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Vitamin D and bone
Metab.
Bone
Dis.
Metabolic Bone Disease 8 Related Research
& Ref. Res. 1, 7-13 (1978)
@ by S.N.P.M.D.
Review
1978)
article
Vitamin
D and Bone
HOWARD
RASMUSSEN
Departments of Internal and the Andre Lichtwiiz Address
versity
(Paris
for
AND
PHILIPPE
Medicine Research
correspondance
School of Medicine,
BORDIER
*
and Cell Biology Yale University Unit, Hdpital LariboisiBre, Paris,
and reprints: H. Rasmussen, Department New Haven, Connecticut 06510
School of Medicine, France.
of Internal
New Haven,
Medicine
and Cell
Connecticut
06570
Biology-Yale Uni-
Abstract
1
The discovery of vitamin D came about as a result of the study of the pathogenesis of clinical and experimental rickets. Hence, from the moment of its discovery attention has been focused on the relationship between vitamin D action and bone physiology. With the recent elucidation of vitamin D metabolism (Deluca, 1974) this interest has been further stimulated. Newly discovered, biologically active vitamin D metabolites are being widely tested as possible therapeutic agents in a variety of metabolic bone diseases ranging from hypoparathyroidism and osteoporosis to renal osteodystrophy.
Key Words: Vitamin Calcification.
Historical
D - 1.25 DHCC - 25 HCC - Bone -
Review
Initial attempts at defining the relationship between vitamin D function and bone focused on the most obvious feature of the bone lesion in vitamin D deficiency: the failure of proper mineralization. However, attempts to demonstrate a direct action of vitamin D on this process were unsuccessful. On the other hand, a marked effect of vitamin D on the intestinal absorption of calcium and phosphate was readily demonstrable. This fact, coupled to the observation that rachitic cartilage became mineralized when incubated in vitro in solutions containing normal amounts of calcium and phosphate, led to the theory that the skeletal changes in vitamin D deficiency are an indirect consequence of a defect in intestinal calcium and phosphate absorption (Nicolaysen and Eeg-Larsen, 1953) (Fig. lA]. This view had to be altered as a result of the work of Carlsson (1952). He and his coworkers demonstrated that the administration of vitamin D to a vitamin D deficient, calcium starved animal led to an increase in the serum calcium concentration and a healing of the rickets. They concluded that vitamin D brought about this change by a direct effect upon bone resorption. Later evidence has confirmed this view and shown that this action of vitamin D is dependent upon the presence of parathyroid hormone (Rasmussen et al., 1963). When the parathyroid glands are intact, the administration of vitamin D to a vitamin D deficient, calcium starved
l
Deceased May 25, 1977.
I, 25(OHI,D,-
25 (OH), D3*
‘D3 I
Fig. 1. The historical
progression of the concept of how vitamin D regulates bone mineralization. A. The first concept in which vitamin D regulated the process of mineralization indirectly by contYolling the mineral ion product of the extracellular fluids by reaulatina the intestinal absorption of calcium and phosphaie. B. The second concept in which the vitamin regulated the mineral ion product by controlling both intestinal mineral absorption and reabsorption of mineral from calcified bone. C.’ The third concept in which the vitamin D metabolite, 1,25 (OH) 203, regulated the mineral ion content by stimulating mineral ion absorption and bone resorption. rat causes an increase in bone resorption with a consequent mobilization of calcium and phosphate from the old mineralized bone and a simultaneous disappearance of the old matrix. The mineral in large part is reincorporated into the previously uncalcified matrix or osteoid leading to a healing of the rickets and osteomalacia. Thus, the total amount of bone matrix decreases, but the percentage of calcified matrix increases. These discoveries were of great importance because they established unequivocally that vitamin D acted directly on bone. Paradoxically, however, this direct
8
Howard Rasmussen et al.: Vitamin
effects was to enhance bone mineral mobilization rather than to increase bone mineral deposition. In fact, these studies provided support for the original view of the action of vitamin D on bone mineralization. In this amended view (Fig. 161, the combination of the actions of vitamin D on intestinal mineral ion absorption and on mineral ion resorption from bone leads to a rise in the calcium times phosphate product in the plasma and extracellular fluids. Once this product exceeds some critical value, bone mineralization ensues. Further support for this widely held point of view has come from the study of the actions of the newly discovered metabolites of vitamin D, particularly I ,25hydroxyvitamin D3 (1,25(OH)zD3). This metabolite is made in the kidney from 25hydroxyvitamin D3 (25(OH)D3). It acts more rapidly and is more pOtWIt biologically than either vitamin D or 25(OH)D 1974; Haussler and McCain, 1977). (DeLuca, Furthermore, it has been shown to stimulate intestinal calcium and phosphate absorption both in vivo and in vitro (DeLuca, 1974; Haussler and McCain. 19771, and to directly stimulate bone resorption in vitro (Raisz et al., 1972). In addition, it has been reported to be antirachitic in animals (Tanaka and Thus, the accepted theory would DeLuca, 1974). appear complete with 1,25(OH)aD3 being the final biologically active form of vitamin D which, by acting on the resorption of bone and on the intestinal absorption of calcium and phosphate, corrects the state .of vitamin D deficiency (Fig. IC). A final, further argument in support of this view is that the infusion of calcium and phosphate into D-deficient humans at a rate sufficient to raise the plasma mineral ion product to normal leads to remineralization of bone (Popovtzer et al., 1973). In spite of this impressive evidence, recent data suggest that the relationship between vitamin D and bone may be more complex. This recent evidence has been obtained largely from studies in man (Bordier et al., 1978) and can best be understood in the context of our present knowledge of bone resorption, bone formation, and bone mineralization.
Vitamin D and Bone Resorption
_
The major vitamin D metabolite concerned with bone resorption is clearly 1,25(OH)zD3. It has been shown to stimulate bone resorption in organ cultures of rat calvaria in vitro, and in D-deficient animals or man in vivo (Haussler and McCain, 1977). For a time, it was considered possible that 1,25(OH)zD3 was the physiologically important regulator of bone resorption, and that the major effect of PTH upon bone resorption was mediated indirectly via its control of 1,25[OH)zD3 synthesis. However, there are three strong arguments against this view: 1) PTH, but not 1,25(OH)zD3, increases the cyclic AMP content of bone; 2) when given in vivo the two agents act synergistically; and 3) when 1,25(OH)zD3 is given to a parathyroid deficient human, it does not increase the number of osteoclasts on bone surfaces although it does increase osteocytic osteolysis (Rasmussen and Bordier, 1974). It is evident that PTH exerts a major control over rate of conversion of osteoprogenitor cells to osteoclasts. Additionally although 1,25(OH)zD3 may enhance this effect and also act to stimulate the resorptive activity of preformed osteoclasts and osteocytes, 1,25(OH)zD3 does not stimulate osteoprogenltor cell activation in the absence of PTH. It is our view that the effect of 1,25(OH)zD3 upon osteocytic osteolysis results in the
D and Bone
mobilization of calcium and phosphate from old mineralized bone and raises the local ion product at the bone surface. This potentiates the effect of PTH on osteoprogenitor cell activation because both CAMP and calcium ion have second messenger functions in the activation of these cells (Rasmussen and Bordier, 1974).
Vitamin D and Bone Formation It is vital in any discussion of bone physiology to distinguish between bone formation and mineralization. The latter is often employed incorrectly as a measure of bone formation since, under normal circumstances, mineralization and formation are closely coupled (Rasmussen and Bordier, 1974). However, this is clearly not the case in states of vitamin D In order for rickets and osteomalacia deficiency. to develop, the rate of bone formation (correctly defined as the rate of bone matrix deposition) must Because of this exceed the rate of mineralization. fact, it is generally held that vitamin D has no effect However, this view is not on bone formation. supported by the available evidence. In their studies of experimental vitamin D deficiency in rats Baylink and associates (1970) have shown that bone formation rate, as well as bone mineralization rate, is decreased. However, the amount of osteoid is increased because the mineralization rate is more severely depressed than the bone formation rate. It is not possible to make precise measurements of bone formation rate in man. What is usually found in the bone biopsy material from D-deficient adults is a slight increase in the extent of the active bone formation surface. However, on such surfaces the histological appearance of the osteoblasts is strikingly abnormal (Rasmussen and Bordier, 1974). They are considerably less basophilic and contain more glycogen. These changes have been taken as an indication of a decrease in the matrix-forming activity of these cells. Evidence supporting an increase in bone formation rate is the finding of an elevation in plasma alkaline phosphatase concentration in the patients with osteomalacia. Under a number of circumstances there is a fairly good correlation between the extent of osteoblastic bone formation surface measured by quantitative analysis of bone biopsy material and the plasma alkaline phosphatase activity (Rasmussen As a consequence plasma and Bordier, 1974). alkaline phosphatase has long been considered a measure of bone formation under all circumstances. Under most circumstances this may be true because there is a coupling between bone formation and the subsequent process of bone mineralization. Hence, when the first is increased the second is also increased. Recent evidence (see below) shows that mineralization is under cellular control, and that membrane bound vesicles derived from osteoid osteocytes are the probable site of initial mineral crystal formation (Anderson et al., 1970; Howell et al., 1976). Germaine to the present discussion is the fact that these vesicles are rich in alkaline phosphatase, and these vesicles form in the bone of D-deficient animals. Thus, it is quite possible that the increase in plasma alkaline phosphatase observed in patients with osteomalacia is a reflection of an increase in osteoid surface and therefore of vesicle number and not an increase in osteoblastic activity. Equally difficult to interpret is the observation that a rise in plasma alkaline phosphatase activity occurs after the administration of vitamin D to patients with
Howard
Rasmussen
et al.:
Vitamin
9
D and Bone
osteomalacia. This may result from an acceleration of the mineralization process, a stimulation of osteoblastic bone formation, or an increase in the turnover of matrix vesicles secondary to the restoration of mineralization rate toward normal.
vesicle formation, or the ability of these vesicles to take up calcium. It is clear, however, that vesicle formation continues in the absence of vitamin D because vesicles are found in the bone and cartilage of D-deficient animals (Howell et al., 19761.
From animal studies it is clear that vitamin D administration to a D-deficient animal stimulates bone formation (Baylink et al.. 1970). Additionally, studies of bone biopsy material in man, show that vitamin D administration leads to a rapid change in the histological appearance of osteoblasts, indicative of an increase in their bone matrix synthetic activity, and a more gradual but significant increase in the extent of the osteoblastic bone formation surface (Rasmussen and Bordier, 1974). A complete discussion of the data is beyond the scope of the present review. However, one additional line of evidence in support of our previous conclusion that plasma phosphate concentration is a determinant of the extent of the osteoblastic bone formation surface is the finding of a reduction in the extent of this surface in patients with a syndrome of hypercalciuria, hypophosphatemia and osteoporosis without hyperparathyroidism. In these patients the defect is presumed to be a primary reduction in the renal phosphate threshold (TmP/GFRI (Bordier et al., 1977).
Vitamin
Vitamin D and Bone Mineralization There is, as discussed above, unequivocal evidence that vitamin D deficiency leads to a reduction in the rate of bone mineralization. The classical view of this process was that once collagen was laid down and underwent some type of maturation, it would then serve as a template for the nucleation of bone mineral crystals. The rate of mineralization was simply a function of the mineral ion product in plasma and extracellular fluids. However, two more recent discoveries argue against this simplistic view. The first is that if sections of bone taken from D-deficient patients who have been treated with calcium and phosphate are compared with similar sections taken from patients treated with vitamin D. there is a crucial difference in the pattern of mineralization (Rasmussen and Bordier, 1974). In the sections from the mineral ion-treated patients, the mineralization is randomly distributed in patches throughout the osteoid with islands of unmineralized osteoid between these patches. On the other hand, the hallmark of vitamin D deficiency is a reduction in the extent of the mineralization front: vitamin D action results in the reinstitution of an orderly process of mineralized bone, and then a progression of this mineralization front toward the bone surface (in reference 81. Thus, a vitamin D metabolite (or metabolites) exerts some directive effect on the process of mineralization. The second discovery is that mineralization is under cellular control (Anderson et al., 1970: Howell et al., 1976); in bone these cells are the osteoid osteocytes and, in cartilage, the cells in the zone of provisional Both types of cells produce small calcification. membrane-bound extracellular matrix vesicles which probably bud off from their respective plasma membranes. The membranes of these vesicles contain high concentrations of alkaline phosphatase and are thought to be the sites of formation of the initial
bone mineral crystals. It seems likely that vitamin D or one of its metabolites affects the activity of these cells, although its precise mode of action is not known. Vitamin D might Influence mineral ion exchange in these cells, the rate or extent of matrix
D and the Kidney
Although the emphasis in the present discussion is on the relationship between vitamin D and bone, a brief discussion of the effects of vitamin D on renal function is necessary to understand the vitamin D-bone relationship. One of the characteristic results of vitamin D administration to D-deficient man is a rise in the Tm for renal phosphate reabsorption (Tm PO~/GFR). Because secondarv hyperparathyroidism is a feature of the vitamin D deficient state, and because plasma PTH concentrations fall after vitamin D administration, this rise in Tm PO~/GFR has been attributed to the fall in PTH. However, recent evidence indicates that 25 (OHID or a metabolite other than I ,25(0H)~D3, can increase the proximal renal tubular reabsorption of calcium and phosphate (Puschett et al., 1972; 19751. Hence, the rise in Tm PO~/GFR is probably a consequence of both a direct renal action of 25IOHID3 (or some unidentified metabolite) and a fall in plasma PTH. Vitamin D Metabolites Osteomalacia
in
Human
Rickets
and
Much of the recent work dealing with vitamin D metabolism and action has been carried out in the vitamin D deficient rat. In general, the work in this species has confirmed the classic view of vitamin D and bone as discussed above. In particular, administration of 1,25(OH)zD3 to the vitamin D deficient rat has been shown to be antirachitic (Tanaka and DeLuca, 1974). However, there is an important difference between rat and man. The rat normally has a plasma phosphate concentration of IO-12 mg/dl whereas infant humans have values approximately half these, and adult men values in the range of 3-4.5 mg/dl. Furthermore, the most common way of inducing rickets in the rat is to reduce the dietary content of both vitamin D and phosphate. In contrast, most human adults with osteomalacia have low phosphate concentration even on a normal phosphate intake and have only a slightly reduced plasma calcium concentration. Based on these differences, it is reasonable to suggest that there may be differences between rat and man as regards the physiology, metabolism, and function of vitamin D and its metabolites. A critical question to be answered is whether or not 1,25IOH)zD3 can cure rickets and/or osteomalacia in man. Vitamin D-dependency rickets was one of the first disorders to be treated with 1,25(OH)aD3 (Reade et al., 1975). This is a disease which presents early in life and clinically is indistinguishable from D-deficiency rickets except that vitmain D intake is normal, plasma 25(OHID3 concentrations are also
normal, and the patients fail to respond to physiological amounts of vitamin D. It was proposed that this condition was due to a specific deficiency of the renal la-25 hydroxycholecalciferol hydroxylase, and that specific replacement therapy could be provided by the administration of 1,25(OH)aD3. Administration of 1,25(OHIzDs was shown to be effective in the treatment of this condition. These results
10
extended the classic model of vitamin D action to man: a specific lack of 1,25(OH)zD~ synthesis leads to rickets clinically similar to that seen in vitamin D deficiency, and this rickets can be cured by the administration of the specific metabolite 1,25(OH)zD3. This would seem to constitute final proof for the model of vitamin D metabolism and action depicted in Fig. 1C. However, other studies in man suggest that the situation is considerably more complex and interesting. Because simple vitamin D deficiency rickets is now rare in the western world, and because vitamin D treatment is so effective, few, if any studies have been carried out to ascertain the effectiveness of 1,25(OH)zD~ in the treatment of this condition. However, a major group of patients with osteomalacia and/or rickets are those with chronic renal disease, particularly those maintained on hemodialysis. It was natural to assume that a major reason for development of osteomalacia or rickets in these patients was an inability of the diseased kindney to synthesize 1,25(OH)zD~ If this were the case, then treatment of these patients with 1,25(OHIzD3 should lead to dramatic improvement in their bone disease. It is not possible in this article to review the many and often conflicting reports concerning the efficiency of 1,25(OH)zD3 treatment in renal osteoHowever, if there is a consensus at dystrophy. present it is this: administration of 1,25[OHlzD3 to patients with azotemic bone disease, presenting primarily as osteitis fibrosa cystica (due presumably to marked secondary hyperparathyroidism) leads to significant clinical improvement; but administration of similar doses of 1,25(OH)zD3 to patients whose bone disease is characterized as predominantly osteomalacia leads to little change in the bone mineralization (Pierides et al., 1976). This rather unexpected experience was the first substantial evidence suggesting that in man deficiency of 1,25(OH)zD~ alone may not be the only factor in the pathogenesis of osteomalacia or rickets. However, there is one weakness in this conclusion. Patients with renal osteodystrophy have a complex set of metabolic alterations, hence there may be something metabolically different about the osteomalacia in these patients as compared to osteomalacia of simple vitamin D deficiency. On the other hand, Eastwood et al. (19761 have recently reported that the occurrence of osteomalacia in patients with chronic renal disease is associated with a decrease in the plasma concentration of 25(OHID3 They reported an inverse correlation between the extent of the mineralization front in bone, and the plasma 25[OH]D3 concentration (Fig. 2). These data raise the possibility that in man metabolites of vitamin D other than 1,25(OH)zD3 are involved in the regulation of events in bone mineralization. This suggestion has gained further credence from a comparative study of the effects of 1,25((OH)zD~, 25[OHlD,, and 24,25(OHI,D, in adult humans with vitr7min D deficiency osteomalacia (Bordier ef al., 1978). Human adults with osteomalacia secondary to vitamin D deficiency usually present with hypophosphatemia, a slightly reduced serum calcium concentration, moderately elevated serum alkaline phosphatase activity, and increased serum immunoreactive PTH. These changes are associated with a reduction in Tm por/GFR and urinary calcium excretion. The three most characteristic changes in bone are a reduction in mineralization front, an increase is osteoid volume, and a decrease in active osteoblastic surface. Depending upon the stage of the disease and the
Howard
Rasmussen
et al.: Vitamin
available surface of mineralized tion surface may be increased
.Iv 0’
iF
. . .
01 0
-0
bone, bone resorpor decreased.
0
0.
.
II and Bone
0
00
0
0
.
0
’
I
IO PLASMA
I
I 20 25(OH)D,
I
0
30
I
1
40
(na/ml)
Fig. 2. A plot of the extent of the mineralization front in bone versus the plasma 25 (OH) DB concentration in patients with simple vitamin D deficiency (O), phenobarbital-induced osteomalacia (01, osteomalacia in chronic renal insufficiency (@I, and primary hyperparathyroidism [A). The data from the latter group of patients is included to illustrate that serum phosphate per se does not determine the extent of the mineralization front because the mean plasma phosphate concentration was similar in this group as compared to the mean values found both in vitamin D deficiency and phenobarbital-induced osteomalacia. The data on the patients with renal osteodystrophy are plotted from those reported by Eastwood et a/. (1976); the data on the patients with drug-induced osteomalacia from Jubiz et a/. (1977); and the remainder from Bordier et al. (1978). The effects of treatment with either 50 pg/d of. 25(OH)D,; l-2.5 pg/d of 1,25(OH),D,; IO-26 pg 24,25(OH),D,; or a combination of 1,25(OH),D, and 24,25(OH),D, in adult humans with vitamin D deficiency osteomalacia are summarized in Table I [see Bordier et al., 1978 for details). If the model of events depicted in figure IC were the correct one, after 25[OH)D3 administration, one would expect to see the conversion of some of the 25(OH]D, to of this latter metabolite 1,25[OH)zD3. Production should then reverse the changes seen in the osteomalacic patient, hence the administration of either 25(OH)D3 or 1,25(OH)zD3 should lead to comparable changes in the bone and in the biochemical indices of mineral metabolism in these patients. This is clearly not the case [Table I). These are three changes following 25[OH)D3 administration that are not seen after 1,25[OH)zD3 therapy: al an increase in Tm POP/GFR; b) an increase in serum alkaline phosphate activity; and c) an increase in the extent of active osteoblastic surface. In addition, neither the increase in serum phosphate nor in extent of the mineralization front are as great after 1,25(OH)zD3 It is possible that these as after 25(OHlD3. differences are due to the differences in amount of the metabolite administered. However, this seems unlikely since: 1) 1,25(OH)~D3 caused a more prompt rise in serum calcium concentration than 25(OH)D3; 21 the 1,25(OH)zD3 induced rise in serum calcium concentration was associated with an increase in the absorption of calcium from the intestine and in. the excretion of calcium in the urine; 31 25[OH)D3 administration also led to an increase in intestinal calcium absorption but to a fall in urinary calcium excretion; 4) the administration of 1-2 pg/d of 1,25(OH)zD3 caused as great a rise in plasma calcium concentration, in intestinal calcium absorption, and in extent
Howard
Rasmussen
et al.:
Vitamin
Table I. Effect of Treatment for Eight Weeks of Vitamin D Deficiency in Adult Humans.
-D
Index
11
D and Bone
-D
with
vitamin
+ 25 (OH) D3
D Metabolites
-D
on Various
+ 1,25(OHlzDs
-D
Osseous
+ 24,25(OH)zDa
and Metabolic
-’
Indices
;bl$5$~S25
Serum Calcium Phosphate Alk Ptase IPTH
sl. decrease decrease increase increase
increase increase increase decrease
increase sl. increase decrease decrease
no change no change increase decrease
increase si. increase increase decrease
Urine Calcium
decrease
decrease
increase
decrease
increase
Tm,&GFR
decrease
increase
no change
no change
no change
Min. Front’ Ost. Vol.” OCRS”’
decrease increase increase
increase decrease increase
sl. increase sl. decrease increase
sl. increase sl. decrease sl. increase
increase decrease increase
OBFS’**’
decrease
increase
increase
sl. to moder. increase
increase
decrease
increase
increase
no change
intestine Calcium absorption
Mineralization Front * Osteoid Volume
l
l
of bone osteoclastic resorption surface as did the administration of 25-50 &g/d of 25(OH)D3; 6) a combination of 24,25(OH)zD3 and 1,25(OH)zD~ led to a return of the mineralization front to normal, but did not cause an increase in extent of active osteoblastic surface nor induce a return of the Tm por/GFR or serum phosphate concentration to normal (Table I); and 6) there were qualitative differences between the effects of 25(OH)D3 and 1,25(OH)zD3 on three important indices : Tm PO~GFR, active osteoblastic surface, and serum alkaline phosphatase activity. These changes cannot be due to a difference in the effect of the three metabolites on serum IPTH. All three caused a fall of IPTH toward or into the normal The correction of Tm PO~/GFR after 25(OH)D3 z?i\istration but not after either 1,25(OH)zD3 or 24,25(OH)zD3 ihdicates that 25(OH)D3, but neither of the other two metabolites, had a direct effect upon the renal tubular reabsorption of phosphate, and possibly of calcium as suggested by the work of Puschett et al. (1972, 1975). Further
evidence in support of the view that deof a metabolite, other than 1,25(OH)zD3, may be involved in the pathogenesis of osteomalacia has recently been reported by Jubiz et al. (19771. Patients receiving long term anticonvulsant therapy with phenobarbital and/or dilantin often develop osteomalacia. Our own studies (unpublished) of the bone histology and 25(OH)D3 concentrations in the plasma of such patients show that plasma 25(OH)D3 concentration is reduced, and the bone changes are indistinguishable from those seen in simple vitamin D In addition, these patients have a slightly deficiency. reduced plasma calcium concentration, moderate secondary hyperparathyroidism, elevated plasma alkaline phosphatase activity, and a low plasma phosphate concentration associated with a reduced TL.C. po4/ GFR, i.e. alterations in their biochemical indices indistinguishable from those seen in vitamin D deficiency. Jubiz et a/. (1977) report that the plasma concentrations of I ,25(OH]aD3 are either normal or increased in such patients. The implication of this result is that a reduction in the extent of the mineralization front and an increase in osteoid volume can occur in states in which the plasma. 1,25(OH)2D.~ concenficiency
* * * Osteoclast * * * * Osteoblast
Resorption Surface Formation Surface
tration is normal. In fact, under a variety of circumstances, we have found a high degree of correlation between the plasma 25(0H)D3 concentration and the extent of the mineralization front (Fig. 2). Of particular note is that this correlation was found in patients with phenobarbital-induced osteomalacia, and that a dose of 25(0HlD~ sufficient to raise the plasma 25(OH)D3 concentration to normal led to a restoration of the mineralization front to normal. These results argue that phenobarbital probably does not induce osteomalacia by a direct effect upon bone, but does so by interfering with the metabolism of vitamin D in such a way as to cause a fall in 25(OH)D3 (and possibly other metabolites), without reducing the concentration of I ,25(OH)zD3 in plasma. Although the data plotted in Figure 2 constitute strong support for some type of direct relationship between plasma 25(OH)D3 concentration and mineralization front, they do raise an additional point. The severity of the osteomalacia (as assessed by the decrease in extent of the mineralization front) seen in patients with renal osteodystrophy is inversely correlated with the plasma 25(OH)D, concentration (Eastwood et al., 1976): but for any given decrease in extent of mineralization front, the 25(OH)D, concentration is significantly higher in patients with renal osteomalacia than in patients with simple D-deficiency osteomalacia. These data mean that an additional factor must be involved in the pathogenesis of the osteomalacia of chronic renal disease. From the results of these human studies it seems reasonable to conclude that the integrated actions of several vitamin D metabolites are necessary to restore the disordered metabolism of calcium and and the multiple changes in bone phosphate, remodeling indices characteristic of the vitamin D deficient state. In terms of bone mineralization a combination of 1,25(OH)zD3 acting to stimulate osteocytic osteolysis and thus supply mineral ions, and 24,25(OH)zD~ acting to stimulate the activity of osteoid osteocytes (Fig. 31 appears necessary. In addition, 1,25(OH)zD3 increases osteoclastic (PTH-
12
dependent) bone resorption tinal absorption of calcium
Howard Rasmussen et al.: Vitamin
and increases and phosphate.
D and Bone
the intes-
The metabolite 25(OH)D3 itself (or possibly an as yet unidentified metabolite) increases the tubular reabsorption. of phosphate, and possibly calcium. These changes are all necessary for a complete biochemical’and osseous response to vitamin D and 25(OH)Ds in osteomalacic man (Fig. 4). Yet to be but determined is why administration of 25(0/i)&, not administration of a combination of 1,25(OH)zD3 and 24,25(OH)zD3 in the doses administered in these studies leads to an increase in active osteoblastic surface. It is possible that this particular effect is mediated indirectly by the 25(0H)DAnduced renal retention of phosphate and the resultant rise in serum phosphato concentration. Such a possibility must receive serious consideration in light of the fact that extent of the active osteoblastic bone surface is reduced in patients with hypophosphatemia due presumably to a renal phosphate leak (Bordier et al., 19771, and the extent of this surface increases as a result of oral phosphate treatment. On the other hand, in several patients given 24,25(OH)zD3 a rise in plasma alkaline phosphatase (unpublished data) has been seen. Only a few patients have been given 24,25(OH)zD3 to date. It is quite possible that additional work will reveal that this particular metabolite is responsible for stimulating bone formation as well as bone mineralization. Returning to a consideration of osteomalacia in renal osteodystrophy, one is struck by the fact that the patients who develop spuren osteomalacia that does not respond to treatment with 1,25(OH)zD3 are nearly all normocalcemic and do not have significant This may play a secondary hyperparathyroidism. significant, but? as yet undefined role in either the pathogenesis or therapeutic unresponsiveness In this regard it is necessary to, i,25(OH)zD3. in humans with complete to point out that hypoparathyroidism, the administration of I ,25(OH)zD3 does not lead to an increase in osteoclast number (Rasmussen and Bordier, 1974). This means that although both PTH and 1,25(OHIzD3 stimulate bone resorption and act synergistically on this process, they do not have similar cellular effects: PTH, but not I ,25(OH)zD3, enhances the rate of osteoprogenitor cell activation to preosteoclasts and osteoclasts and thereby regulates the overall rate of bone remodeling. The lack of this stimulus to remodeling in the patients with osteomalacia secondary to long standing renal disease may play a significant role in their lack of response to 1,25[OH)zD3.
Fig. 3. The proposed roles of vitamin D metabolites in controlling the process of bone mineralization. The metabolite I,25 (OH) 2D3 acts to stimulate osteolysis and thereby provide mineral ions for the formation of the mineralization front, and 24,25 (OH) 2D3 acts to stimulate the activity of the osteoid osteocytes in forming this front. This latter metabolite may also have an additional effect on surface osteoblasts (not shown).
NTESTINE
I
,
KIDNEY
Fig. 4. An integrated view of the roles of vitamin d metabolites in regulating mineral metabolism and bone remodeling processes in man. The two major non-osseous effects are: al a 1,25 (OH) 2D3- mediated increase in intestinal mineral absorption, and b) a 25 (OH) D3-mediated increase in renal tubular phosphate [and possibly calcium) reabsorption. The major actions on bone are: cl a 1.25 (OH) zD3-mediated increase in both osteocytic osteolysis and ostoclastic osteolysis [PTH-dependent); d) a 24,25 (OH) zh-mediated regulation of the activity of osteoid osteocytes at the mineralization front; and el a stimulation of osteoblastic bone formation either by the rise in plasma and extracellular fluid phosphate concentration, an increase in 24,25 (OH) 2D3, and/or a direct action of 25 (OH) D3.
Conclusion Our present
understanding of the effects of vitamin D Nevertheless, it has upon bone are incomplete. become increasingly clear that vitmain D, or more its recently discovered metabolites, correctly, influence. at least three processes in bone: a) formation; b) mineralization: and cl resorption. Recent studies in man indicate that 1,25(OH)zD3 is the major metabolite regulating intestinal calcium and phosphate absorption and bone resorption, but not bone formation ; that a combination of 1,25[OH)aDs and 24,25(OH)zD3. but neither alone, can stimulate the process of bone mineralization, as can their immediate metabolic precursor, 25(OH)D3; and that 25(OH)D3 stimulates bone formation. This latter effect may be mediated by a further metabolite, or an indirect consequence of a rise in serum phosphate concentration secondary to a 25(OHIDAnduced
This renal effect of rise in renal Tm PO~/&FR. 25(OH)D3 is unique in the sense that neither 1,25(OH)zD3 or 24,25(OH)zD3 alone or in combination have this action. Further work is necessary to define the precise mechanism of the specific stimulatory effect of 25(OH)D3 on bone formation. Definition of this mechanism is of more than academic interest because of the implication it has for our understanding of the possible therapeutic utility of vitamin D metabolites in the treatment of human metabolic bone disease.
Supported
by grants from the National Institutes of Health and by lnstitut National de la Sante’ et de la Recherche M&kale.
(AM-19813). the Kroc Foundation
Howard Rasmussen et al.: Vitamin
13
D and Bone
Acknowledgement: This was the last scientific manuscriot uoon which Doctor Bordier and I worked before his un$nejy death. As such it represents part of his legacy to research in human metabolic bone disease and is a fitting contribution to the journal he helped found.
References Anderson, H.C.. Matsuzawa. T., Sajdera. S.W., Ali, S.Y.. Membranous partices in calcifying cartilage matrix. Trans. N.Y. Acad. Sci. 32: 619-630. 1970. Baylink, D., Stauffer, M., Wergedal, J., Rich. C.: Formation, mineralization, and resorption of bone in vitamin D-deficient rats. J. C/in. Invest. 49: 1122-1139. 1970. Bordier, P., Rasmussen, H., Marie, P., Miravet. L., Gueris, J., Ryckewaert, A.: Vitamin D metabolites and bone mineralization in man. J. C/in. Endocrine/. Metab. 46 : 284-294, 1978. Bordier, P., Ryckewaert, A., Gueris, J., Rasmussen, H.: On the pathogenesis of so-called idiopathic hypercalciuria. Am. J. Med. 63: 398409, 1977. Carlsson, A.: Tracer experiments on the effect of vitamin D on the skeletel metabolism of calcium and phosphorus. Acta Physiol. [Stand). 26: 212-220, 1952. DeLuca, H.F.: Vitamine D: The vitamin Fed. Proc. 33: 221-229, 1974.
and the
hormone.
Eastwood, J.B.. Harris, E., Stamp. T.C.B., dewardener, Vitamin-D deficiency renal failure. Lancet.
in the osteomalacia of 2: 1209-1211, 1976.
H.E.: chronic
Haussler, MR., McCain, T.P.: Basic and clinical related to vitamin Eng. J. Med. 297:
concepts D metabolism and action. New 974-983. and 1041-1050, 1977.
Howell, D., Pita, J.C., Alvarez, cellular matrix vesicles healing rachitic cartilage. 1976.
J.: Possible role of extrain initial calcification of Fed. Proc. 35: 122-126.
Jubiz, W.. Haussler, M.R., McCain, T.A., Tolman, K.G.: Plasma 1,25-dihydroxyvitamin D levels in patients receiving anticonvulsant drugs. Metab. 44: 617-62.1. 1977.
Nicolaysen, siology
J.
C/in.
Pierides, A.M., Simpson, W., Ward, M.K., Ellis, H.A., Dewar. J.H., Kerr, D.N.S.: Variable response to long term lrhydroxycholecalciferol in haemodialysis osteodystrophy. Lancet 1: 1092-1095. 1976. Popovtzer, M.M., Malthay, R., Alfrey. A.C., Block, M., Beck, P., Miles, J., Reeve, E.B.: Vitamin D deficiency osteomalacia-Healing of the bone disease in the absence of vitamin D with intravenous calcium and phosphorus infusion. In: Clinical Aspects of Metabolic Bone Disease. B., Frame, A.M., Parfitt. H., Duncan, eds.. Amsterdam: Excerpta Medica. pp. 382-387. 1973. Puschett, J.B., Beck, W.S. Jr., Jelonels, A.: Parathyroid hormone and 25-hydroxyvitamin D3: Synergistic and antagonistic effects on renal phosphate transport. Science. 190: 473-475. 1975. Puschett, J.B., Moranz, J., Kurnick, W.S.: Evidence for a direct action of cholecalciferol and 25-hydroxycholecalciferol on the renal transport of phosphate, sodium 51: 373-385, 1972. and calcium. J. C/in. Invest. Raisz. L.G., Trummel, CL., Holick, M.F., DeLuca, H.F.: 1.25dehydroxycholecalciferol: A potent stimulator of bone resorption in tissue culture. Science. 175: 768-769, 1972. Rasmussen, H.. Bordier, P.J.: The Physiological lar Basis of Metabolic Bone Disease. Williams and Wilkins, 1974.
and CelluBaltimore:
Rasmussen, H.. DeLuca, H., Arnaud, C., Hawker, C.H., von Stedina. K.M.: The relationshio between vitamin D and parathyroid hormone. J. k/in. Invest. 42: .t-9401946, 1963. Reade, T.M., Striver, C.R., Glorieux, F.H., Nogrady, B., Delvin, E., Poirier, R., Holick. M.F., DeLuca, H.F.:
Response to crystalline la-hydroxyvitamin D3 in vitamin D dependency. Ped. Res. 9: 593-599, 1975. Tanaka, Y., DeLuca. H.F.: Role of 1.25-dehydroxyvitamin D3 in maintaining serum phosphorus and curing rickets. Proc.
Nat.
Acad.
Sci.
USA.
71:
1040-1044,
1974.
Endocrine/.
R.. Eeg-Larsen. N.: The biochemistry and phyof vitamin D. Vitam. Horm. Ii: 29-69. 1953.
Received: Accepted:
April May
3. 1978 14, 1978
Bone
Dis.
Metabolic Bone Disease 8 Related Research
& Ref. Res. 1, 7-13 (1978)
@ by S.N.P.M.D.
Review
1978)
article
Vitamin
D and Bone
HOWARD
RASMUSSEN
Departments of Internal and the Andre Lichtwiiz Address
versity
(Paris
for
AND
PHILIPPE
Medicine Research
correspondance
School of Medicine,
BORDIER
*
and Cell Biology Yale University Unit, Hdpital LariboisiBre, Paris,
and reprints: H. Rasmussen, Department New Haven, Connecticut 06510
School of Medicine, France.
of Internal
New Haven,
Medicine
and Cell
Connecticut
06570
Biology-Yale Uni-
Abstract
1
The discovery of vitamin D came about as a result of the study of the pathogenesis of clinical and experimental rickets. Hence, from the moment of its discovery attention has been focused on the relationship between vitamin D action and bone physiology. With the recent elucidation of vitamin D metabolism (Deluca, 1974) this interest has been further stimulated. Newly discovered, biologically active vitamin D metabolites are being widely tested as possible therapeutic agents in a variety of metabolic bone diseases ranging from hypoparathyroidism and osteoporosis to renal osteodystrophy.
Key Words: Vitamin Calcification.
Historical
D - 1.25 DHCC - 25 HCC - Bone -
Review
Initial attempts at defining the relationship between vitamin D function and bone focused on the most obvious feature of the bone lesion in vitamin D deficiency: the failure of proper mineralization. However, attempts to demonstrate a direct action of vitamin D on this process were unsuccessful. On the other hand, a marked effect of vitamin D on the intestinal absorption of calcium and phosphate was readily demonstrable. This fact, coupled to the observation that rachitic cartilage became mineralized when incubated in vitro in solutions containing normal amounts of calcium and phosphate, led to the theory that the skeletal changes in vitamin D deficiency are an indirect consequence of a defect in intestinal calcium and phosphate absorption (Nicolaysen and Eeg-Larsen, 1953) (Fig. lA]. This view had to be altered as a result of the work of Carlsson (1952). He and his coworkers demonstrated that the administration of vitamin D to a vitamin D deficient, calcium starved animal led to an increase in the serum calcium concentration and a healing of the rickets. They concluded that vitamin D brought about this change by a direct effect upon bone resorption. Later evidence has confirmed this view and shown that this action of vitamin D is dependent upon the presence of parathyroid hormone (Rasmussen et al., 1963). When the parathyroid glands are intact, the administration of vitamin D to a vitamin D deficient, calcium starved
l
Deceased May 25, 1977.
I, 25(OHI,D,-
25 (OH), D3*
‘D3 I
Fig. 1. The historical
progression of the concept of how vitamin D regulates bone mineralization. A. The first concept in which vitamin D regulated the process of mineralization indirectly by contYolling the mineral ion product of the extracellular fluids by reaulatina the intestinal absorption of calcium and phosphaie. B. The second concept in which the vitamin regulated the mineral ion product by controlling both intestinal mineral absorption and reabsorption of mineral from calcified bone. C.’ The third concept in which the vitamin D metabolite, 1,25 (OH) 203, regulated the mineral ion content by stimulating mineral ion absorption and bone resorption. rat causes an increase in bone resorption with a consequent mobilization of calcium and phosphate from the old mineralized bone and a simultaneous disappearance of the old matrix. The mineral in large part is reincorporated into the previously uncalcified matrix or osteoid leading to a healing of the rickets and osteomalacia. Thus, the total amount of bone matrix decreases, but the percentage of calcified matrix increases. These discoveries were of great importance because they established unequivocally that vitamin D acted directly on bone. Paradoxically, however, this direct
8
Howard Rasmussen et al.: Vitamin
effects was to enhance bone mineral mobilization rather than to increase bone mineral deposition. In fact, these studies provided support for the original view of the action of vitamin D on bone mineralization. In this amended view (Fig. 161, the combination of the actions of vitamin D on intestinal mineral ion absorption and on mineral ion resorption from bone leads to a rise in the calcium times phosphate product in the plasma and extracellular fluids. Once this product exceeds some critical value, bone mineralization ensues. Further support for this widely held point of view has come from the study of the actions of the newly discovered metabolites of vitamin D, particularly I ,25hydroxyvitamin D3 (1,25(OH)zD3). This metabolite is made in the kidney from 25hydroxyvitamin D3 (25(OH)D3). It acts more rapidly and is more pOtWIt biologically than either vitamin D or 25(OH)D 1974; Haussler and McCain, 1977). (DeLuca, Furthermore, it has been shown to stimulate intestinal calcium and phosphate absorption both in vivo and in vitro (DeLuca, 1974; Haussler and McCain. 19771, and to directly stimulate bone resorption in vitro (Raisz et al., 1972). In addition, it has been reported to be antirachitic in animals (Tanaka and Thus, the accepted theory would DeLuca, 1974). appear complete with 1,25(OH)aD3 being the final biologically active form of vitamin D which, by acting on the resorption of bone and on the intestinal absorption of calcium and phosphate, corrects the state .of vitamin D deficiency (Fig. IC). A final, further argument in support of this view is that the infusion of calcium and phosphate into D-deficient humans at a rate sufficient to raise the plasma mineral ion product to normal leads to remineralization of bone (Popovtzer et al., 1973). In spite of this impressive evidence, recent data suggest that the relationship between vitamin D and bone may be more complex. This recent evidence has been obtained largely from studies in man (Bordier et al., 1978) and can best be understood in the context of our present knowledge of bone resorption, bone formation, and bone mineralization.
Vitamin D and Bone Resorption
_
The major vitamin D metabolite concerned with bone resorption is clearly 1,25(OH)zD3. It has been shown to stimulate bone resorption in organ cultures of rat calvaria in vitro, and in D-deficient animals or man in vivo (Haussler and McCain, 1977). For a time, it was considered possible that 1,25(OH)zD3 was the physiologically important regulator of bone resorption, and that the major effect of PTH upon bone resorption was mediated indirectly via its control of 1,25[OH)zD3 synthesis. However, there are three strong arguments against this view: 1) PTH, but not 1,25(OH)zD3, increases the cyclic AMP content of bone; 2) when given in vivo the two agents act synergistically; and 3) when 1,25(OH)zD3 is given to a parathyroid deficient human, it does not increase the number of osteoclasts on bone surfaces although it does increase osteocytic osteolysis (Rasmussen and Bordier, 1974). It is evident that PTH exerts a major control over rate of conversion of osteoprogenitor cells to osteoclasts. Additionally although 1,25(OH)zD3 may enhance this effect and also act to stimulate the resorptive activity of preformed osteoclasts and osteocytes, 1,25(OH)zD3 does not stimulate osteoprogenltor cell activation in the absence of PTH. It is our view that the effect of 1,25(OH)zD3 upon osteocytic osteolysis results in the
D and Bone
mobilization of calcium and phosphate from old mineralized bone and raises the local ion product at the bone surface. This potentiates the effect of PTH on osteoprogenitor cell activation because both CAMP and calcium ion have second messenger functions in the activation of these cells (Rasmussen and Bordier, 1974).
Vitamin D and Bone Formation It is vital in any discussion of bone physiology to distinguish between bone formation and mineralization. The latter is often employed incorrectly as a measure of bone formation since, under normal circumstances, mineralization and formation are closely coupled (Rasmussen and Bordier, 1974). However, this is clearly not the case in states of vitamin D In order for rickets and osteomalacia deficiency. to develop, the rate of bone formation (correctly defined as the rate of bone matrix deposition) must Because of this exceed the rate of mineralization. fact, it is generally held that vitamin D has no effect However, this view is not on bone formation. supported by the available evidence. In their studies of experimental vitamin D deficiency in rats Baylink and associates (1970) have shown that bone formation rate, as well as bone mineralization rate, is decreased. However, the amount of osteoid is increased because the mineralization rate is more severely depressed than the bone formation rate. It is not possible to make precise measurements of bone formation rate in man. What is usually found in the bone biopsy material from D-deficient adults is a slight increase in the extent of the active bone formation surface. However, on such surfaces the histological appearance of the osteoblasts is strikingly abnormal (Rasmussen and Bordier, 1974). They are considerably less basophilic and contain more glycogen. These changes have been taken as an indication of a decrease in the matrix-forming activity of these cells. Evidence supporting an increase in bone formation rate is the finding of an elevation in plasma alkaline phosphatase concentration in the patients with osteomalacia. Under a number of circumstances there is a fairly good correlation between the extent of osteoblastic bone formation surface measured by quantitative analysis of bone biopsy material and the plasma alkaline phosphatase activity (Rasmussen As a consequence plasma and Bordier, 1974). alkaline phosphatase has long been considered a measure of bone formation under all circumstances. Under most circumstances this may be true because there is a coupling between bone formation and the subsequent process of bone mineralization. Hence, when the first is increased the second is also increased. Recent evidence (see below) shows that mineralization is under cellular control, and that membrane bound vesicles derived from osteoid osteocytes are the probable site of initial mineral crystal formation (Anderson et al., 1970; Howell et al., 1976). Germaine to the present discussion is the fact that these vesicles are rich in alkaline phosphatase, and these vesicles form in the bone of D-deficient animals. Thus, it is quite possible that the increase in plasma alkaline phosphatase observed in patients with osteomalacia is a reflection of an increase in osteoid surface and therefore of vesicle number and not an increase in osteoblastic activity. Equally difficult to interpret is the observation that a rise in plasma alkaline phosphatase activity occurs after the administration of vitamin D to patients with
Howard
Rasmussen
et al.:
Vitamin
9
D and Bone
osteomalacia. This may result from an acceleration of the mineralization process, a stimulation of osteoblastic bone formation, or an increase in the turnover of matrix vesicles secondary to the restoration of mineralization rate toward normal.
vesicle formation, or the ability of these vesicles to take up calcium. It is clear, however, that vesicle formation continues in the absence of vitamin D because vesicles are found in the bone and cartilage of D-deficient animals (Howell et al., 19761.
From animal studies it is clear that vitamin D administration to a D-deficient animal stimulates bone formation (Baylink et al.. 1970). Additionally, studies of bone biopsy material in man, show that vitamin D administration leads to a rapid change in the histological appearance of osteoblasts, indicative of an increase in their bone matrix synthetic activity, and a more gradual but significant increase in the extent of the osteoblastic bone formation surface (Rasmussen and Bordier, 1974). A complete discussion of the data is beyond the scope of the present review. However, one additional line of evidence in support of our previous conclusion that plasma phosphate concentration is a determinant of the extent of the osteoblastic bone formation surface is the finding of a reduction in the extent of this surface in patients with a syndrome of hypercalciuria, hypophosphatemia and osteoporosis without hyperparathyroidism. In these patients the defect is presumed to be a primary reduction in the renal phosphate threshold (TmP/GFRI (Bordier et al., 1977).
Vitamin
Vitamin D and Bone Mineralization There is, as discussed above, unequivocal evidence that vitamin D deficiency leads to a reduction in the rate of bone mineralization. The classical view of this process was that once collagen was laid down and underwent some type of maturation, it would then serve as a template for the nucleation of bone mineral crystals. The rate of mineralization was simply a function of the mineral ion product in plasma and extracellular fluids. However, two more recent discoveries argue against this simplistic view. The first is that if sections of bone taken from D-deficient patients who have been treated with calcium and phosphate are compared with similar sections taken from patients treated with vitamin D. there is a crucial difference in the pattern of mineralization (Rasmussen and Bordier, 1974). In the sections from the mineral ion-treated patients, the mineralization is randomly distributed in patches throughout the osteoid with islands of unmineralized osteoid between these patches. On the other hand, the hallmark of vitamin D deficiency is a reduction in the extent of the mineralization front: vitamin D action results in the reinstitution of an orderly process of mineralized bone, and then a progression of this mineralization front toward the bone surface (in reference 81. Thus, a vitamin D metabolite (or metabolites) exerts some directive effect on the process of mineralization. The second discovery is that mineralization is under cellular control (Anderson et al., 1970: Howell et al., 1976); in bone these cells are the osteoid osteocytes and, in cartilage, the cells in the zone of provisional Both types of cells produce small calcification. membrane-bound extracellular matrix vesicles which probably bud off from their respective plasma membranes. The membranes of these vesicles contain high concentrations of alkaline phosphatase and are thought to be the sites of formation of the initial
bone mineral crystals. It seems likely that vitamin D or one of its metabolites affects the activity of these cells, although its precise mode of action is not known. Vitamin D might Influence mineral ion exchange in these cells, the rate or extent of matrix
D and the Kidney
Although the emphasis in the present discussion is on the relationship between vitamin D and bone, a brief discussion of the effects of vitamin D on renal function is necessary to understand the vitamin D-bone relationship. One of the characteristic results of vitamin D administration to D-deficient man is a rise in the Tm for renal phosphate reabsorption (Tm PO~/GFR). Because secondarv hyperparathyroidism is a feature of the vitamin D deficient state, and because plasma PTH concentrations fall after vitamin D administration, this rise in Tm PO~/GFR has been attributed to the fall in PTH. However, recent evidence indicates that 25 (OHID or a metabolite other than I ,25(0H)~D3, can increase the proximal renal tubular reabsorption of calcium and phosphate (Puschett et al., 1972; 19751. Hence, the rise in Tm PO~/GFR is probably a consequence of both a direct renal action of 25IOHID3 (or some unidentified metabolite) and a fall in plasma PTH. Vitamin D Metabolites Osteomalacia
in
Human
Rickets
and
Much of the recent work dealing with vitamin D metabolism and action has been carried out in the vitamin D deficient rat. In general, the work in this species has confirmed the classic view of vitamin D and bone as discussed above. In particular, administration of 1,25(OH)zD3 to the vitamin D deficient rat has been shown to be antirachitic (Tanaka and DeLuca, 1974). However, there is an important difference between rat and man. The rat normally has a plasma phosphate concentration of IO-12 mg/dl whereas infant humans have values approximately half these, and adult men values in the range of 3-4.5 mg/dl. Furthermore, the most common way of inducing rickets in the rat is to reduce the dietary content of both vitamin D and phosphate. In contrast, most human adults with osteomalacia have low phosphate concentration even on a normal phosphate intake and have only a slightly reduced plasma calcium concentration. Based on these differences, it is reasonable to suggest that there may be differences between rat and man as regards the physiology, metabolism, and function of vitamin D and its metabolites. A critical question to be answered is whether or not 1,25IOH)zD3 can cure rickets and/or osteomalacia in man. Vitamin D-dependency rickets was one of the first disorders to be treated with 1,25(OH)aD3 (Reade et al., 1975). This is a disease which presents early in life and clinically is indistinguishable from D-deficiency rickets except that vitmain D intake is normal, plasma 25(OHID3 concentrations are also
normal, and the patients fail to respond to physiological amounts of vitamin D. It was proposed that this condition was due to a specific deficiency of the renal la-25 hydroxycholecalciferol hydroxylase, and that specific replacement therapy could be provided by the administration of 1,25(OH)aD3. Administration of 1,25(OHIzDs was shown to be effective in the treatment of this condition. These results
10
extended the classic model of vitamin D action to man: a specific lack of 1,25(OH)zD~ synthesis leads to rickets clinically similar to that seen in vitamin D deficiency, and this rickets can be cured by the administration of the specific metabolite 1,25(OH)zD3. This would seem to constitute final proof for the model of vitamin D metabolism and action depicted in Fig. 1C. However, other studies in man suggest that the situation is considerably more complex and interesting. Because simple vitamin D deficiency rickets is now rare in the western world, and because vitamin D treatment is so effective, few, if any studies have been carried out to ascertain the effectiveness of 1,25(OH)zD~ in the treatment of this condition. However, a major group of patients with osteomalacia and/or rickets are those with chronic renal disease, particularly those maintained on hemodialysis. It was natural to assume that a major reason for development of osteomalacia or rickets in these patients was an inability of the diseased kindney to synthesize 1,25(OH)zD~ If this were the case, then treatment of these patients with 1,25(OHIzD3 should lead to dramatic improvement in their bone disease. It is not possible in this article to review the many and often conflicting reports concerning the efficiency of 1,25(OH)zD3 treatment in renal osteoHowever, if there is a consensus at dystrophy. present it is this: administration of 1,25[OHlzD3 to patients with azotemic bone disease, presenting primarily as osteitis fibrosa cystica (due presumably to marked secondary hyperparathyroidism) leads to significant clinical improvement; but administration of similar doses of 1,25(OH)zD3 to patients whose bone disease is characterized as predominantly osteomalacia leads to little change in the bone mineralization (Pierides et al., 1976). This rather unexpected experience was the first substantial evidence suggesting that in man deficiency of 1,25(OH)zD~ alone may not be the only factor in the pathogenesis of osteomalacia or rickets. However, there is one weakness in this conclusion. Patients with renal osteodystrophy have a complex set of metabolic alterations, hence there may be something metabolically different about the osteomalacia in these patients as compared to osteomalacia of simple vitamin D deficiency. On the other hand, Eastwood et al. (19761 have recently reported that the occurrence of osteomalacia in patients with chronic renal disease is associated with a decrease in the plasma concentration of 25(OHID3 They reported an inverse correlation between the extent of the mineralization front in bone, and the plasma 25[OH]D3 concentration (Fig. 2). These data raise the possibility that in man metabolites of vitamin D other than 1,25(OH)zD3 are involved in the regulation of events in bone mineralization. This suggestion has gained further credence from a comparative study of the effects of 1,25((OH)zD~, 25[OHlD,, and 24,25(OHI,D, in adult humans with vitr7min D deficiency osteomalacia (Bordier ef al., 1978). Human adults with osteomalacia secondary to vitamin D deficiency usually present with hypophosphatemia, a slightly reduced serum calcium concentration, moderately elevated serum alkaline phosphatase activity, and increased serum immunoreactive PTH. These changes are associated with a reduction in Tm por/GFR and urinary calcium excretion. The three most characteristic changes in bone are a reduction in mineralization front, an increase is osteoid volume, and a decrease in active osteoblastic surface. Depending upon the stage of the disease and the
Howard
Rasmussen
et al.: Vitamin
available surface of mineralized tion surface may be increased
.Iv 0’
iF
. . .
01 0
-0
bone, bone resorpor decreased.
0
0.
.
II and Bone
0
00
0
0
.
0
’
I
IO PLASMA
I
I 20 25(OH)D,
I
0
30
I
1
40
(na/ml)
Fig. 2. A plot of the extent of the mineralization front in bone versus the plasma 25 (OH) DB concentration in patients with simple vitamin D deficiency (O), phenobarbital-induced osteomalacia (01, osteomalacia in chronic renal insufficiency (@I, and primary hyperparathyroidism [A). The data from the latter group of patients is included to illustrate that serum phosphate per se does not determine the extent of the mineralization front because the mean plasma phosphate concentration was similar in this group as compared to the mean values found both in vitamin D deficiency and phenobarbital-induced osteomalacia. The data on the patients with renal osteodystrophy are plotted from those reported by Eastwood et a/. (1976); the data on the patients with drug-induced osteomalacia from Jubiz et a/. (1977); and the remainder from Bordier et al. (1978). The effects of treatment with either 50 pg/d of. 25(OH)D,; l-2.5 pg/d of 1,25(OH),D,; IO-26 pg 24,25(OH),D,; or a combination of 1,25(OH),D, and 24,25(OH),D, in adult humans with vitamin D deficiency osteomalacia are summarized in Table I [see Bordier et al., 1978 for details). If the model of events depicted in figure IC were the correct one, after 25[OH)D3 administration, one would expect to see the conversion of some of the 25(OH]D, to of this latter metabolite 1,25[OH)zD3. Production should then reverse the changes seen in the osteomalacic patient, hence the administration of either 25(OH)D3 or 1,25(OH)zD3 should lead to comparable changes in the bone and in the biochemical indices of mineral metabolism in these patients. This is clearly not the case [Table I). These are three changes following 25[OH)D3 administration that are not seen after 1,25[OH)zD3 therapy: al an increase in Tm POP/GFR; b) an increase in serum alkaline phosphate activity; and c) an increase in the extent of active osteoblastic surface. In addition, neither the increase in serum phosphate nor in extent of the mineralization front are as great after 1,25(OH)zD3 It is possible that these as after 25(OHlD3. differences are due to the differences in amount of the metabolite administered. However, this seems unlikely since: 1) 1,25(OH)~D3 caused a more prompt rise in serum calcium concentration than 25(OH)D3; 21 the 1,25(OH)zD3 induced rise in serum calcium concentration was associated with an increase in the absorption of calcium from the intestine and in. the excretion of calcium in the urine; 31 25[OH)D3 administration also led to an increase in intestinal calcium absorption but to a fall in urinary calcium excretion; 4) the administration of 1-2 pg/d of 1,25(OH)zD3 caused as great a rise in plasma calcium concentration, in intestinal calcium absorption, and in extent
Howard
Rasmussen
et al.:
Vitamin
Table I. Effect of Treatment for Eight Weeks of Vitamin D Deficiency in Adult Humans.
-D
Index
11
D and Bone
-D
with
vitamin
+ 25 (OH) D3
D Metabolites
-D
on Various
+ 1,25(OHlzDs
-D
Osseous
+ 24,25(OH)zDa
and Metabolic
-’
Indices
;bl$5$~S25
Serum Calcium Phosphate Alk Ptase IPTH
sl. decrease decrease increase increase
increase increase increase decrease
increase sl. increase decrease decrease
no change no change increase decrease
increase si. increase increase decrease
Urine Calcium
decrease
decrease
increase
decrease
increase
Tm,&GFR
decrease
increase
no change
no change
no change
Min. Front’ Ost. Vol.” OCRS”’
decrease increase increase
increase decrease increase
sl. increase sl. decrease increase
sl. increase sl. decrease sl. increase
increase decrease increase
OBFS’**’
decrease
increase
increase
sl. to moder. increase
increase
decrease
increase
increase
no change
intestine Calcium absorption
Mineralization Front * Osteoid Volume
l
l
of bone osteoclastic resorption surface as did the administration of 25-50 &g/d of 25(OH)D3; 6) a combination of 24,25(OH)zD3 and 1,25(OH)zD~ led to a return of the mineralization front to normal, but did not cause an increase in extent of active osteoblastic surface nor induce a return of the Tm por/GFR or serum phosphate concentration to normal (Table I); and 6) there were qualitative differences between the effects of 25(OH)D3 and 1,25(OH)zD3 on three important indices : Tm PO~GFR, active osteoblastic surface, and serum alkaline phosphatase activity. These changes cannot be due to a difference in the effect of the three metabolites on serum IPTH. All three caused a fall of IPTH toward or into the normal The correction of Tm PO~/GFR after 25(OH)D3 z?i\istration but not after either 1,25(OH)zD3 or 24,25(OH)zD3 ihdicates that 25(OH)D3, but neither of the other two metabolites, had a direct effect upon the renal tubular reabsorption of phosphate, and possibly of calcium as suggested by the work of Puschett et al. (1972, 1975). Further
evidence in support of the view that deof a metabolite, other than 1,25(OH)zD3, may be involved in the pathogenesis of osteomalacia has recently been reported by Jubiz et al. (19771. Patients receiving long term anticonvulsant therapy with phenobarbital and/or dilantin often develop osteomalacia. Our own studies (unpublished) of the bone histology and 25(OH)D3 concentrations in the plasma of such patients show that plasma 25(OH)D3 concentration is reduced, and the bone changes are indistinguishable from those seen in simple vitamin D In addition, these patients have a slightly deficiency. reduced plasma calcium concentration, moderate secondary hyperparathyroidism, elevated plasma alkaline phosphatase activity, and a low plasma phosphate concentration associated with a reduced TL.C. po4/ GFR, i.e. alterations in their biochemical indices indistinguishable from those seen in vitamin D deficiency. Jubiz et a/. (1977) report that the plasma concentrations of I ,25(OH]aD3 are either normal or increased in such patients. The implication of this result is that a reduction in the extent of the mineralization front and an increase in osteoid volume can occur in states in which the plasma. 1,25(OH)2D.~ concenficiency
* * * Osteoclast * * * * Osteoblast
Resorption Surface Formation Surface
tration is normal. In fact, under a variety of circumstances, we have found a high degree of correlation between the plasma 25(0H)D3 concentration and the extent of the mineralization front (Fig. 2). Of particular note is that this correlation was found in patients with phenobarbital-induced osteomalacia, and that a dose of 25(0HlD~ sufficient to raise the plasma 25(OH)D3 concentration to normal led to a restoration of the mineralization front to normal. These results argue that phenobarbital probably does not induce osteomalacia by a direct effect upon bone, but does so by interfering with the metabolism of vitamin D in such a way as to cause a fall in 25(OH)D3 (and possibly other metabolites), without reducing the concentration of I ,25(OH)zD3 in plasma. Although the data plotted in Figure 2 constitute strong support for some type of direct relationship between plasma 25(OH)D3 concentration and mineralization front, they do raise an additional point. The severity of the osteomalacia (as assessed by the decrease in extent of the mineralization front) seen in patients with renal osteodystrophy is inversely correlated with the plasma 25(OH)D, concentration (Eastwood et al., 1976): but for any given decrease in extent of mineralization front, the 25(OH)D, concentration is significantly higher in patients with renal osteomalacia than in patients with simple D-deficiency osteomalacia. These data mean that an additional factor must be involved in the pathogenesis of the osteomalacia of chronic renal disease. From the results of these human studies it seems reasonable to conclude that the integrated actions of several vitamin D metabolites are necessary to restore the disordered metabolism of calcium and and the multiple changes in bone phosphate, remodeling indices characteristic of the vitamin D deficient state. In terms of bone mineralization a combination of 1,25(OH)zD3 acting to stimulate osteocytic osteolysis and thus supply mineral ions, and 24,25(OH)zD~ acting to stimulate the activity of osteoid osteocytes (Fig. 31 appears necessary. In addition, 1,25(OH)zD3 increases osteoclastic (PTH-
12
dependent) bone resorption tinal absorption of calcium
Howard Rasmussen et al.: Vitamin
and increases and phosphate.
D and Bone
the intes-
The metabolite 25(OH)D3 itself (or possibly an as yet unidentified metabolite) increases the tubular reabsorption. of phosphate, and possibly calcium. These changes are all necessary for a complete biochemical’and osseous response to vitamin D and 25(OH)Ds in osteomalacic man (Fig. 4). Yet to be but determined is why administration of 25(0/i)&, not administration of a combination of 1,25(OH)zD3 and 24,25(OH)zD3 in the doses administered in these studies leads to an increase in active osteoblastic surface. It is possible that this particular effect is mediated indirectly by the 25(0H)DAnduced renal retention of phosphate and the resultant rise in serum phosphato concentration. Such a possibility must receive serious consideration in light of the fact that extent of the active osteoblastic bone surface is reduced in patients with hypophosphatemia due presumably to a renal phosphate leak (Bordier et al., 19771, and the extent of this surface increases as a result of oral phosphate treatment. On the other hand, in several patients given 24,25(OH)zD3 a rise in plasma alkaline phosphatase (unpublished data) has been seen. Only a few patients have been given 24,25(OH)zD3 to date. It is quite possible that additional work will reveal that this particular metabolite is responsible for stimulating bone formation as well as bone mineralization. Returning to a consideration of osteomalacia in renal osteodystrophy, one is struck by the fact that the patients who develop spuren osteomalacia that does not respond to treatment with 1,25(OH)zD3 are nearly all normocalcemic and do not have significant This may play a secondary hyperparathyroidism. significant, but? as yet undefined role in either the pathogenesis or therapeutic unresponsiveness In this regard it is necessary to, i,25(OH)zD3. in humans with complete to point out that hypoparathyroidism, the administration of I ,25(OH)zD3 does not lead to an increase in osteoclast number (Rasmussen and Bordier, 1974). This means that although both PTH and 1,25(OHIzD3 stimulate bone resorption and act synergistically on this process, they do not have similar cellular effects: PTH, but not I ,25(OH)zD3, enhances the rate of osteoprogenitor cell activation to preosteoclasts and osteoclasts and thereby regulates the overall rate of bone remodeling. The lack of this stimulus to remodeling in the patients with osteomalacia secondary to long standing renal disease may play a significant role in their lack of response to 1,25[OH)zD3.
Fig. 3. The proposed roles of vitamin D metabolites in controlling the process of bone mineralization. The metabolite I,25 (OH) 2D3 acts to stimulate osteolysis and thereby provide mineral ions for the formation of the mineralization front, and 24,25 (OH) 2D3 acts to stimulate the activity of the osteoid osteocytes in forming this front. This latter metabolite may also have an additional effect on surface osteoblasts (not shown).
NTESTINE
I
,
KIDNEY
Fig. 4. An integrated view of the roles of vitamin d metabolites in regulating mineral metabolism and bone remodeling processes in man. The two major non-osseous effects are: al a 1,25 (OH) 2D3- mediated increase in intestinal mineral absorption, and b) a 25 (OH) D3-mediated increase in renal tubular phosphate [and possibly calcium) reabsorption. The major actions on bone are: cl a 1.25 (OH) zD3-mediated increase in both osteocytic osteolysis and ostoclastic osteolysis [PTH-dependent); d) a 24,25 (OH) zh-mediated regulation of the activity of osteoid osteocytes at the mineralization front; and el a stimulation of osteoblastic bone formation either by the rise in plasma and extracellular fluid phosphate concentration, an increase in 24,25 (OH) 2D3, and/or a direct action of 25 (OH) D3.
Conclusion Our present
understanding of the effects of vitamin D Nevertheless, it has upon bone are incomplete. become increasingly clear that vitmain D, or more its recently discovered metabolites, correctly, influence. at least three processes in bone: a) formation; b) mineralization: and cl resorption. Recent studies in man indicate that 1,25(OH)zD3 is the major metabolite regulating intestinal calcium and phosphate absorption and bone resorption, but not bone formation ; that a combination of 1,25[OH)aDs and 24,25(OH)zD3. but neither alone, can stimulate the process of bone mineralization, as can their immediate metabolic precursor, 25(OH)D3; and that 25(OH)D3 stimulates bone formation. This latter effect may be mediated by a further metabolite, or an indirect consequence of a rise in serum phosphate concentration secondary to a 25(OHIDAnduced
This renal effect of rise in renal Tm PO~/&FR. 25(OH)D3 is unique in the sense that neither 1,25(OH)zD3 or 24,25(OH)zD3 alone or in combination have this action. Further work is necessary to define the precise mechanism of the specific stimulatory effect of 25(OH)D3 on bone formation. Definition of this mechanism is of more than academic interest because of the implication it has for our understanding of the possible therapeutic utility of vitamin D metabolites in the treatment of human metabolic bone disease.
Supported
by grants from the National Institutes of Health and by lnstitut National de la Sante’ et de la Recherche M&kale.
(AM-19813). the Kroc Foundation
Howard Rasmussen et al.: Vitamin
13
D and Bone
Acknowledgement: This was the last scientific manuscriot uoon which Doctor Bordier and I worked before his un$nejy death. As such it represents part of his legacy to research in human metabolic bone disease and is a fitting contribution to the journal he helped found.
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Received: Accepted:
April May
3. 1978 14, 1978