peptides. This has led to considerable confusion, but should ultimately clarify problems of the metabolism of parathyroid hormone. Berson and Yalow (1968) found that plasma samples from certain human subjects (measured against a single extract as standard) frequently showed greater immunoreactivity in two antisera (272 and 273) than in a third (C329). The rate of disappearance of hormone after parathyroidectomy appeared more rapid when measured with C239 than with 273. They were thus the first to provide evidence that plasma hormone did differ from standard hormone, but the explanation of the heterogeneity was not then apparent. A different type of evidence for heterogeneity was provided more recently by Cohn, Sherwood, Arnaud and their colleagues (Hamilton and Cohn, 1969; Sherwood, Rodman and Lundberg, 1970; Arnaud, Tsao and Oldham, 1970). Studying PTH secretion in vitro from parathyroid explants maintained as surviving organ cultures, these investigators postulated that the hormonal polypeptide present in the glands is cleaved into smaller fragments prior to release into the medium. They provided strong evidence that immunoreactive PTH found in the tissue culture medium was smaller in size than the 1-84 polypeptide extractable from bovine parathyroids, whose structure has been determined. This led to the speculation that the hormone is cleaved prior to release in vivo as well as in vitro (Sherwood et ai, 1970).

63

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

More recent clinical studies have indicated that this concept is not correct. When analysed by gel filtration and immunoassay of column effluent with several antisera, the hormone found in the peripheral circulation did indeed prove to consist primarily of one or more peptide fragments smaller in size than the 1-84 polypeptide extracted from parathyroid glands or adenomas. However, samples were also obtained by catheterisation from the small thyroid veins directly draining the parathyroids (Figure 13). Analysis of these thyroid vein samples indicated that the hormone released from the gland is at least as large as the 1-84 polypeptide (Habener et al, 1971; Figure 14).

PATIENT J. RIGHT

PATIENT R. LEFT

RIGHT

LEFT

Figure 13. Plasma PTH concentrations in samples obtained at different points in the venous circulation in two patients. Veins are jugular (J), innominate (I), superior vena cava (SVC), superior thyroid (STV) and inferior thyroid veins (lTV, IT). In Patient J, samples also were obtained from medial and lateral branches of the right inferior thyroid vein (ITV-M and ITV-L). Sites of sampling indicated by.; adjacent numbers indicate PTH concentration in ng/ml. (From Potts et al, 1971b, by courtesy of the publishers).

Therefore cleavage to peptide fragments must occur after the hormone has entered the peripheral circulation. Present evidence suggests that the cleaving enzyme does not circulate in the plasma. It may be present in the circulatory bed of one or more organs, such as the kidney, liver or lung. Further evidence on the metabolism of parathyroid hormone has been obtained by characterising the antisera used for analysis of plasma samples and Sephadex eluates (Habener et ai, 1972). It appears that the overall conformation of hormonal polypeptides has little to do with determining antigenic recognition sites. Quite short regions of the sequence of the PTH molecule appear to satisfy the binding requirements of the various antibodies in an antiserum. For example, the antibodies present in one antiserum (GP-l), widely used in clinical studies, can be shown to react as well with an equimolar mixture of peptides 1-29 plus 53-84 from the bovine sequence as with intact 1-84 bovine PTH (Figure 15).

1.0

2.5r

HPTH

A.

J

THYROID] 5 VENOUS

f{\.

2 .0 :-

~H

I"

B. 0 .3

( \

,

BPTHi-34

I

YO

• '.....

I

"~

I

0 .6

0.4

I

I

/

01:

\

'

r

'

\

i \.

-1 1

!

I

I

I

\

I

j ~....

..J O

O~ 12

I

I

....... I

I

28

!

~

\ \

1

\ \

,

J I

:

..

~

'

I

"

I

I

36

44

0

I

~

'....

0'

..

20

Cl.

------/---4------------------- .

\,\

I

l

;

\

I

~

~

BPTH 1-34

~

I

0 .2

" \

~

\

,

------ -------

- ---4 ---1------ - -~ - -

"

~

~

\

I

N'

b

2

\

II

VI'

:

OL

t

J

2:

\

I

BPTH '·34

t

I

~

:;:.

I \ J,

2

i

V,

'l" \

I

.

!

\I ~

f

0 2

0 5 f--

I

"

\

f

3

1.0

.

0 B

I

'

1.~

~

~

I

.j::>.

ADENOMA t ~ EXTRACT

: '

~ ~

3

~

,

c

,

:

4

0\

MIXED VENOUS

I

I

'2

20

I

I

28

I

I

-J O I

36

FRACrlON NVMBCR

Figure 14. Bio-gel P-IO filtration patterns of radioimmunoassayable parathyroid hormone in samples obtained simultaneously by venous catheterisation of (A) inferior thyroid vein (lTY) and (B) superior vena cava (SYC) in a patient with a parathyroid adenoma. This is compared to (C) filtration of a partially purified standard hormone extract prepared from human parathyroid adenomas. Parathyroid hormone concentration in ITY plasma was 150 ng/ml, Indicated hormone concentration in SYC samples is only approximate becau se of non-parallel response in assay. 1251-labelled bovine parathyroid hormone was co-chromatographed in each filtration as a marker (dotted lines). Arrows mark elution positions of first fraction containing plasma proteins (yO), and additional added markers human parathyroid hormone (HPTH) and the synthetic bovine 1-34 amino-terminal peptide (BPTH 1-34). Dashed horizontal line indicates sensitivity limit of the radioimmunoassay (Antiserum : G P-l; dilution, 1-250000). Note that the thyroid venous sample resembles the adenoma extract in eluting before the markers of HPTH and 1251-BPTH. In contrast, activity in the mixed venous sample elutes later than the markers. (From Habener et al, 1971, by courtesy of the publishers.)

?> ;;'

'"~

tl z o ..... >

;3

'8

~~

'-

?"

65

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

0.6

~0.4

....J:

GP- 1

a:l H

el-84 (), 1-29

a..

~ 0.2

• 53-84 o 1-29+53-84

o 0.1

1.0

10

100

Moles PEPTIDE x 10- 15 Figure 15. Immunological reactivity of native parathyroid hormone (1-84), peptide subfragments (1-29 and 53-84) and an equimolar mixture of 1-29 and 53-84 equivalent in concentration to that of the native 1-84 intact molecule. The curves show the decline in binding of 125 IPTH (BjF) due to displacement by unlabelled peptides at a series of concentrations (in moles x to- 15) . (From Habener et ai, 1972, by courtesy of the publishers.)

It has therefore been possible by adsorption methods to prepare antisera which recognise exclusively a limited region of the sequence (Habener et al, 1972). The large circulating fragment of human PTH has been found to react strongly with antibodies directed against the middle or carboxyl end of the molecule but not with an antibody that recognises the N-terminal portion, specifically the region between residues 14 and 19. As noted above, existing information on the structural requirements for biological activity makes it probable that any fragment which does not contain the aminoterminal 21 residues in intact form will be biologically inert. It is therefore probable that this large fragment, having a molecular weight in the region of 7000 in contrast to the 9400 of the 1-84 sequence and accounting for the greater part of the immunoassayable PTH in plasma, represents hormone whose biological activity has been destroyed. These considerations may explain disagreements in published reports concerning the diagnostic significance of the levels of PTH measured in the plasma. Reiss and his colleagues concluded that all patients with primary hyperparathyroidism have a peripheral hormone concentration exceeding that found in normal subjects (Reiss and Canterbury, 1969; Reiss, 1970). On the other hand, Berson and Yalow (1968) and Potts et al (1971b) found that the concentration of hormone in the blood of some patients with primary hyperparathyroidism did not exceed the concentration found in some normals. The way in which differences in the characteristics of antisera used in different laboratories could account for such disagreement is illustrated in

J. A.

66

PARSONS AND

J. T.

POTTS, JR.

Figure 16. Note the extraordinary difference in detection of the large circulating fragment of PTH, the bulk of the immunoreactive hormone found in the blood, when antisera sensitive to the N-terminal or C-terminal regions of the molecule are used. 4r-------,-----,------.-------, o

-•.

• GP-144 2

0

~

GP-I

-l

3

°

E

~

LO C\J

Q Ol c

r<'l

'0

2

><

0l

~

/

I

0...

u

°

b= I

I

i

0

l ~o

I-l

lO

0,

~

/°

0 Fraction Number

Figure 16. Rio-gel P-1O elution of immunoreactive parathyroid hormone (HPTH) in peripheral plasma obtained from a patient with hyperparathyroidism. HPTH in identical aliquots of the eluted fractions was measured by radioimmunoassay using two different guinea pig anti-PTH antisera: -0-, GP-I (dilution 1:250 000); -e-, GP-144 (dilution 1:10 000). 125I-labelled BPTH (-e-) was co-chromatographed with the plasma samples to mark the elution position of intact 84-amino acid PTH and it can be seen that the activity detected by both antisera eluted later than this marker. (From Habener et ai, 1972, by courtesy of the publishers.)

Similar disagreement over the question of calcium-dependence or autonomy of secretion from parathyroid adenomas may be similarly explained. There is evidence that the large fragment disappears from blood much more slowly than it is formed. Accordingly, an antiserum reacting with the middle and carboxy-terminal portions of the molecule will detect little change in the blood level of immunoreactive hormone after calcium infusion, even if secretion of new hormone from the gland has been suppressed. Thus the impression might be gained that the production of hormone by a parathyroid adenoma is not responsive to elevation of serum calcium. This is what has been reported by Reiss (1970). However, Potts et al (1971b) observed a sudden reduction of serum immunoreactive PTH on infusing calcium to patients with surgicaIly proved parathyroid adenomas. This contrasting finding is probably explained by the fact that Potts et al used an antiserum sensitive to the N-terminal region of the molecule. Such an N-terminal immunoassay may closely reflect the concentration of intact biologicaIly active hormone if subsequent metabolic clearance of the smaIl fragment from the amino terminus is rapid in relation to the rate at which the intact hormone is cleaved.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

67

The heterogeneity of circulating PTH at present makes it impossible to measure absolute concentrations of hormone in the plasma. Absolute measurements must await further studies of the peripheral metabolism of PTH to determine the clinical significance of the fragments which might be measured, and will require provision of appropriate standards and thorough characterisation of antisera to guard against misleading cross-reactivity .

Control of parathyroid secretion Although the present immunoassays respond to a vari ety of parathyroid peptides, their application to whole animal studies has pro vided a great deal of information on the control of secretion . This has confirmed and extended earlier knowledge obtained by perfusing the parathyroids with high- and lowcalcium blood (Patt and Luckhardt , 1942; Care et al , 1966). Infusing calcium, EDTA or phosphate into cows, Potts et al (196~a) found that the circulating level of immunoreactive PTf-{ increased steeply whenever the free calcium concentration fell. Phosphate infusion stimulated secretion if it led to hypocalcaemia, but was without effect if the plasma calcium level was maintained (Figure 17). Some information on the mode of control was obtained by plotting the simultaneous concentrations of calcium and parathyroid hormone for all samples from the animals which received these infusions. A linear inverse correlation was seen between PTH concentration and plasma calcium as calcium varied between 4 and 12 mgj 100 ml. This supported a working hypothesis that control was predominantly proportional. However, there was evidence of derivative control during the earl y phases of some EDTA infusions. In addition, it could not be decided whether secretion of hormone really ceased completely at calcium concentrations above 12 mg/IOO ml. Details of the control mechanism will require further study with the newer, more specific radioimmunoassay methods. There have been reports that magnesium ion also influences parathyroid hormone production (Buckle et aI, 1968), but the changes in ion concentration used greatly exceeded the range of magnesium concentration found physiologicall y. Clinical applications of the radioimmunoassay Despite the fact that our present knowledge of what is measured is incomplete, the radioimmunoassay provides reliable information to assist diagnosis in two principal situations. In patients with chronic hypercalcaemia, estimation of plasma PTH can be extremely helpful (Figure 18). The hypercalcaemia of vitamin D intoxication, sarcoidosis, malignant metastases in bone, and multiple myeloma leads to suppression of parathyroid glandular activity and PTH becomes undetectable in the plasma (Potts et ai, 1971b). Although some of the fragments which apparently result from peripheral cleavage of the hormone disappear from blood only slowly, the half-time is measured in hours, not days. Hence, all immunoreactive hormone disappears from blood in patients with chronic hypercalcaemia. On the other hand, in patients with hypercalcaemia caused by hyperparathyroidism, PTH is readily detectable in the blood. The other present application of the assay is its use to analyse samples obtained by catheterising the large veins draining the neck and the medias-

68

J. A. 4.0

e 3.O

PARSONS AND

A.

.c.

K-13

K-15

J. T. POTTS,

JR.

<,

g'2.0

2

e

3.0

.a

.Q.

K-41

K-41

468 HOURS

~2.0

e

E

Calcium

.......

->

Cl

8

a.
o

6

4

6

8

HOURS

10

~

4

6 HOURS

8

10

I

24

Figure 17. Effect of phosphate infusion on plasma hormone concentration . A and B demonstrate the results seen in two animals in which the phosphate infusion caused a delayed hypocalcaemia; an increase in hormone coincided with the nadir in calcium. In C and D, no hypocalcaemia occurred (in D hypocalcaemia was prevented by an infusion of calcium). In the latter situation (C and D) no significant change in hormone concentration occurred despite the marked and rapid changes in blood phosphate. (From Potts et aI, 1968a, by courtesy of the publishers.)

69

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

tinum (Reitz et at, 1969) or, preferably, the smaller veins draining the thyroid venous plexus (see Figure 13). It has been established that the concentration of hormone in such veins is 10 to 50 times higher than at any other site. When an adenoma was present, no production of PTH by the remaining normal parathyroid glands could be detected. In contrast, several patients with chief-cell hyperplasia showed an increased concentration of hormone at multiple sites on both sides of the plexus. This indicates that venous catheterisation and immunoassay will be useful for distinguishing preoperatively between chief-cell hyperplasia and adenomatous hyperparathyroidism (Potts et al, 1971 b). It may also be useful in clinical situations where the ectopic PTH syndrome must be considered (e.g. with a relatively short history of illness and symptoms suggesting a malignancy). If a concentration difference ofimmunoassayable hormone is found between the periphery and some point in the veins draining the thyroid plexus, neck or mediastinum, the diagnosis of primary hyperparathyroidism rather than ectopic production ofPTH is almost assured (Potts et al, 197Ib). 6 r---,------,

•

51-

..... l::

"

~ ~

~

Q..

4e-

• • I

3-

•

-

-

• •

•

•

-

21-

tl-

0

• 8

Normals

• A ••

-

+ Hyper pora· thyroid

•

•

•

B

. ... .

•

• • • °o~ t.-I • r I 9

to

1t

•

1

I

12

13

• 14

CALCIUM, mg/fOOml

Figure 18. Plasma parathyroid hormone concentrations in normal subjects, patients with hyperparathyroidism and non-parathyroid hypercalcaemia. The overlap between normals and those with hyperparathyroidismis minimised by plotting PTH in relation to the blood calcium concentrations; all patients with non-parathyroid hypercalcaemia have undetectable levels (below dotted lines). (From Potts et ai, 1971b, by courtesy of the publishers.)

One must conclude that the true nature of the defect in primary hyperparathyroidism has not been defined. It seems established that production of parathyroid hormone by adenomas is not autonomous but responds to changes in plasma calcium, although in an abnormal way. This subtle type of defect may represent what is called a 'set point error' in control system analysis. It may be that the abnormal parathyroid cells found in an adenoma continue to secrete hormone even after the plasma calcium concentration reaches 14 or 15 mg/IOO ml or higher. Again, this question cannot be resolved until more is known about the circulating forms of parathyroid hormone and immunoassay methods are developed which are sensitive only to biologically active peptides.

70

J. A.

PARSONS AND

J. T.

POTTS, JR.

Further development and refinement of the radioimmunoassay are likely to make it an even more valuable tool for the diagnosis of all types of parathyroid disorder.

REFERENCES Addison, G. M. et al (1971) Immunoradiometric assay of parathyroid hormone. Journal of Endocrinology, 49, 521-530. Albright, F. et al (1932) Studies in parathyroid physiology, III. The effect of phosphate ingestion in clinical hyperparathyroidism. Journal of Clinical Investigation, 11,411-435 Albright, F. & Ellsworth, R. (1929) Studies on the physiology of the parathyroid glands, I. Calcium and phosphorus studies on a case of idiopathic hypoparathyroidism. Journal of Clinical Investigation, 7, 183-201. Albright, F. & Reifenstein, E. C. (1948) The Parathyroid Glands and Metabolic Bone Disease. Baltimore: Williams & Wilkins. Allardyce, W. J. (1931) Studies on the chemistry and the physiology of the parathyroid hormone. American Journal of Physiology, 98, 417-429. Amer, M. S. (1968) An improved assay of parathyroid hormone. Endocrinology, 82, 166-170. Andreyer, L. & Pugsley, L. I. (1933) The effect of parathyroid hormone and of irradiated ergosterol upon the calcium content of parotid saliva in the dog. Journal ofPhysiology, 80,96-100. Arnaud, C. D., Tsao, H. S. & Littledike, E. T. (1971) Radioimmunoassay of human parathyroid hormone in serum. Journal of Clinical Investigation, 50, 21-34. Arnaud, C. D., Tsao, H. S. & Oldham, S. B. (1970) Native human parathyroid hormone: an immunochemical investigation. Proceedings of the National Academy of Sciences, U.S.A., 67, 415-422. Askanazy, M. (1904) Ueber Osteitis deformans ohne osteides Gewebe. Arbeiten auf dem Gebiete der pathologischen Anatomie und Bacteriologie, Pathologisch-anatomisches Institut der Universitdt Tiibingen, 4, 398-422. Au, W. & Raisz, L. G. (1967) Restoration of parathyroid responsiveness in vitamin Ddeficient rats by parenteral calcium or dietary lactose. Journal of Clinical Investigation, 46, 1572-1578. Aubert, J-P. et al (1964) Etude du metabolisme du calcium chez Ie rat a l'aide de calcium 45. V La thyro-parathyroidectomie et I'effet de la thyroxine et de la parathormone. Biochemical Pharmacology, 13, 31-44. Aurbach, G. D. (1959a) Isolation of parathyroid hormone after extraction with phenol. Journal of Biological Chemistry, 234, 3179-3181. Aurbach, G. D. (1959b) The influence of gelatin in the assay of purified parathyroid extracts. Endocrinology, 64, 296-298. Aurbach, G. D., Beck, R. N. & Astwood, E. B. (1958) Further purification of parathyroid extract. Federation Proceedings, 17, 7. Aurbach, G. D. & Potts, J. T. (1964) Partition of parathyroid hormone on Sephadex G-IOO. Endocrinology, 75, 290-292. Avioli, L. V. et al (1965) A new oral isotopic test of calcium absorption. Journal of Clinical Investigation, 44, 128-139. Barnicot, N. A. (1948) The local action of the parathyroid and other tissues on bone in intracerebral grafts. Journal of Anatomy, 82, 233-248. Bartter, F. C. (1961) The effect of the parathyroid on phosphate excretion. In The Parathyroids, ed. Greep, R. O. & Talmage, R. V. pp. 388-405. Springfield, lllinois: Thomas. Bates, W. K., McGowen, J. & Talmage, R. V. (1962) Influence of the parathyroids on plasma hydroxyproline levels. Endocrinology, 71, 189-195. Baud, C. A. (1966) The fine structure of normal and parathormone-treated bone cells. In IV European Symposium on Calcified Tissues, ed. Gaillard, P. & van den Hoof, A., pp. 4-6. Amsterdam: Excerpta Medica. Belanger, L. F. (1969) Osteocytic osteolysis. Calcified Tissue Research, 4, 1-12. Belanger, L. F. et al (1963) Resorption without osetoclasts (osetolysis). In Mechanisms of Hard Tissue Destruction, ed. Sognnaes, R. F., pp. 531-556. Washington, D.C.: American Association for the Advancement of Science.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

71

Bernstein, D., Kleeman, C. R. & Maxwell, M. H. (1963) The effect of calcium infusions, parathyroid hormone and vitamin D on renal clearance of calcium. Proceedings of the Society for Experimental Biology & Medicine, 112, 353-355. Berson, S. A. & Yalow, R. S. (1968) Immunochemical heterogeneity of parathyroid hormone in plasma. Journal of Clinical Endocrinology & Metabolism, 28,1037-1047. Berson, S. A. et al (1963) Immunoassay of bovine and human parathyroid hormone. Proceedings of the National Academy of Sciences, U.S.A., 49, 613~617. Bethune, J. E., Inoue, H. & Turpin, R. A. (1967) A bio-assay for parathyroid hormone in mice. Endocrinology, 81, 67-70. Biering, A. (1950) Bioassay of parathyroid hormone on rats. Acta Pharmacologica Toxicologica, 6, 40--73. Bingham, P. (1968) Studies of the cells of the osteogenic connective tissues using autoradiographic techniques. D. Phil. Thesis, Oxford, Cited by Vaughan (1970). Bingham, P., Brazell, 1. & Owen, M. (1969) The effect of parathyroid extract on cellular activity and plasma calcium levels in vivo. Journal of Endocrinology, 45, 387-400. Birge, S. J. et al (1969) Study of calcium absorption in man: a kinetic analysis and physiologic model. Journal of Clinical Investigation, 48, 1705-1713. Bliss, C. I. & Rose, C. L. (1940) The assay of parathyroid extract from the serum calcium of dogs. American Journal of Hygiene, 31A, 79-98. Borle, A. B., Keutmann, H. T. & Neuman, W. F. (1963) Role of parathyroid hormone in phosphate transport across rat duodenum. American Journal of Physiology, 204, 705-709. Boyle, 1., Gray, R. & de Luca, H. F. (1972) Calcium control of the in vivo biosynthesis of 1,25-dihydroxyvitamin D3: Nicolaysen's endogenous factor? Endocrinology 1971, ed. Taylor, S. F. London: Heinemann Medical Books (in press). Brazell, 1. & Owen, M. (1971) Some effects of actinomycin D on ribunucleic acid and protein synthesis in osteogenic cells. Clinical Orthopaedics, 79, 173-186. Brewer, H. B. & Ronan, R. (1970) Bovine parathyroid hormone: amino acid sequence. Proceedings of the National Academy of Sciences, U.S.A., 67, 1862-1869. Bronner, F. (1960) Effect of parathyroid extract on metabolism of sulphate in immature rats. American Journal of Physiology, 198, 605-608. Buckle, R. M. et al (1968). The influence of plasma magnesium concentration on parathyroid hormone secretion. Journal of Endocrinology, 42, 529-534. Biilbring, E. (1931) Uber die Beziehungen zwischen Epithelkorperchen, Calciumstoffwechsel und Knochenwachstum. Archiv fur Experimentelle Pathologie & Pharmakologie, 162, 209-248. Burrows, R. B. (1938) Variations produced in bones of growing rats by parathyroid extracts. American Journal of Anatomy, 62, 237-290. Candlish, J. K. & Taylor, G. F. (1970) The response-time to the parathyroid hormone in the laying fowl. Journal of Endocrinology, 48, 143-144. Care, A. D. et al (1966) Perfusion of the isolated parathyroid gland of the goat and sheep. Nature, 209, 55-57. Causton, A., Chorlton, B. & Rose, G. A. (1965) An improved assay for parathyroid hormone, observing the rise of serum calcium in thyroidectomized rats. Journal of Endocrinology, 33, 1-12. Chang, H. (1951) Grafts of parathyroid and other tissues to bone. Anatomical Record, 111,23-48. Charbon, G. A. (1968a) A rapid and selective vasodilator effect of parathyroid hormone. European Journal of Pharmacology, 3, 275-278. Charbon, G. A. (1968b) Parathormone-a selective vasodilator. In Parathyroid hormone and Thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp. 475-484. Amsterdam: Excerpta Medica. Chase, L. R. & Aurbach, G. D. (1968) Cyclic AMP and the mechanism of action of parathyroid hormone. In Parathyroid Hormone and Thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp. 247-257. Amsterdam: Excerpta Medica. Chase, L. R., Fedak, S. A. & Aurbach, G. D. (1969a) Activation of skeletal adenyl cyclase by parathyroid hormone in vitro. Endocrinology, 84, 761-768. Chase, L. R., Melson, G. L. & Aurbach, G. D. (1969b) Pseudohypoparathyroidism: defective excretion of 3',5'-AMP in response to parathyroid hormone. Journal of Clinical Investigation, 48, 1832-1844.

72

J. A.

PARSONS AND

J. T.

POTTS, JR.

Clark, J. J. (1939) The effect of parathyroid hormone on the permeability of the lens capsule to calcium. American Journal of Phvsiology, 126, 136-141. Collip, J. B. (1925) Extraction of a parathyroid hormone which will prevent or control parathyroid tetany and which regulates the level of blood calcium. Journal ofBiological Chemistry, 63, 395-438. Collip, J. B. & Clark, E. P. (1925) Further studies on the parathyroid hormone. Second paper. Journal of Biological Chemistry, 66, 133-137. Copp, D. H. (1969) Calcitonin and Parathyroid Hormone. Annual Reviews ofPharmacology, 9,327-344. Cramer, C. F. (1963) Participation of parathyroid glands in control of calcium absorption in dogs. Endocrinology, 72, 192-196. Cuisinier-Gleizes, P., Mathieu, H. & Royer, P. (1967) Influence de l'acidose metabolique sur l'augmentation de la calcemie induite par la parathormone. Revue Francaise d'Etudes cliniques et biologiques, 12,907-911. Davies, D. R., Dent, C. E. & Watson, L. (1968) Tertiary hyperparathyroidism. British Medical Journal, 3, 395-399. Davies, B. M. A., Gordon, A. H. & Mussett, M. V. (1954) A plasma calcium assay for parathyroid hormone, using parathyroidectomised rats. Journal of Physiology, 125, 383-395. Deftos, L. J. & Potts, J. T. (1969) Radioimmunoassay for parathyroid hormone and calcitonin. British Journal of Hospital Medicine, 2, 1813-1827. DeGrazia, J. A. et al (1965) A double isotope method for measurement of intestinal absorption of calcium in man. Journal ofLaboratory & Clinical Medicine, 66, 822-829. Eisenberg, E. (1968) Renal effects of parathyroid hormone. In Parathyroid Hormone and Thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp. 465-474. Amsterdam: Excerpta Medica. Ellsworth, R. & Howard, J. E. (1934) Studies on the physiology of the parathyroid glands, VII. Some responses of normal human kidneys and blood to intravenous parathyroid extract. Bulletin of the Johns Hopkins Hospital, 55, 296-308. Erdheim, J. (1911) Zur Kenntnis der parathyreopriven Dentin Veranderung, Frankfurter Zeitschrift fur Pathologie, 7, 238-248. Erlichman, J., Hirsch, A. H. & Rosen, O. M. (1971) Interconversion of cyclic nucleotideactivated and cyclic nucleotide-independent forms of a protein kinase from beef heart. Proceedings of the National Academy of Sciences, U.S.A., 68, 731-735. Evanson, J. M. (1966) The response to the infusion of parathyroid extract in hypocalcaemic states. Clinical Science, 31, 63-75. Flanagan, B. & Nichols, G. (1964) Parathyroid inhibition of bone collagen synthesis Endocrinology, 74, 180-186. Fourman, P. & Royer, P. (1968) Calcium metabolism and the Bone, 2nd edn. Oxford: Blackwell Scientific Publications. Frick, A. et al (1965) Microperfusion study of calcium transport in the proximal tubule of the rat kidney. Pflugers Archiv fur die gesamte Physiologie des Menschen und der Tiere, 286, 109-117. Gaillard, P. J. (1955) Parathyroid gland tissue and bone in vitro. Experimental Cell Research, Supplement 3, 154-169. Gaillard, P. J. (1961) Parathyroid and bone in tissue culture. In The Parathyroids, ed. Greep, R. O. & Talmage, R. V., pp. 20-45. Springfield, Illinois: Thomas. Gaillard, P. J. (1965) Protein metabolism and the effect of parathyroid hormone on bone. Texas Reports on Biology & Medicine, 23, 259-277. Garel, J-M. (1969) Dosage biologique de la parathormone chez Ie jeune rat de 3 jours. Comptes Rendus Hebdomadaires des Seances de I'Academie des Sciences, 268, 29322933. Goldberg, M. (1972). Mode of phosphaturic action of parathyroid hormone: micropuncture studies. In Parathyroid hormone and the Calcitonins, ed. Talmage, R. V. Amsterdam: Excerpta Medica (in press). Goldhaber, P. (1963) Some chemical factors influencing bone resorption in tissue culture. In Mechanisms ofHard Tissue Destruction, ed. Sognnaes, R. F., pp. 609-636. Washington, D.C.: American Association for the Advancement of Science.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

73

Gran, F. C (1960) Studies on calcium and strontium-90 metabolism in rats . Acta Physiologica Scandinavica, 48, Supplement 167, 1-109. Greenwald, 1. (1911) The effect of parathyroidectomy upon metabolism. American Journal ofPhysiology, 28, 103-132 . Greenwald, 1. (1926) The effect of the administration of calcium salts and of sodium phosphate upon the calcium and phosphorus metabolism of thyroparathyroidectomised dogs, with a consideration of the nature of the calcium compounds of blood and their relation to the pathogenesis of tetany. Journal of Biological Chemistry, 67, 1-28. Greep, R. O. (1948) The physiology and chemistry of the parathyroid hormone. In The Hormones , ed. Pincus, G. & Thimann, K . V., Vol. 1, pp . 255-299. New York : Academic Press. Greep, R. O. (1963) Parathyroid hormone. In Comparati ve Endocrinology , ed. Von Euler , U. S. & Heller, H., Vol. I, pp . 325-370. New York: Academic Press. Greep, R. O. & Kenny, A. D. (1955) Physiology and chemistry of the parathyroids. In The Hormones, ed. Pincus, G . & Thimann, K. V., Vol. III, pp. 153-174. New York: Academic Press. Habener, J . F. et al (1971) Parathyroid hormone : Secretion and metabolism in vivo. Proceedings of the National Academy of Sciences, U.S.A., 68, 2986-2991. Habener, J. F. et al (1972) Characterization of the immunoreactive parathyroid hormone in the circulation of man. Nature (in press). Hamilton, J. W. & Cohn, D. V. (1969) Studies on the biosynthesis in vitro of parathyroid hormone, 1. Synthesis of parathyroid hormone by bovine parathyroid gland slices and its control by calcium. Journal of Biological Chemistry, 244, 5421-5429 . Handler, P., Cohn, D . V. & Dratz, A. F. (1954) In Transactions ofthe 5th Macy Conference on Metabolic Interrelations, ed. Reifenstein, E. C, pp . 320-330. Caldwell, New Jersey: Progress Associates. Harris, E. D. & Sjoerdsma, A. (1966) Effect of parathyroid extract on collagen metabolism. Journal of Clinical Endocrinology & Metabolism, 26, 358-359. Hirsch, P. F. & Munson, P. L. (1969) Thyrocalcitonin. Physiological Reviews, 49, 548--{i22. Hueper, W. (1927) Metastic calcifications in the organs of the dog after injections of parathyroid extract. Archives of Pathology, 3, 14-25. Jaffe, H. L. (1933) Hyperparathyroidism (Recklinghausen's disease of bone). Archives of Pathology, 16,63-112. Johnston, C. C., Deiss, W. P. & Holmes, L. B. (1961) Effect of parathyroid extract on bone matrix hexosamine. Endocrinology, 68, 484-491. Kalu , D. N. et al (1970) Parathyroid hormone and experimental osteosclerosis. Lancet, i, 1363-1366. Keiser, H. R. et al (1964) Relation between urinary hydroxyproline and parathyroid function. Journal of Clinical Investigat ion, 43, 1073-1082. Keutmann, H. T. (1971) Isolation and characterization of the bovine parathyroid hormones. Biochemistry, 10, 2779-2787 . Keutmann, H. T. et al (1972) A biologically active amino-terminal fragment of bovine parathyroid hormone prepared by dilute acid hydrolysis. Biochemistry, (in press). Kimberg, D. V., Schachter, D. & Schenker, H. (1961) Active transport of calcium by intestine: effects of dietary calcium. American Journal of Physiology, 200, 1256-1262. Kleeman, C. R. et al (1961) Studies on the renal clearance of diffusible calcium and the role of the parathyroid glands in its regulation. In The Parathyroids, ed. Greep, R. O. & Talmage, R. V. Springfield, Illinois : Thomas. Kodicek, E. (1972) The intermediary metabolism of vitamin D: 1,25-dihydroxycholecalciferol, a kidney hormone affecting calcium metabolism. In Endocrinology 1971, ed. Taylor, S. F. London : Heinemann Medical Books (in press). Kraintz, L., Kraintz, F . W. & Talmage, R. V. (1965) Effect of parathyroidectomy on calcium concentration of the rat submandibular glands. Proceedings of the Society for Exp erimental Biology & Medicine, 120, 118-119. Krebs, E. G. et al (1966) Activation of skeletal muscle phosphorylase. Pharmacological Reviews, 18, 163-171. Kuo, J. F. & Greengard, P. (1969) Cyclic nucleotide-dependent protein kinases, IV. 3',5'-monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom. Proceedings of the National Academy of Sciences, U.S.A., 64, 13491355.

74

J. A.

PARSONS AND

J. T.

POTTS, JR.

MacCallum, W. G. & Voegtlin, C. (1909) On the relation of tetany to the parathyroid glands and to calcium metabolism. Journal of Experimental Medicine, 11, 118-151. MacIntyre, I., Boss, S. & Troughton, V. A. (1963) Parathyroid hormone and magnesium homeostasis. Nature, 198, 1058-1060. MacIntyre, I. & Robinson, C. J. (1969) Magnesium and the gut: experimental and clinical observations. Annals of the New York Academy of Sciences, 162, 865-873. McLean, F. C. & Bloom, W. (1937) Mode of action of parathyroid extract on bone (abstract) Science, 85, 24. Mandl, F. (1926a) Therapeutischer Versuch bei einem Faile von Ostitis fibrosa generalisata mitteIs Extirpation eines Epithelkorperchentumors. Zentralblatt fur Chirurgie, 53, 260-264. Mandl, F. (1926b) Klinisches und Experimentelles zur Frage der lokalisierten und generalisierten Ostitis fibrosa. Zweiter Teil; Die generalisierter Form. Archiv fur klinische Chirurgie, 143, 245-284: Marcus, R. & Aurbach, G. D. (1969) Bioassay of parathyroid hormone in vitro with a stable preparation of adenyl cyclase from rat kidney. Endocrinology, 85, 801-810. Mayer, G. P. et al (1969) Plasma parathyroid hormone concentration in hypocalcaemic parturient cows. American Journal of Veterinary Research, 30, 1587-1597. Melick, R. A. et al (1967) Antibodies and clinical resistance to parathyroid hormone. New England Journal of Medicine, 276, 144-147. Milhaud, G., LeDu & Perault Staub, A. M. (1971) Mechanism of the rapid hypercalcaemic effect of parathormone: inhibition of bone accretion. Revue Europeenne d' Etudes Cliniques et Biologiques, 16, 451--454. Milhaud, G., Perault, A. M. & Moukhtar, M. S. (1965) Etude du mecanisme de I'action hypocalcemiante de la thyrocalcitonine. Comptes Rendus Hebdomadaires des Seances de l'Academie des Sciences. 261, Serie D, 813-816. Miller, L. C. (1938) The dose-response relationship in the U.S.P. XI parathyroid assay. Journal of the American Pharmaceutical Association, 27, 90-95. Munson, P. L. (1955) Studies on the role of the parathyroids in calcium and phosphorus metabolism. Annals of the New York Academy of Sciences, 60, 776-796. Munson, P. L. (1961) Biological assay of parathyroid hormone. In The Parathyroids, ed Greep, R. O. & Talmage, R. V., pp. 94-113. Springfield, Illinois: Thomas. Neer, R. M. et al (1967) Multicompartmental analysis of calcium kinetics in normal adult males. Journal of Clinical Investigation, 46, 1364-1379. Neufeld, A. H. & Collip, J. B. (1942) The primary action of the parathyroid hormone. Endocrinology, 30, 135-141. Niall, H. D. (1971) Automated sequence analysis of proteins and peptides. Journal of Agricultural & Food Chemistry, 19, 638-644. Niall, H. D. et al (1970) The amino acid sequence of bovine parathyroid hormone I. Hoppe-Seyler's Zeitschrift fur physiologische Chemie, 351, 1586-1588. Nordin, B. E. C., Hodkinson, A. & Peacock, M. (1967) The measurement and the meaning of urinary calcium. Clinical Orthopaedics, 52, 293-322. Nordin, B. E. C. & Peacock, M. (1969) Role of kidney in regulation of plasma-calcium. Lancet, ii, 1280-1283. Olson, E. B., De Luca, H. F. & Potts, J. T. (1972) The effect of calcitonin and parathyroid hormone on calcium transport of isolated intestine. In Parathyroid Hormone and the Calcitonins, ed. Talmage, R. V. Amsterdam: Excerpta Medica (in press). O'Riordan, J., Aurbach, G. D. & Potts, J. T. (1969) Immunological reactivity of purified human parathyroid hormone. Proceedings ofthe National Academy ofSciences, U.S.A., 63, 692-698. O'Riordan, J. L. H., Potts, J. T. & Aurbach, G. D. (1971) Isolation of human parathyroid hormone. Endocrinology, 89, 234-239. Owen, M. & Bingham, P. (1968) The effect of parathyroid extract on RNA synthesis in osteogenic cells in vivo. In Parathyroid hormone and thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp. 216-225. Amsterdam: Excerpta Medica. Owen, M. & Shetlar, M. R. (1968) Uptake of 3H glucosamine by osteoclasts. Nature, 220, 1335-1336. Parsons, J. A. (1968) Effects of added protein on apparent potency of thyrocalcitonin. In Calcitonin: proceedings of a symposium on thyrocalcitonin and the C-cells, ed. Taylor, S., pp. 36--41. London: Heinemann Medical Books.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

75

Parsons, J. A. et al (1972a) A rapid and sensitive hypercalcaemic assay for parathyroid hormone using the chicken. Journal of Endocrinology, 52, Proceedings (in press). Parsons, J. A., Neer, R. M. & Potts, J. T. (1971) Initial fall of plasma calcium after intravenous injection of parathyroid hormone. Endocrinology, 89, 735-740. J. (l972b) Potentiation by divalent cations of the Parsons, J. A., Reit, B. & Robinson, response to intravenously injected parathyroid hormone. Journal of Physiology, 222, Proceedings (in press). Parsons, J. A. & Robinson, C. J. (1968) A rapid indirect hypercalcemic action of parathyroid hormone demonstrated in isolated blood-perfused bone. In Parathyroid Hormone and Thyrocalcitonin. ed. Talmage, R. V. & Belanger, L. F., pp. 329-331. Amsterdam: Excerpta Medica. Parsons, J. A. & Robinson, C. J. (1971) Calcium shift into bone causing transient hypocalcaemia after injection of parathyroid hormone. Nature, 230, 581-582. Parsons, J. A. & Robinson, C. J. (l972a) The earliest effects of parathyroid hormone and calcitonin on blood-bone calcium distribution. In Parathyroid Hormone and the Calcitonins, ed. Talmage, R. V. Amsterdam: Excerpta Medica (in press). Parsons, J. A. & Robinson, C. J. (l972b) Further evidence that the initial calcium shift into bone is a primary response to parathyroid hormone. In Endocrinology 1971, ed. Taylor, S. London: Heinemann Medical Books (in press). Patt, H. M. & Luckhardt, A. B. (1942) Relationship of a low blood calcium to parathyroid secretion. Endocrinology, 31, 384-392. Peacock, M., Robertson, W. G. & Nordin, B. E. C. (1969) Relation between serum and urinary calcium with particular reference to parathyroid activity. Lancet, i, 384-386. Pinnell, S. R. & Krane, S. M. (1972) Collagen turnover: a new method of analysis utilizing unique urinary breakdown products. In Parathyroid Hormone and the Calcitonins, ed. Talmage, R. V. Amsterdam: Excerpta Medica (in press). Polin, D., Sturkie, P. D. & Hunsaker, W. (1957) The blood calcium response of the chicken to parathyroid extracts. Endocrinology, 60, 1-5. Potts, J. T., Aurbach, G. D. & Sherwood, L. M. (1966) Parathyroid hormone: chemical properties and structural requirements for biological and immunological activity. Recent Progress in Hormone Research, 22, 101-151. Potts, J. T. et al (l968a) Control of secretion of parathyroid hormone. In Parathyroid Hormone and Thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp. 407-416. Amsterdam: Excerpta Medica.. Potts, J. T. et al (l968b) Covalent structure of bovine parathyroid hormone in relation to biological and immunological activity. In Parathyroid Hormone and Thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp, 44-53. Amsterdam: Excerpta Medica. Potts, J. T. et al (I 972a) The chemistry of parathyroid hormone and the calcitonins. Vitamins and Hormones, 29, 41-93. Potts, J. T. et al (l972b) Structural relations to biological and immunological activity in the parathyroid hormones. in Endocrinology 1971, ed. Taylor, S. London: Heinemann (in press), Potts, J. T. et al (l97Ia) Synthesis of a biologically active N-terminal tetratriacontapeptide of parathyroid hormone. Proceedings of the National Academy of Sciences, U.S.A., 68,63-67. Potts, J. T. et al (1971b) Parathyroid hormone: sequence, synthesis, immunoassay studies. American Journal of Medicine, 50, 639-649. Pugsley, L. I. (1932) The effect of parathyroid hormone and of irradiated ergosterol on calcium and phosphorus metabolism in the rat. Journal of Physiology, 76, 315-328. Pugsley, L. I. & Selye, H. (1933) The histological changes in the bone responsible for the action of parathyroid hormone on the calcium metabolism of the rat. Journal of Physiology, 79, 113-117. Pullman, T. N. et al (1960) Direct renal action of a purified parathyroid extract. Endocrinology, 67, 570-582. Raisz, L. G., Au, W. Y. W. & Tepperman, J. (1961) Effect of changes in parathyroid activity on bone metabolism in vitro. Endocrinology, 68, 783-794. Raisz, L. G. et al (1968) Interactions of parathyroid hormone and thyrocalcitonin on bone resorption in tissue culture. In Parathyroid Hormone and thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, L. F., pp. 370-380. Amsterdam: Excerpta Medica.

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J. T.

POTTS, JR.

Rauol , Y., Marnay-Gulat, C. & Aldbais, A. (1971) Recherche d'une methode biologique de titrage de l'hormone parathyroidienne. Bulletin de I'Ordre des Pharmaciens, 138, 1259-1282. Rasmussen, H. (1959) The influence of parathyroid function upon the transport of calcium in isolated sacs of rat small intestine. Endocrinology, 65, 517-519. Rasmussen, H. (1970) Cell communication, calcium ion, and cyclic adenosine monophosphate. Science, 170,404-412. Rasmussen , H., Arnaud, C. & Hawker, C. (1964) Actinom ycin D and the response to parathyroid hormone. Science, 144, 1019-1021. Rasmussen , H. & Craig, L. C. (1959) Purification of parathyroid hormone by use of countercurrent distribution. Journal of the Am erican Chemical Society, 81, 5003. Ra smussen, H. & Craig, L. C. (1962) Purification of bovine parathyroid hormone by gel filtration . Biochimica et Biophysica Acta , 56, 332-338. Rasmussen, H. & De Luca, H. F. (1963) Calcium homeostas is. Ergebnisse der Phys iologie, biologischen Chemie und experimenteller Pharmakologie, 53, 108-173. Rasmussen, H., Sze, Y. L. & Young, R. (1964) Further studies on the isolation and characterization of parathyroid peptides. Journal of Biological Chemistry , 239, 28522857. Rasmussen, H. & Tenenhouse, A. (1970) Parathyroid hormone and calcitonin. In Biochemical Actions of Hormones, ed. Litwack, G., pp. 365-413. New York: Academic Press. Reiss, E. (1970) Primary hyperparathyroidism: a simplified approach to diagnosis. Medical Clinics of North Am erica, 54,131-139. Reiss, E. & Canterbury, J. M. (1968) Radioimmunoassay for parathyroid horm one in man. Proceedings of the Society for Experimental Biology and Medicine, 128, 501-504. Reiss, E. & Canterbury, J. M. (1969) Primary hyperparathyroidism: application of radioimmunoassay to differentiation of adenoma and hyperplasia and to preoperati ve localization of hyperfunctioning parathyroid glands. New England Journal of Medicine, 280, 1381-1385. Reitz, R. E. et al (1969) Localization of parathyroid adenomas by selective venous catheterization and radio immunoassay. New England Journal of Medicine, 281, 348-351. Robinson, C. J., Berryman, I. & Parsons , J. A. (1972a) Research standard for bovine PTH : summary of an international collaborative study. In Parathyroid Hormone and the Calcitonins, ed. Talmage, R. V. Amsterdam: Excerpta Medica (in press). Robinson, C. J., Rafferty, B. & Parsons, J. A. (i972b) Calcium shift into bone : a calcitoninresistant primar y action of parathyroid hormone, studied in rats. Clinical Science, 42, 235-241. Samiy, A. H., Hirsch, P. F. & Ramsa y, A. G. (1965) Localisation of the phosphaturic effect of parathyroid hormone in the nephron of the dog. American Journal ofPhysiology 208,73-77. Schajowicz, F. & Cabrini , R. L. (1954) Histochemical studies of bone in normal and pathological conditions with especial reference to alkaline phosphatase , glycogen and mucopol ysaccharidcs. Journal of Bone & Joint Surgery , 36B, 474-489. Schajowicz, F. & Cabrini, R. L. (1958) Histochemical localisation of acid phosphatase in bone tissue. Science, 127, 1447-1448. Selye, H. (1932) On the stimulation of new bone-formation with parathyroid extract and irradiated ergosterol. Endocrinology, 16, 547-558. Shah, B. G. & Draper, H. H. (1966) Depre ssion of calcium absorption in parathyroidectomised rats . American Journal of Physiology, 211, 963-966. Sherwood , L. M. et al (1966) Intravenous infusions of calcium and EDTA in the cow and goat. Nature, 209, 52-55. Sherwood, L. M., Rodm an, J. S. & Lundberg, W. B. (1970) Evidence for a precursor to circulating parathyroid hormone. Proceedings of the National Academy of Sciences, U.S.A., 67, 1631-1638. Stewart . G. S. & Bowen, H. F. (1952) The urinary pho sphate excretion factor of parathyroid gland extracts : a hormone or an artefact ? Endocrinology , 51, 80-86. Strickler, J. C. et al (1964) Micropuncture study of inorganic phosphate excretion in the rat. Journal of Clinical Investigation, 43,1596-1607.

PHYSIOLOGY AND CHEMISTRY OF PARATHYROID HORMONE

77

Sutherland, E. W. (1970) On the biological role of cyclic AMP Journal of the American Medical Association, 214, 1281-1288. Swan, K. C. & Salit, P. W. (1941) Lens opacities associated with experimental calcium deficiency. American Journal of Ophthalmology, 24, 611-614. Talbot, N. B. et al (1952) Functional Endocrinology from Birth through Adolescence. Cambridge, Massachusetts: Harvard University Press. Talmage, R. V., Cooper, C. W. & Park, H. Z. (1970) Regulation of calcium transport in bone by parathyroid hormone. Vitamins and Hormones, 28, 103-140. Talmage, R. V. et al (1965)Cytological and biochemical changes resulting from fluctuations in endogenous parathyroid hormone levels. In The Parathyroid Glands: Ultrastructure, Secretion and Function, ed. Gaillard, P. J., Talmage, R. V. & Budy, A. M., pp. 107124. Chicago: University of Chicago Press. Talmage, R. V. & Elliott, J. R. (1956) Changes in extracellular fluid levels of calcium, phosphate and citrate ions in nephrectomized rats following parathyroidectomy. Endocrinology, 59, 27-33. Talmage, R. V. & Elliott, J. R. (1958a) Parathyroid function as studied by continuous peritoneal lavage in nephrectomized rats. Endocrinology, 61, 256-263. Talmage, R. V. & Elliott, J. R. (1958b) Influence of parathyroids on intestinal absorption of radiocalcium and radiostrontium. Federation Proceedings, 17, 160. Talmage, R. V. & Kraintz, F. W. (1954) Progressive changes in, renal phosphate and calcium excretion in rats following parathyroidectomy or parathyroid administration. Proceedings of the Society for Experimental Biology & Medicine, 87, 263-267. Talmage, R. V., Kraintz, F. W. & Buchanan, G. D. (1955) Effect of parathyroid extract and phosphate salts on renal calcium and phosphate excretion after parathyroidectomy. Proceedings of the Society for Experimental Biology & Medicine, 88, 600-604. Tao, M., Salas, M. L. & Lipmann, F. (1970) Mechanism of activation by adenosine 3',5'cyclic monophosphate of a protein phosphokinase from rabbit reticulocytes. Proceedings of the National Academy of Sciences, U.S.A., 67, 408-414. Tashjian, A. H., Frantz, A. G. & Lee, J. B. (1966) Pseudohypoparathyroidism: assays of parathyroid hormone and thyrocalcitonin. Proceedings of the National Academy of Sciences, U.S.A., 56, 1138-1142. Thomson, D. L. & Collip, J. B. (1932) The parathyroid glands. Physiological Reviews, 12, 309-383. Tregear, G. W. (1972) Solid phase peptide synthesis on graft copolymer supports. In Battelle Seattle Research Center, ed. Hoffman, A. S. (in press). Toverud, S. U. & Munson, P. L. (1956) The influence of the parathyroids on the calcium concentration of milk. Annals of the New York Academy of Sciences, 64, 336. Tweedy, W. R. & Chandler, S. B. (1929) Studies on the blood plasma calcium of normal and parathyroidectomised albino rats. American Journal of Physiology, 88, 754-760. Vaes, G. (1967) La resorption osseuse et l'hormone parathyroidienne. Paris: Maloine. Vaes, G. (1968) The role of Iysosomes and of their enzymes in the development of bone resorption induced by parathyroid hormone. In Parathyroid Hormone and Thyrocalcitonin (Calcitonin), ed. Talmage, R. V. & Belanger, R. F., pp. 318-328. Amsterdam: Excerpta Medica. Vaes, G. & Nichols, G. (1961) Metabolic studies of bone in vitro III. Citric acid metabolism and bone mineral solubility. Effects of parathyroid hormone and estradiol. Journal of Biological Chemistry, 236, 3323-3329. Vaughan, J. M. (1970) The Physiology of Bone. Oxford University Press. Wasserman, R. H. & Comar, C. L. (1961) The parathyroids and the intestinal absorption of calcium, strontium and phosphate ions in the rat. Endocrinology, 69, 1074-1079. Wasserman, R. H. & Taylor, A. N. (1969) Some aspects of the intestinal absorption of calcium, with special reference to Vitamin D. In Mineral Metabolism, ed. Comar, C. L. & Bronner, F., Vol. III, pp. 321-403. New York: Academic Press. Widrow, S. H. & Levinsky, N. G. (1962) The effect of parathyroid extract on renal tubular calcium reabsorption in the dog. Journal of Clinical Investigation, 41, 2151-2159. Wills, M. R. et al (1970) The role of parathyroid hormone in the gastro-intestinal absorption of calcium. Clinical Science, 39, 89-94. Woodhead, J. S. et al (1971) Isolation and chemical properties of porcine parathyroid hormone. Biochemistry, 10, 2787-2792.

78

J. A.

PARSONS AND

J. T.

POTIS, JR.

Woods, J. F. & Nichols, G. (1965) Collagenolytic activity in rat bone cells. Characteristics and intracellular location. Journal of Cell Biology, 26, 747-757. ZanelIi, J. M., Lea, D. J. & Nisbet, J. A. (1969) A bioassay method in vitro for parathyroid hormone. Journal of Endocrinology, 43, 33-46 . Ziegler, R . et al (1967) Ober die biologische Bestirnmung von Parathormon mit Hilfe der Ausscheidung von 32P durch die parathyroidektomierte Ratte in Athanolnarkose. Methodik, Empfindlichkeit, Genauigkeit. Endokrinologie, 51, 54-66.