1,25(OH)2D3Is Not the Only D Metabolite Involved in the Pathogenesis of Osteomalacia HOWARD RASMUSSEN, M.D. ROLAND BARON, M.D. ARTHUR BROADUS. M.D. RALPH DeFRONZO, M.D. ROBERT LANG, M.D. New Haven, Connecticut RONALD HORST, M.D. Ames, lowo From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut: and the U.S. Department of Agri- culture Scientific and Educational Administra- tion, Ames, Iowa. This work was supported by an NIH SCOR Grant [AM 20570). Clinical studies were performed in the Clinical Research Center of the Yale-New Haven Hospital and were supported by a grant (RP-1251 from the General Clinical Research Centers Branch, Division of Research Resources, NIH. Requests for reprints should be addressed to Dr. Howard Rasmussen, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. Manu- script accepted March 17,198O. Three patients are described in whom there was no simple correla- tion between plasma 1,25(OHhD3 concentration and the occurrence of osteomalacia. One patient had severe osteomalacia with high plasma 1,25(OHbD3 and normal mineral ion product; the second had a normal mineral ion product and no evidence of osteomalacia even though plasma 1,25(OH)zD3was undetectable; and the third had os- teomalacia, low plasma 1,25(OH)zD3 and a reduced mineral ion product. In considering these data in the light of presently available information, it is concluded that osteomalacia can occur as a conse- quence of a lack of a vitamin D metabolite other than l$5(OHbD3, or as a consequence of a reduced mineral ion product, but not as a consequence of 1,25(OHbD3 lack if the mineral ion product is nor- mally maintained and other D metabolites are present. However, a deficiency of 1,25(OHhD3 normally leads to a reduction in the mineral ion product hence 1,25(OHhD3 deficiency may play a role in the development of certain forms of osteomalacia. It has become widely accepted that l,Z&dihydroxyvitamin DB [1,25(0H)~D~] is the only important biologically active form of vitamin D3 in man [l-8]. However, there is evidence that other metabolites of vitamin D3 may also be important in the biologic effects of vitamin DS [g-16]. In the present article, we present data from three patients which support the notion that metabolites of vitamin DJ, other than 1,25(OHJzD3, are important in the pathogenesis of osteomalacia in adult man. SUBJECTS AND METHODS Case Reports Patient 1 was a 52 year old man with a 25 year history of regional enteritis ne- cessitating multiple surgical procedures and courses of adrenal steroids. For the previous five years he had had no operations and his enteritis had been stable. However, he had daily diarrhea only partially controlled by opiates. For the year prior to admission he had noted progressive muscle weakness, at first confined to the proximal muscles but eventually becoming generalized to the point that he was unable to walk and was confined to bed. He had been treated with vitamin Dz, 10,000 U/day, without clinical improvement. Roent- genograms revealed demineralization and pseudofractures of both femurs. He was found to have hypocalcemia (7.7 mg/dl), hypoalbuminemia (2.5 mg/dl). an elevated alkaline phosphatase level (196 ILJ], but normal serum phosphorus (5.2 mg/dl] and magnesium (2.3 mg/dl) levels. His creatinine clearance was normal (105 ml/min). He was admitted to the clinical research center for de- tailed evaluation. 360 September 1960 The American Journal of Medicine Volume 69
Transcript
1,25(OH)2D3 Is Not the Only D Metabolite Involved in the Pathogenesis of Osteomalacia
HOWARD RASMUSSEN, M.D. ROLAND BARON, M.D.
ARTHUR BROADUS. M.D. RALPH DeFRONZO, M.D.
ROBERT LANG, M.D.
New Haven, Connecticut
RONALD HORST, M.D.
Ames, lowo
From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut: and the U.S. Department of Agri- culture Scientific and Educational Administra- tion, Ames, Iowa. This work was supported by an NIH SCOR Grant [AM 20570). Clinical studies were performed in the Clinical Research Center of the Yale-New Haven Hospital and were supported by a grant (RP-1251 from the General Clinical Research Centers Branch, Division of Research Resources, NIH. Requests for reprints should be addressed to Dr. Howard Rasmussen, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. Manu- script accepted March 17,198O.
Three patients are described in whom there was no simple correla- tion between plasma 1,25(OHhD3 concentration and the occurrence of osteomalacia. One patient had severe osteomalacia with high plasma 1,25(OHbD3 and normal mineral ion product; the second had a normal mineral ion product and no evidence of osteomalacia even though plasma 1,25(OH)zD3 was undetectable; and the third had os- teomalacia, low plasma 1,25(OH)zD3 and a reduced mineral ion product. In considering these data in the light of presently available information, it is concluded that osteomalacia can occur as a conse- quence of a lack of a vitamin D metabolite other than l$5(OHbD3, or as a consequence of a reduced mineral ion product, but not as a consequence of 1,25(OHbD3 lack if the mineral ion product is nor- mally maintained and other D metabolites are present. However, a deficiency of 1,25(OHhD3 normally leads to a reduction in the mineral ion product hence 1,25(OHhD3 deficiency may play a role in the development of certain forms of osteomalacia.
It has become widely accepted that l,Z&dihydroxyvitamin DB [1,25(0H)~D~] is the only important biologically active form of vitamin D3 in man [l-8]. However, there is evidence that other metabolites of vitamin D3 may also be important in the biologic effects of vitamin DS [g-16]. In the present article, we present data from three patients which support the notion that metabolites of vitamin DJ, other than 1,25(OHJzD3, are important in the pathogenesis of osteomalacia in adult man.
SUBJECTS AND METHODS
Case Reports
Patient 1 was a 52 year old man with a 25 year history of regional enteritis ne- cessitating multiple surgical procedures and courses of adrenal steroids. For the previous five years he had had no operations and his enteritis had been stable. However, he had daily diarrhea only partially controlled by opiates. For the year prior to admission he had noted progressive muscle weakness, at first confined to the proximal muscles but eventually becoming generalized to the point that he was unable to walk and was confined to bed. He had been treated with vitamin Dz, 10,000 U/day, without clinical improvement. Roent- genograms revealed demineralization and pseudofractures of both femurs. He was found to have hypocalcemia (7.7 mg/dl), hypoalbuminemia (2.5 mg/dl). an elevated alkaline phosphatase level (196 ILJ], but normal serum phosphorus (5.2 mg/dl] and magnesium (2.3 mg/dl) levels. His creatinine clearance was normal (105 ml/min). He was admitted to the clinical research center for de- tailed evaluation.
360 September 1960 The American Journal of Medicine Volume 69
Patient 2 was a 32 year old woman who had tetany at age seven, and who was found to have hypocalcemia (7.1 mg/dl) and hyperphosphatemia (8.5 mg/dl] at the Vanderbilt Uni- versity Hospital. She underwent an Ellsworth-Howard test and, on the basis of a fivefold increase in urinary phosphorus excretion after the infusion of parathyroid extract, a diagnosis of idiopathic hypoparathyroidism was made. She was treated over the next 23 years with dihydrotachysterol (DHT) in doses of 0.25 to 0.75 mg/dl. On this regimen, her serum calcium ranged from 10.3 to 11.6 mg/dl. In 1966, at the time of her first pregnancy, DHT therapy was discontinued. She went through her pregnancy without incident, and DHT therapy was rein- stituted even though the serum calcium concentration was normal. In 1970, during her second pregnancy, DHT therapy was again discontinued without incident. This treatment was reinstituted three months after delivery because of muscle cramps even ihough her serum calcium concentration at the time was 10.1 mg/dl. She was first seen at the Yale-New Haven Hospital in 1977 because of a 10 year history of hypertension and an increase in blood urea nitrogen (36 mg/dl). At that time she was receiving DHT (0.25 mg/day), had a blood pressure of 150/100 mm Hg and mild hypercalcemia (10.3 to 11.6 mg/dl]. Her creatinine clearance was 60 mg/min. Vitamin D therapy was stopped. Six to nine months later the serum calcium con- centration ranged from 9.6 to 10.2 mg/dl. At this time a serum immunoreactive parathyroid hormone (iPTH] was found to be 580 Fleq/ml (normal < 100 ~leq/ml]. Her creatinine clearance increased to 75 ml/min six months after cessation of DHT therapy. On the basis of these findings, she was ad- mitted to the clinical research center for further study.
Patient 3 was a 32 year old man with familial hypophospha- temic rickets who had a history of rickets in childhood com- plicated by multiple fractures. No medical therapy was given. At age 10 he had an operation to straighten his legs. At age 12. reoperation to straighten the right leg was performed, At age 21 he sustained a traumatic fracture of the left femur and he was in a body case for six months. However, there was poor healing of the fracture and shortly after discharge the femur refractured. Reoperation and insertion of a rod was required. An additional operation on this leg was necessary 10 years later. In the meantime, at age 29 he sustained a fracture of the right femur following a grand ma1 seizure. This was treated by open reduction and plating. In the five years prior to admission he experienced increasing pain in both thighs and hips asso- ciated with muscle weakness. He was able to walk only with the aid of two canes. The only other medical problem was the onset of grand ma1 epilepsy at age 25. Over the period of the next four years, he had six seizures for which he was given diphenylhydantoin but he did not take this medication regu- larly. He had no seizures for four years prior to admission. At the time of admission he had hypophosphatemia, normocal- cemia, increased plasma alkaline phosphatase and normal creatinine clearance (128 ml/min].
METHODS
Study Protocol. All three patients underwent a detailed evaluation in the Clinical Research Center after informed written consent. Each was placed on a diet containing 400 mg calcium, 800 mg phosphorus and 100 meq sodium. On the first
l.25(OHJ2D3 AND OSTEOMALACIA-RASMUSSEN ET AL.
two days serum levels of creatinine, electrolytes, calcium, phos- phorus, alkaline phosphatase, 25(OH]Ds, 1,25(OH)zD3, were determined and 24 hour urine collections were made for the determination of creatinine, calcium and phosphate excretion. On the third day each underwent an oral calcium tolerance test [17]. The following day, a transileal bone biopsy specimen was obtained as well as skeletal roentgenograms. Repeat x-ray films and bone biopsy specimens were obtained in Patients 1 and 3 nine and 11 months after institution of treatment.
After initial evaluation, Patient 1 was started on 50 pg/day of 25(OH)D3. Patient 2 was started on 1.0 pg/day of 1,25(OH)zD3 and Patient 3 on 2.5 pg/day of 1,25(OH)zDa and 2.5 g of phosphate orally in four divided doses. They were re- evaluated at monthly intervals, and readmitted for detailed reevaluation after approximately nine to 12 months. Analytical Procedure. Serum and urine calcium were mea- sured by atomic absorption spectrophotometry, phosphorus by the phosphomolybdate reaction and creatinine by the method of Jaffe. Nephrogenous cyclic adenosine monophos- phate (AMP] was measured by a previously described method [la]. iPTH was measured at the Nichols Institute for Endo- crinology (San Pedro, CA] using a radioammunoassay (RIA] employing GP-101 antibody. Both 25-hydroxyvitamin D [25(OH)Ds] and 1,25-dihydroxyvitamin D [1,25(0H)2D3] were measured by previously published methods [19]. Fasting and postabsorptive urinary calcium excretion were expressed in terms of glomerular filtrate as previously described [u]. Fasting plasma and urine phosphorus and creatinine con- centrations were used to calculate the maximum tubular reabsorptive capacity for phosphorus in terms of glomerular filtration rate (TmP/GFR] as described by Bijvoet [20].
Transileal bone biopsy was carried out by the Bordier method (211. The various parameters of bone remodeling were assessed by the methods of Frost [22]. Tetracycline double labeling [23] was performed in Patients 2 and 3 before treat- ment and in Patient 2 after treatment by the administration of Declomycin@ 150 mg every 8 hours for two days, then 12 days later Terramycinm 250 mg every 8 hours for four days. The biopsy was performed two days after the last dose of Terra- mycin was given. Appropriate sections from these biopsies were viewed by fluorescent microscopy to estimate the extent of the mineralization front and the rate of bone mineraliza- tion.
RESULTS
The results of the laboratory studies in the three patients are summarized in Table I, and the histomorphometric analysis of the bone biopsy specimens is presented in Table II. The histologic appearance of the trabecular bone from these patients are shown in Figures 1,2 and 3.
The first patient had hypocalcemia, hyperphospha- temia, secondary hyperparathyroidism as indicated by an elevated iPTH level and nephrogenous cyclic CAMP, hypocalciuria and an elevated alkaline phosphatase level. Despite the secondary hyperparathyroidism, he had an increased TmP/GFR. His plasma calcium times phosphate ion product was normal. The plasma
September 1980 The American Journal of Medicine Volume 69 361
Normal 9.5-10.3 3.0-3.4 35 f 5 10-70 <90 30f 15 45f 10 60-160 0.3-2.8
NOTE: Patient 1 = Crohn’s disease with severe osteomalacia treated with 25(OH)Ds, 50 pg/day. Patient 2 = Pseudohypoparathyroidism treated with 1,25(OH)2Ds, 1 to 1.5 pg/day. Patient 3 = Hypophosphatemic osteomalacia treated with oral phosphate, 2.5 g every day and 1,25(OH)2Ds, 3.0 pglday.
TmPlGFR (mg/ldO ml)
5.0 2.7
3.4 2.6
1.9 1.7
2.4-4.5
1,25(OH)zD3 concentration was increased, but the 25(OH)D3 concentration was very low.
The clinical and laboratory data in this patient were consistent with a diagnosis of osteomalacia, and this diagnosis was established by analysis of a transileal boric biopsy (Table II and Figure 1A). Severe osteoma- lacia was present (Figure 1AJ: the osteoid surface and thickness were markedly increased and 19 percent of the total trabecular bone was unmineralized. Consistent with the presence of secondary hyperparathyroidism was an increase in the extent of active osteoclastic re- sorption surface (7.1 versus normal of 0.5 f 0.1 per- cent).
Treatment of this patient with 25(0H)2D3 led to a marked clinical improvement within weeks, and a sig- nificant healing of his osteomalacia in approximately
eight months (Figure 1B and Table II). The repeat lab- oratory value after eight months of therapy are shown in Table I. The parathyroid hormone and nephrogenous cyclic AMP values were normal. The ion product was still normal. The plasma 1,25(OH)~Ds had declined to a high normal value and the 25(OH)Ds had increased to the mid-normal range. The plasma alkaline phos- phatase level, which rose even higher after initiation of 25(OH)Ds treatment, declined into the normal range after eight months of therapy. The bone biopsy speci- men showed nearly complete healing of the osteoma- lacia (Figure 1B and Table II].
The second patient had normal values of plasma calcium and phosphate but extremely high iPTH. Based on the fact that the bone showed signs of increased re- modeling consistent with hyperparathyroidism [Table
TABLE II Hlstomorphometrlc Analysis of Bone Biopsy Data
l TBV = trabecular bone volume as percent of total biopsy volume; AOV = absolute osteoid volume as percent of total biopsy volume; ROV = relative osteoid volume as percent of bone volume; OS = osteoid surface as percent of trabecular bone surface; OT = osteoid thickness in microns; AOBS = active osteoblastic surface as percentage of trabecular bone surface; ROBS = relative osteoblastic surface as percent of osteoid surface; MF = mineralization front as percent of osteotd surface; hM = mineralizaUon rate in p/day as the mean distance between the two labels treated for obliquity of the cations; AOCS = active osteoclastic surface as percent of trabecular bone surface; and Revs = reversal surface as percent of trabecular bone surface.
362 September 1960 The American Journal of Medicine Volume 69
1,25(0H)~D~ AND OSTEOMALACIA-RASMUSSEN ET AL.
Figure 1. therapy with 25(OH)Ds. The dark area represents calcified bone and the light homogeneous area uncalcified osteoid. In A the arrows indicate large volumes of osteoid overlain with inactive bone formation surfaces. In B there is a marked decrease in extent of osteoid, and the osteoid present is covered by active osteoblasts (arrows). Magnification X250, reduced by 11 per- cent.
Figure 2. Sections of undecalcified bone from the transileal biopsy specimen obtained from Patient 2. A regular section showing on the right concave surface (straightarrow) a normal area of bone formation with a small thin osteoid surface and active os- teoblasts, and on the convex surface (heavy curved arrows) a surface of active bone formation. A section of bone viewed by fluorescence microscopy showing two double tetracycline labels. The one on the left is deep within the bone and is the label resulting from pretreatment administration of tetracyclines. The ones on the left are the result of post-treatment tetracycline administration (see Table II). Magnification X250, reduced by 11 percent.
September 1989 The American Journal of Medicine Volume 69 363
1,25(OH)zD3 AND OSTEOMALACIA-RASMUSSEN ET AL
Figure 3. Stained sections of*zd:calcified bone from the transileal biopsy specimen obtained from Patient 3. Note the ap- pearance of severe osteomalacia with a marked in&ease in osteoid volume and largely inactive (arrows) osteoblastic surfaces. Also seen are partially mineralized regions of dark granular material deep within the matrix. Appearance after nine months of treatment with a combination of 1,25(OH)2D3 and oral phosphate. Note that small layers of osteoid are still seen upon both surfaces, but that width of osteoid seams has been markedly reduced (thin arrow). Also, a considerable portion of the osteoid surface is covered with active osteoblasts (heavy arrows). Magnification X250, reduced by 11 percent.
II and Figure 2) and that the rate of urinary calcium excretibn was low even though there was no detectable nephrogenous cyclic AMP, we concluded that this pa- tient had a form of pseudohypoparathyroidism in which the proximal renal tubule was unresponsive to PTH, but the distal tubule was responsive. Of particular note was the finding that plasma 1,25(OHJzD3 was undetectable. Despite the absence of 1,25(OH)zD~, there was no his- tologic evidence of osteomalacia (Table II, Figure 2A) and the mineralization front as measured by tetracycline labeling was normal (Figure 2Bj. To determine whether the absence of 1,25(OH)zDa was correlated with a re- diction in intestinal caldium absorption, a calcium tol- erance test was performed before and several weeks after treatment with 1.5 pg/day of 1,25(OH)zDs. Before treatment, there was no significant increase in either plasma calcium concentration (9.7 - 9.7 mg/dl) or uri- nary calcium excretion (0.02 - 0.03 mg/lOO ml GFR] after the ingestion of a 1 g calcium load. However, after several weeks of treatment with 1,25(OH)zD3, there was a significant change in both plasma calcium concen- tration (9.8 - 10.5 mg/dl) and urinary calcium excretion (0.06 - 0.23 mg/lOO GFR). It is of interest that the TmP/GFR was lower (2.9 versus 3.3) after treatment with 1,25(OH)zDs even though iPTH decreased from 412 to 413 pleq/ml. These data indicate that the patient had
significant malabsorption of calcium associated with undetectable amounts of 1,25(OH)zD3 in her plasma and that this malabsorption was corrected by the adminis- tration of 1,25(OH)zDs. Long-term treatment of this pa- tient with 1,25(OH)zD~ led to a marked reduction in plasma iPTH, an increase in urinary calcium excretion with little change in plasma calcium concentration or mineral ion product, a normal response to the oral cal- cium tolerance test, an increase in plasma 1,25(OH)2D3 to detectable but still low values and a significant de- crease in rate of bone remodeling (Table II). The tra- becular bone turnover fell in this patient, from 56 per- cent before treatment to 18 percent per year after treatment, indicating a correlation between the decline in iPTH and the decrease in the rate of bone turn- over.
The third patient presented with normocalcemia, severe hypophosphatemia, an elevated alkaline phos- phatase level without evidence of secondary hyper- parathyroidism, normal plasma 25(OH)D3 but a low plasma 1,25(OH)zDa concentration (Table I]. These biochemical changes were associated with severe os- teomalacia with only a slightly increased bone resorp- tion (Table II and Figure 3A). Treatment with a combi- nation of 1,25(OH)zD3 and oral phosphate led to clinical, biochemical (Table I) and marked morphologic im-
364 September 1980 The American Journal of Medicine Volume 69
1,25(OH)zD3 AND OSTEOMALACIA-RASMUSSEN ET AL.
TABLE III A Summary of the Findings In States of Altered Vitamin D Metabolism
State Mineral Ion Product Plasma PTH Plasma 25(OH)Ds Plasma 1,25(OH)2D3 Mineralization Front
Vitamin D deficiency* 1 N 1 Malabsorption+ Drug-Induced osteomalacia’ i i SL Hypophosphatemic osteomalacia+ r N i Pseudohypoparathyroidism+ N N f N
l From [ll]. + Present patient. t From [lo] and [ 141.
provement in the osteomalacia (Figure 3B and Table II]. It is of interest that the extent of active osteoclastic sur- face increased significantly during treatment (1.4 to 6.5 percent of trabecular surface).
COMMENTS
A number of factors thought to be important in the pathogenesis of osteomalacia are listed in Table III. A consideration of these factors in relation to the present patients raises a number of interesting points.
The first is the relationship between the presence of osteomalacia and the changes in the so-called mineral ion product (calcium X phosphorus] of plasma which has often been used as a clinical index of the saturation of plasma with these ions [24]. In osteomalacia this product is usually reduced [24]. It increases when vi- tamin D is given to a D-deficient patient, and an increase in this product by mineral ion infusions in patients with osteomalacia secondary to vitamin D deficiency has been reported to induce healing of the osteomalacia [25]. However, a comparison of bone biopsy material from patients treated with mineral ion infusion with that from those treated with vitamin D reveals a critical difference [14,21]. Following treatment with vitamin D, a normal mineralization front appears on the osteoid surface immediately adjacent to the remaining mineralized bone, and the mineralization proceeds as an orderly process from the deepest regions of osteoid to the sur- face. In contrast, following mineral ion therapy, min- eralization takes place in a random fashion throughout the extent of the osteoid volume; when completed, it leaves behind islands of unmineralized osteoid. Hence, it would appear that simply increasing the mineral ion product does not reestablish the normal mineralization sequence.
In the present group of three patients, the most striking finding was that in Patient 1 there was vitamin D defi- ciency as indicated by low plasma 25(OH)Ds, but a high plasma 1,25(OH)zDs. However, a normal plasma min- eral ion product was found to coexist with osteomalacia. In this patient neither a decrease in mineral ion product nor in plasma l,25(OH)zD3 concentration could account for the development of osteomalacia. This means that 25(OH)Ds or another metabolite influences the process of bone mineralization directly [12,14,16]. There is ev- idence both in man [12,14] and in the chick [16] that
24,25(OH)zDs is important in the process of healing rickets and/or osteomalacia. In both species, the com- bined administration of the two metabolites produces greater healing than the administration of either alone.
The findings in the second patient illustrate that, under exceptional circumstances, the mineral ion product can be maintained ‘at a normal value in the complete absence of 1,25(0H)~Da. Under these cir- cumstances osteomalacia does not appear even though the rate of bone remodeling is increased: a situation which would predispose to the development of osteo- malacia if there were any significant delay in mineral- ization rate because of the marked increase in matrix formation. Considered in isolation, the findings in this patient would support the concept that if the plasma mineral ion product is maintained at a normal value then osteomalacia does not occur. However, when the findings in this patient are contrasted with those in the first patient, this conclusion is untenable because the mineral ion product (calculated either as total calcium X phosphorus, or as the ion product) in the plasma of these two patients is nearly identical. Hence, the lack of osteomalacia in Patient 2 and its presence in Patient 1, both of whom have evidence of an effect of excess PTH on bone and increased bone turnover, must be explicable in terms of some other variable. The most likely one is plasma 25(OH)D3 or another of its metab- olites. It is low in Patient 1 and normal in Patient 2.
The finding in Patient 1 of an association of severe osteomalacia with a low plasma level of 25(OH)Ds and a high level of l,25(OH)zDs is similar to that described by Eastwood et al. [ll] in three patients with nutritional osteomalacia, and by Jubiz et al. [lo] in patients with drug-induced osteomalacia. The finding in Patient 2 of an undetectable concentration of plasma 1,25(OH)zDs without evidence of osteomalacia is similar to the sit- uation described in certain anephric patients [9]. Thus, severe osteomalacia secondary to vitamin D deficiency can occur when plasma 1,25(OH)zD3 concentrations are either normal or high. Conversely, Bordier et al. [l2] showed that administration of 1 to 5 pg/day of Ia(O or l,25(OH)zD3 to patients with vitamin D-deficiency osteomalacia did not result in a restoration of normal plasma phosphate concentrations nor normal bone mineralization rates. In toto, these data indicate that a
September 1980 The American Journal of Medicine Volume 89 385
1,25(OH)2DS AND OSTEOMALACIA-RASMUSSEN ET AL.
deficiency of 1,25(OH)zDs is not solely responsible for the development of osteomalacia in vitamin D-deficient man.
A possibility that should be considered in our Patient 1 and in the subject with nutritional osteomalacia de- scribed by Eastwood et al. [ll] is that a decrease in vi- tamin Ds and 25(OH)Ds concentrations increases the binding of 1,25(OH)sDs to the serum transport protein and thereby reduces the free concentration of 1,25(OH)2Ds even though the total is normal. This probably is not the case. First, the total amount of this protein is in the range of 10h5 M in blood plasma whereas the total concentration of all D metabolites is
-in the range of lop7 M [26]. This means that only 1 to 2 percent of the protein binding sites are normally occu- pied. Also the Kd for the binding of 1,25(OH)2Ds to this protein is 3 X 10T7 M. Thus, all but 2 to 3 percent of the 1,25(OH)sDs is normally bound. A decrease in vitamin D3 plus 25(OH)D3 content in plasma would have little effect upon the binding sites available to 1,25(OH)sD3 and hence have little influence on the distribution of this metabolite between free and bound forms.
Even though in some patients with osteomalacia the plasma 1,25(OH)xDs concentration is normal, it is clear that 1,25(OH)zDs deficiency can lead to the development of osteomalacia in patients with vitamin D-dependency rickets. Rickets develops in these patients as a conse- quence of an altered renal la-hydroxylase [4]. They present with rickets, a normal plasma concentration of 25 (OHIDs, a decreased concentration of 1,25(OHhD3 and a reduced mineral ion product. Most importantly, they respond to treatment with 1,25(OH)zDs. Charac- teristically their plasma ion product is low, and 1,25(OH)zDs brings this back to normal. If we contrast their situation with the situatiqn in the patients in the present study, we conclude that osteomalacia or rickets can develop when the mineral ion product is low even when levels of 25(OH)Ds.and metabolites other than 1,25(OH)zDs are nosmal and that 1,25(OH)zDs is not necessary if another mechanism for maintaining the ion product is operating.
The results in the third patient in the present study support this conclusion. In this case, the ion product was low because of an extremely low plasma phosphate concentration, and osteomalacia developed even though 25(OH)Ds concentrations were normal. By giving a combination of oral phosphate and 1,25(OHhD3 it was possible to increase the ion product, initiate mineral- ization and bring about a striking reduction in the se- verity of the osteomalacia (Tables I and II, Figure 3).
The present and other recently reported data [ll-151 raise another interesting point: the question of whether or not 1,25(OH)zDs is the only metabolite of vitamin D active in the intestine. Unfortunately, both in Patient 1 of the present study and in the three patients with nu- tritional (vitamin D-deficiency) osteomalacia described by Eastwood and co-workers [ll], no direct measure of intestinal calcium absorption was carried out. Never-
theless, based on data from previous studies in similar patients [24], it is likely that intestinal calcium absorption was reduced in these patients. If so, it occurred at a time when the plasma 1,25(OH)zDs and was either normal or high: a circumstance in which plasma 1,25(OHhD3 concentration and rate of intestinal calcium absorption are not correlated. In our Patient 1, treatment with 25(OH)Ds obviously led to a positive calcium balance at a time when the plasma 1,25(OH)zDs level was falling, and this balance could only have been a consequence of an increase in intestinal calcium absorption. Also, in the patient described by Zerwekh et al. [15], severe os- teomalacia was present at a time when plasma 1,25(OH)2Ds concentration was high (212 pg/ml). At that
time, true fractional calcium absorption was a low normal value, 0.26 (normal range 0.26 to 0.56) and the patient was in marked negative calcium balance (-232 mg/day) primarily because of a marked loss of endog- enous fecal calcium (approximately 160 mg/day). The plasma 25(OH)D was 18.6 pg/ml (normal = 15 to 40 pg/ml). When the patient was given a small dose of 25(OH)Ds (20 pg/day), the plasma 25(OH)Ds increased slightly (24.5 mg/ml), calcium balance changed by 280 mg becoming positive (-t-48 mg/day) but fractional cal- cium absorption rose only to 0.30. These changes oc-l curred at a time when 1,25(OH)zDs concentration de- creased from 212 to 169 pg/ml. Hence, physiologic concentrations of 25(OH)Ds were responsible for in- ducing this change. Their data suggest that either 25(OH)Ds itself or one of its other metabolites acts syn- ergistically with 1,25(OH)zDs to regulate intestinal cal- cium absorption or that 25(OH)Ds or one of its other metabolites acts directly upon bone formation and/or mineralization and, as a consequence, a bone derived product is generated that acts upon the intestine.
In regard to the first possibility, it is of interest that a 5s binding protein highly specific for 25(OH)D3 is found in the intestinal mucosa [27-301. Likewise, considerable quantities of 25(OH)Ds are present in intestinal mucosa cell [3l]. In regard to the second possibility, if one ana- lyzes the data presented by Zerwekh et al. [15] the major effects of 25(OH)Ds on the intestine during the first three months of therapy was to decrease endogenous fecal calcium loss. This might mean that vitamin D metabo- lites (or bone derived factor) control bidirectional in- testinal calcium fluxes. It is of interest that our recent studies [32] in growth hormone deficient dwarfs given human growth hormone show that the anabolic effects of growth hormone and the associated retention of cal- cium occur without a significant change in plasma 1,25(OH)zDs values or parathyroid function. Thus, fac- tors other than plasma 1,25(OH)zDs determine the net absorption of calcium from the gut. An attractive pos- sibility is a bone derived “hormone” that coordinates the rate of intestinal absorption with the needs of the skeleton.
Three recent studies raise the interesting possibility that this hormone may be either 24,25(OHlzD3 or a
366 September 1960 The American Journal of Medicine Volume 69
1,25(OHbDs AND OSTEOMALACIA-RASMUSSEN ET AL.
product derived from the action of this hormone. First, Garabedian et al. [33] showed that cartilage produces 24,25(OH)zD3 from 25(OH)Ds. Second, Kanis et al. [13] showed that administration of 24,25(OH)sDs to normal controls or to patients with renal osteodystrophy, in- cluding anephric patients, led to a net retention of orally administered 47Ca. The degree of this retention was comparable to that produced by 1,25(OH)zDs but oc- curred without an increase in plasma calcium concen- tration or in urinary calcium excretion as seen after the administration of 1,25(OH)zDs. Third, Szymendera and Galus [34] reported that in patients with chronic renal disease, the administration of 24,25(OH)zDs did not in- crease intestinal calcium absorption as measured by the concurrent use of oral and intravenous calcium tracers. At first glance, these data would appear to contradict those of Kanis et al. [13]. However, there is no contra- diction if 24,25(OHlzD3 or some product derived from its action stimulates net calcium retention in some distal part of the intestinal tract rather than in the proximal portion. Since plasma 24,25(OHhDs did not increase after the administration of growth hormone [32], even though calcium retention increased, it may be that 24,25(OH)zDs is not directly involved in regulating in- testinal calcium retention. Recent data derived from studies in D-deficient man [l2] and the D-deficient rat
5.
6.
10.
11.
12.
[35] indicate that the administration of 25(OH)Ds, but neither 1,25(OH)sDs or 24,25(OH)zDs, to the D-deficient organism induces an increase in plasma alkaline phosphatase activity. These data are consistent with the concept that 25(OH)D3 acts directly on bone formation and/or mineralization. Hence, it is possible that this metabolite may also be important in the induction of a bone-derived factor involved in regulating intestinal calcium absorption.
Clearly, more data are necessary before the patho- physiology of vitamin D metabolism in man is com- pletely understood. Our interest here is to call attention to the compelling clinical evidence that 1,25(OH)zDa is not the only important metabolite of vitamin D, and to raise the interesting possibility that events in bone in- fluence net intestinal calcium retention by a mechanism independent of 1,25(OHbDs.
ACKNOWLEDGMENT
The 25(OH)D3 employed in these studies was supplied by Rouse11 UCLAF, Paris, France; the 1,25(OH)zDs by the Hoffmann-LaRoche Company, Nutley, New Jersey. We are indebted to Dr. M. Perianu of Rousell, and to Dr. Zane Gaut of Hoffmann-LaRoche for the supply of these drugs.
DeLuca HF: Vitamin D metabolism. Clin EndocrinoI7 1977; (suppl): 15.
Stanbury SW: The role of vitamin D in renal bone disease. Clin Endocrinoi 1977; 7 (suppl): 255.
DeLuca HF: Vitamin D. The vitamin and the hormone. Fed Proc 1974; 33: 221.
Fraser D. Kooh SW, Kid HP, Holick MF, Tanaka Y, DeLuca HF: Pathogenesis of hereditary vitamin D-dependent rickets: an inborn error of vitamin D metabolism involving defective conversion of 25-hydroxyvitamin D to 101,25- dihydroxyvitamin D. N Engl J Med 1973; 289: 817.
Tanaka Y. DeLuca HF: Role of 1,2t%dihydroxyvitamin Ds in maintaining serum nhosuhorus and curine rickets. Proc Nat1 Acad &i USA i974171: 1640. y
Mawer EB. Backhouse J, Taylor CM, Lumb GA, Stanbury SW Failure of formation of 1.25-dihydroxycholecalciferol in chronic renal insufficiencv. Lancet 1973: 1: 626.
Eastwood JB. Hanes E, Stamp TCB, de Wardener HE: Vita. min D deficiencv in the osteomalacia of chronic renal failure. Lancet 1976: 2: 1209.
Drezner MK, Feinglos MN: Osteomalacia due to la.25- dihydroxycholecalciferol deficiency. J Clin Invest 1979; 60: 1046.
Bordier P, Tun Chot S, Eastwood JB, Fournier A, de War- dener HE: Lack of histological evidence of vitamin D ab- normality in the bones of anephric patients. Clin Sci 1973; 44: 33.
Jubiz W, Haussler MR. McCann TR. Tolman KG: Plasma 1,25-dihydroxyvitamin D levels in patients receiving anticonvulsant drugs. J Clin Endocrinoi Metabolism 1977; 44: 617.
Eastwood JR, de Wardener HE, Gray RW, Lemann J Jr: Normal plasma 1,25(0H)s vitamin D concentrations in nutritional osteomalacia. Lancet 1979: 1: 1377.
Bordier P, Rasmussen H. Marie P. Miravet L, Gueris J. Ryckewaert A: Vitamin D metabolites and bone mineral- ization in man. J Chn Endocrinol Metab 1978; 46: 284.
13. Kanis JA, Cundy T, Bartlett M, Smith R, Heynen G. Warner GT, Russell RGG: Is 24,25-dihvdroxvcholecalciferol a calcium-regulating hormone in man? Br Med J 1978; 1: 1382.
14. Rasmussen H, Bordier P: Vitamin D and bone. Metab Bone Dis Rel Res 1978; 1: 7.
15. Zerwekh JE, Glass K. Jowsey J. Pak CY: An unique form of osteomalacia associated with endorgan refractoriness to 1,25-dihydroxyvitamin D and apparent defective synthesis of 25-hydroxyvitamin D. J Clin Endocrinol Metab 1979; 49: 171.
16. Stamp TCB, Flanagan RJ, Rechens A, et al.: Anticonvulsant osteomalacia. In: CODD DH. Talmaae RV. eds. Endocri- nology of calcium metabolism. Amsterdam: Excerpta Medica, 1978; 16.
17. Broadus AE, Domenguez M, Bartter FC: Pathophysiological studies in idiopathic hypercalciuria: use of an oral calcium tolerance test to characterize distinctive hypercalciuric subgroups. J Clin Endocrinol Metab 1978; 47: 731.
18. Broadus AE, Mahaffey JE, Bartter FC, Neer RM: Nephro- genous cyclic AMP as a parathyroid function test. J Clin Invest 1977: 60: 771.
19. Horst RL. Shepard RM, Jorgenson NA, DeLuca HF: Assay of vitamin D metabolites: changes in milk fever disease. Arch Biochem Biophys 1979; 192: 512.
20. Bijvoet OLM: Kidney function in calcium and hosphate metabolism. In: Avioli LV, Krane SM. eds. Meta g olic bone disease, Vol I. New York: Academic Press, 1977: 49.
21. Rasmussen H, Bordier I? The cellular and physiological basis of metabolic bone disease. Baltimore: Williams &Wilkins, 1974.
22. Frost HM: A method of analysis of trabecular bone dynamics. In: Meunier PJ. ed. Bone histomorphometry. Paris: Lab Armour-Montagu, 1977; 445.
23. Frost HM: Tetracycline based analysis of bone remodeling. Calcif Tissue Res 1969; 3: 211.
24 Nordin BEC: Metabolic bone and stone disease. Baltimore:
September 1980 The American Journal of Medicine Volume 69 367
1.25[OH)2D3 ANU OSTEOMALACIA-RASMIJSSEN ET AI,
Williams & Wilkins, 1973. 25. Popvtzer MM, Mallbay R, Alfrey AC, et al.: Vitamin D de-
ficiencv osteomalacia. Healing of the bone disease in the absenck of vitamin D with-intravenous calcium and nhosohorus infusion. In: Frame B. Parfitt AM, Duncan H, eds. Clinical aspects of metabolic bone disease. Amster- dam: Exceruta Medica. 1973: 382.
26. Haddad JG, Walgate J: 25-Hydroxyvitamin D transport in human plasma. J Biol Chem 1976; 251: 4803.
27. Haddad JG, Birge SJ: Widespread, specific binding of 25- hydroxycholecalciferol in rat tissues. J Biol Chem 1975; 250: 299.
28. Lawson DEM, Charman H, Wilson PW. Edelstein S: Some characteristics of new tissue-binding proteins for metab- olites of vitamin D other than 1.25dihydroxyvitamin D. Biochem Biophys Acta 1976; 437: 403.
29. Kream BE, Reynolds RD, Knudson JC. Ersnam JA, DeLuca HF: Intestinal cytosol binders of 1,25-dihydroxyvitamin Ds and 25-hydroxyvitamin Ds. Arch Biochem Biophys 1976; 176: 779.
30. Cooke NE, Walgate J, Haddad JG: Human serum binding protein for vitamin D and its metabolites. II. Specific high affinity association with a protein in nucleated tissue. 1 Biol Chem 1979: 254: 5965.
31. Haussler MR, Rasmussen H: Metabolism of vitamin Ds in the chick. J Biol Chem 1972: 247: 2328.
32. Gertner JM, Horst RL, Broadus AE. Rasmussen H, Gene1 M: Parathyroid function and vitamin D metabolism during human growth hormone replacement. ] Clin Endocrinol Metab 1979; 49: 185.
33. Garabedian M, Bailly Du Bois M, Corvol MT, Pezant E, Balsan S: Vitamin D and cartilage. I. In vitro metabolism of 25-hydroxycholecalciferol by cartilage. Endocrinology 1978; 102: 1262.
34. Szymendera J. Galus K: Effect of 24.25dihydroxycholecal- ciferol on calcium absorption in proximal small intestine in uraemia. Br Med J 1978: 2: 1465.
35. Lieberhen M, Pezant E, Garabedian M. Balsan S: Phospha- tase content of rat calvaria after in vivo administration of vitamin D metabolites. Calcif Tissue Res 1977; 23: 235.
368 September 1980 The American Journal of Medicine Volume 69
