Osteomalacia and celiac disease

CASE REPORTS

Osteomalacia and Cel iac Disease Response to 25-Hydroxyvitamin D

GERSHON W. HEPNER. M.D.’ Hershey, Pennsylvania JENIFER JOWSEY.

Ph.D.

CLAUDE ARNAUD,

M.D.+

Rochester, Minnesota STANLEY GORDON, M.D. JOSEPH BLACK, M.D.1

In this 54 year old woman with celiac disease, osteomalacia developed while she was on a gluten-free diet which had caused regression of her steatorrhea. She was not responsive to large doses of parenterally administered dihydrotachysterol and calcium, but she was responsive to the oral administration of 25hydroxyvitamin D3 (25-0HD3). The data suggest that 250HD3 is the treatment of choice for patients with vitamin D deficiency due to intestinal malabsorption.

Hershey, Pennsylvania MARTIN ROGINSKY, M.D. HING FAI MOO, Ph.D. East Meadows, New York JAMES F. YOUNG, M.D. Lancaster, Pennsylvania

From the Departments oof Medicine and Surgery, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania; the Department of Endocrinology and Orthopedic Research, Mayo Clinic, Rochester, Minnesota; the Department of Endocrinology, Nassau County Medical Center, East Meadows, New York; and St. Joseph’s Hospital, Lancaster, Pennsylvania. Requests for reprints should be addressed to Dr. Gershon W. Hepner. Manuscript accepted May 22, 1978. Present address: Division of Gastroenterology. Harbor General/UCLA Medical Center, Torrance, California 90509. + Present address: Division of Endocrinology, VA Hospital, San Francisco, California 94 12 1. z Present address: Department of Neurology, Washington University Medical School, St. Louis, Missouri 83110. l

Patients with celiac disease (nontropical sprue, gluten-sensitive enteropathy, idiopathic steatorrhea) frequently have osteomalacia [l-3]. This complication may be the major problem in patients in whom other abnormalities are absent or occult [ 4-71. The osteomalacia has been related to vitamin D deficiency, because antirachitic activity is low in the serum of celiac patients with osteomalacia and frequently returns to normal after treatment with vitamin D or withdrawal of gluten [3-81. Malabsorption of vitamin D occurs in celiac patients [9], but this may not be the only cause of vitamin D deficiency since there is no correlation between osteomalacia and the degree of steatorrhea, and osteomalacia may be present in the absence of steatorrhea [ 51. In addition, some celiac patients are resistant to oral or parenteral therapy with vitamin D [lo]. We have recently encountered a patient with celiac disease in whom osteomalacia and proximal muscle weakness persisted during a period when she was responding well in other ways to a gluten-free diet. She failed to respond to large doses of orally-administered vitamin D2 and to a parenteral dose of vitamin DS, but she showed dramatic improvement when treated with orally-administered 25-hydroxyvitamin D3 (25-0HD3). METHODS Serum PS-Hydroxyvitamin D (25OHD). Serum 25-OHD was measured by a modification of the competitive protein-binding assay of Beisey et al. [ 111, as described by Rosen et al. [ 121.This assay utilizes a specific binding protein isolated from rachitic rat serum with a high affinity for 25-OHD. Since reference 25-hydroxyvitamin DP(25XM-Q) was not available for comparison of its displacement potency with that of 25-OHD3the plasma levels of 25-OHD reported represent total 25-OHD, 25-OHD2as well as 25-O&. Other vitamin D metabolites, including 1,25-(0H$, are not measured by this method. Crystalline 250HD3 was used as the reference standard, and the tracer was (26,27-3H) 25-0HD3. The sensitivity of the method is 1 ng/mi. in our healthy control subjects, the serum 25-OHD is 29.2 f 9.9 ng/mi (mean f SD).

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Serum Parathyroid Hormone (PTH). Serum PTH was assayed with 1 ml of guinea pig antiserum (GPIM) to PTH by the method of Arnaud et al. [ 131. Bone Biopsy. All biopsy specimens were obtained from the iliac crest after local anesthesia. Bone biopsy specimens were taken six days before treatment with parenteral vitamin Ds, and 17 days and five months after treatment with oral 25-DHDs. Quantitative histology was performed as previously described [ 141. All measurements were made on 100-p thick undecalcified sections cut from a bone biopsy specimen embedded in methyl methacrylate. At a magnification of X 100, osteoid appeared as a longitudinally streaked, semitranslucent, glossy material lying on a bone surface; osteocytes were clearly visible, having been incorporated into slit-like lacunas in the osteoid [ 151. Thin layers of collagenous material, only a few microns thick, were not considered osteoid. Paired measurements were made on each border midway between the center-point of maximum thickness and the tapered lateral edge, with care taken to avoid obvious distortions. Each section was examined until 10 paired observations were obtained or the specimen was completely scanned. Osteoid tissue on periosteal surfaces was not measured. Inactive osteoid, namely, osteoid with no adjacent osteoblasts, was evaluated in the same sections. The results were expressed as the percentage of the total and of the mineralized surface of the biopsy specimen covered by inactive osteoid. The calcification front was also evaluated. The calcification front, indicating new mineral deposition, appears as a granular deposit at the line of demarcation between the unmineralized osteoid and the mineralized bone. The percentage of this line showing the granular deposit was evaluated: 15 measurements were made, and the mean and range are presented. The percentage of bone surface occupied by bone resorption and bone formation was also measured. The measurements were made from the microradiographic appearance of the bone surface, and the histologic appearance of osteoclasts and osteoblasts. The results were expressed as the length of forming or resorbing surfaces as a percentage of the total and of the mineralized surface in the bone biopsy specimen. The calculation showing the resorbing and forming surfaces in terms of mineralized bone surfaces was carried out because bone covered with inactive osteoid may not be available for resorption, so that this value is preferred when assessing resorption activity. Electrophysiology. Quantitative electromyography (EMG) was performed by the method of Buchthal [ 161. The following were measured: mean duration of at least 20 different muscle action potentials elicited during minimal efforts, frequency and type of polyphasic potentials during mild voluntary contraction, spontaneous activity at rest, and pattern of action potentials on full voluntary contraction. Muscle Biopsy. Biopsies were performed with a muscle clamp after local anesthesia. Tissue was fixed in formalin and embedded in paraffin wax. Transverse sections were stained with hematoxylin and eosin. Tissue for histochemistry was frozen in isopentane and cooled with liquid nitrogen. Ten micron cryostat sections were prepared for Gomori trichrome, reduced diphosphopyridine nucleotidase (DPNG),

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myofibrillar ATPase (pH 9.4) oil red 0, and periodic acidSchiff stains. CASE REPORT The patient is a white married woman, born December 13, 19 18. She has always been teetotal and enjoyed being out of doors, gardening and swimming. At age six she had occasional abdominal cramps of unknown cause for which a laparotomy was performed. She was then asymptomatic until 1962, age 44, when she first noted diarrhea with foul-smelling stools. In 1964, age 46, she was hospitalized and found to pass 12 to 50 g fat in stools per day. Moreover, anemia developed with a hemoglobin level of 7.5 g/d1 thought to be due to combined iron, folic acid and vitamin B12deficiency. Serum calcium was low (7.0 mg/dl) and so was alkaline phosphatase (4.4 King-Armstrong units/liter). A jejunal biopsy specimen showed subtotal villous atrophy. A gluten-free diet without alteration of milk intake was prescribed, and she adhered rigorously to the regimen over the ensuing years. As a result of the diet, the diarrhea disappeared and she gained 30 pounds in weight. In 1971, age 53, still on the gluten-free diet and without bowel symptoms, she had pain in her left leg and groin which, over the next 18 months, spread to her back and her left hip. In addition, she noted difficulty in walking, climbing stairs and rising out of a chair. At this time, the serum calcium was 8.1 mg/dl, serum inorganic phosphate 3.3 mg/dl and alkaline phosphatase twice the upper limit of normal. Roentgenograms of her bones were within normal limits with periosteal erosions suggestive of secondary hyperparathyroidism. Inguinal node and liver biopsy specimens were also within normal limits. It was thought that the bone pain, as well as hypocalcemia and the increased serum alkaline phosphatase activity, were due to vitamin D deficiency. The patient was given oral doses of 1.2 mg crystalline dihydrotachysterol and 8 g calcium lactate in May 1972. This treatment was maintained until her first visit to the Hershey Medical Center in January 1974. At no time during her clinical course was she taking drugs, such as phenytoin, or abusing alcohol. Physical examination at that time revealed a chronically ill-appearing woman who weighed only 52 kg. She had a slow-waddling gait and weakness of the proximal muscles of both the upper and lower extremities. There was also slight weakness of the extensors and flexors of the wrists and ankles. All deep tendon reflexes were normal, and there was no Babinski sign. There were no signs of muscle wasting or fasciculation. No involuntary tongue or limb movements were present. Sensation, coordination and cranial nerve examination were within normal limits. A positive Chvostek sign was present. Laboratory investigations disclosed the following (normal values are given in parentheses: Hemoglobin level 10 g/dl (12 to 16 g/dl), white blood cells 6,500/mm3 (5,000 to 10,000/mm3), serum folate 7.0 ng/ml(4 to 16 ng/ml), vitamin 8,s 546 pg/ml(300 to 1,000 pg/ml), iron 17 pgldl(40 to 140 pg/dl), total iron-binding capacity 534 pg/dl (220 to 380 pgldl), iron saturation 3 per cent (22 to 25 per cent), calcium 8.1 mgldl(9.0 to 10.5 mg/dl), magnesium 2.4 mg/dl(2.4 to 4.8 mg/dl), phosphorus 3.2 mgIdl(3.0 to 4.6 mg/dl), alkaline phosphatase 242 U/liter (30 to 110 U/liter), 5’-nucleotidase

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0.1 U/liter (0.1 to 1.6 U/liter), bilirubin 0.3 mg/dl (0.1 to 1.2 mg/dl), aspartate transaminase 35 U/liter (5 to 40 U/liter, lactic dehydrogenase 195 U/liter (50 to 180 U/liter), creatinine phosphokinase 138 U/liter (10 to 90 U/liter), aldolase 27 U/liter (0 to 20 U/liter), albumin 4.4 g/dl(3.5 to 5.0 g/dl), globulin 3.1 g/d1 (2.0 to 3.5 g/dl). lmmunoglobulins were normal. Creatinine clearance was 108 ml/min. Aminopyrine breath test [ 171 was 4.8 per cent (>4.4 per cent). The prothrombin time value was equal to the control value. Serum carotene was 26 pgldl (60 to 200 pg/dl) and cu-tocopherol was 15 U/ml (10 to 20 U/ml). A 1Bhour postprandial serum gave the following values: clear serum, no chylomicrons, cholesterol 210 mg/dl (160 to 330 mg/dl), triglycerides 82 mg/dl (10to 190 mg/dl) and low density lipoproteins 40 mg/dl (80 to 120 mg/dl). Serum 25-OHD was 5.4 ng/ml (20 to 50 ng/ml). Serum PTH was 49 ~1 eq/ml(<40). Absorption test results were as follows: xylose, 3.7 g of 25 g dose excreted in 5 hours (>5 g); vitamin B12 17.4 per cent excreted in 24 hours (> 10 per cent); fecal fat on 100 g fat diet 1.8-5.7 g/ day. Skeletal roentgenograms showed no pathologic fractures, no subperiosteal bone erosions and only minimal thinning of bones. Jejunal biopsy specimen showed partial villous atrophy, with normal surface epithelial cells. A bone biopsy specimen taken on January 25, before treatment with parenteral vitamin D3 or oral 25-OHD3, showed bone formation surfaces to be greater than normal. Bone resorption surfaces were twice normal, or four times normal when the results were expressed in terms of the available mineralized surface only (Table I). The surface covered by inactive osteoid was increased and the width of unmineralized osteoid was three times normal. The calcification front was absent or appeared only partially in the demarcation line

TABLE

I

AND CELIAC DISEASE--HEPNER

Quantitative

ET At

on the Bone Biopsies

Histology

1st 2nd 3rd Control* Biopsy* Biopsy7 Biopsy* (mean f SD)

___ Formation Per cent total surface Per cent mineralized surface Resorption Per cent total surface Per cent mineralized surface Inactive osteoid Per cent total surface Per cent mineralized surface Osteoid width (II) Mean SD Calcification front (%) Mean Range

4.3 10.2

1.8 3.4

28.1 29.0

2.4 f

1.8

8.9 21.1

15.8 30.0

11.6 11.9

4.1 f

1.6

57.6 136.0

47.4 90.1

2.8 2.9

45.9 7.7

31.4 10.9

15.0 3.3

15.4 f

53 O-90

80 50-100

80f

13 O-40

0

1.8

12

Performed six days before D3 therapy. 7 Performed after 17 days of 25-OH therapy. i Performed after five months of 24-OH therapy. 5 Control subjects 45 to 60 years of age. l

between osteoid and mineralized bone. The latter three findings are diagnostic of osteomalacia (Figure 1A). EMG performed on February 7, after therapy with parenteral vitamin D3 but before therapy with oral 25-OH&, showed the following: Left deltoid: mean action potential duration 6.63 msec (56 per cent decreased); 57 per cent polyphasic potentials (increased); no spontaneous acfivity at rest; amplitude 1 to 5 mV (normal) and mixed to interference pattern at full effort. Left triceps: mean action potential duration 6.95 msec (49

Figure 1. Unstained, m&calcified section of iliac crest. A, first biopsy specimeh obtained before parentem/& therapy. Large areas of the bone biopsy specimen consist of osteoid tissue which are broad and cover a significant propoftion of bone surface. Magnification X 100. B, second biopsy specimen; the amount of osteoid has decreased in the second biopsy specimen taken after 17 ckys of treatment with 25 Olil&. Magnification X 160. C, third biopsy specimen; further decrease in osteoti after further treatment with 25-0HD3. Magnification X 700. (All reduced by 28 per cent).

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-t-o 31,

I I

5

IO

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I I

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per cent decreased); 65 per cent polyphasic potentials (increased); no spontaneous activity at rest; amplitude 0.5 to 2.5 mV (decreased) and mixed to interference pattern at full effort. Left quadriceps: mean action potential duration 8.52 msec (37 per cent decreased); 29 per cent polyphasic potentials (increased): no spontaneous activity at rest; amplitude 1 to 2 mV (decreased) and mixed pattern at full effort. EMG of all muscles was myopathic. There was no electromyographic evidence of tetany. A right quadriceps biopsy was performed on January 29. Muscle fiber diameter ranged from 30 to 110 CLwith the majority of fibers between 10 and 110 ~1.No degeneration, regeneration, necrosis or inflammation was noted. A normal checkerboard pattern of both histochemical fiber types was seen on the myofibrillar ATPase and DPNH stains. Neither target fibers nor group atrophy was observed. The biopsy specimen was within normal limits. Nine months after the patient discontinued 25-OHDs therapy, muscle weakness redeveloped. Serum biochemical tests became abnormal, with a decrease in serum calcium and phosphate and an increase in alkaline phosphatase; serum 25OHD decreased to 11 ng/ml. She was treated with 20 pg of 250HDs for eight weekly intervals; muscle strength and serum biochemical test results returned to normal. Serum 25OHD increased to 38 ng/ml. RESULTS

The patient was given, intravenously, 120 pg of vitamin D3 dissolved in propylene glycol on January 31, 1974. There was no change in biochemical values except for an increase in serum phosphate during this regimen (Figure 2). Therefore, treatment with oral 25OHD3 was begun on February 11. This therapy was continued until March 8, by which time she had received 4.08 mg of 25-OHDs. Some laboratory values changed dramatically as a result of 25-OHDs therapy (Figure 2). Serum 25-OHD, which was 5.4 ng/ml on admission and remained approximately at this level during the course of treatment with parenteral vitamin D3, increased to 18 ng/ml within

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27, ,I , __-.. APKIL

1

Figure 2. Serum calcium phosphorus, alkaline phosphatase, magnesium and 25-OHD during therapy with 120 pg of parenteral vitamin 4 (January 3 1) and successive oral doses of 25-OHD (February 7 1 through March 8).

48 hours of administration of 40 pg of 25OHD and to 54 ng/ml 24 hours after the administration of another 200 pg of the vitamin. The serum inorganic phosphate level, which had begun to rise during the course of the treatment with parenteral vitamin Ds, continued to rise throughout the course of treatment during treatment with 25OHDs. Serum calcium did not change during treatment with parenteral vitamin Ds but increased 48 hours after oral 25OHDs therapy was started. Serum alkaline phosphatase began to increase about four weeks after therapy with 25OHDs was commenced. It continued to increase until the middle of April, and then began to decline. Serum PTH responded rapidly to therapy with 25OHDs. It was 49 ~1 eq/ml on January 31, increased to 62 ~1 eq/ml on February 13 and decreased to 32 ~1 on February 21. On March 1 it was 28 ~1 eq/ml, and on March 13 it was 17 ~1 eq/ml. Two bone biopsies were performed after treatment with 25-OHD had been instituted. The percentage of osteoid decreased after 17 days of 25-OHD therapy (Figure 1B) and after five months (Figure 1C). The quantitative histologic measurements are shown in Table I. Bone formation surfaces decreased initially; they had increased substantially by the time of the third blopsy, perhaps due to remodelling, since the third biopsy was adjacent to the first biopsy site. Bone resorption surfaces increased initially, perhaps in response to the increase in available mineralized surface. After five months of 25-OHD therapy, the value had decreased but was still above normal. The surface of bone covered with inactive osteoid decreased to just above normal (2.8 per cent) whereas the width of the osteoid decreased to within the control range; the calcification front values also became normal. The appearance generally changed from one of gross osteomalacia to one which was relatively normal.

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EMG performed four months after 25OHD therapy (June 26) showed the following: Left deltoid: mean action potential duration 7.9 msec (47 per cent decreased); 21 per cent polyphasic potentials (increased); occasional fibrillation potentials rest; amplitude 2 to 5 mV (normal and mixed pattern at full effort). Left triceps: mean action potential duration 8.7 msec (37 per cent decreased); 29 per cent polyphasic potentials (increased); no spontaneous activity at rest; amplitude 1 to 2 mV (decreased) with mixed pattern at full effort. Left quadriceps: mean action potential duration 9.5 msec (30 per cent decreased); 27 per cent polyphasic potentials (increased); no spontaneous activity at rest; amplitude 1 to 2 mV (decreased) with mixed pattern at full effort. EMG was myopathic but there was marked improvement in mean action potential duration in all muscles. COMMENTS The response to 25-OHDs in our patient was documented clinically, biochemically, histologically and electromyographically, and contrasted strikingly with her failure to respond to large doses of dihydrotachysterol. After three months of therapy with 25OHDs, she had no problem walking, climbing stairs or rising out of the chair, and she had no bone pain. Biochemically, the serum calcium, phosphate, alkaline phosphatase, PTH and serum 25-OHD had returned to normal. Histologically, the osteomalacia healed; and electromyographically the mean action potential of all tested muscles had improved. This patient may have had osteomalacia when she first presented with steatorrhea and a serum calcium of 7.0 mg/dl in 1964. Vitamin D deficiency after she had responded to gluten withdrawal became her principal clinical problem despite the correction of steatorrhea. Although the jejunal biopsy specimen was not completely normal, and she had evidence or proximal small bowel dysfunction as manifested by iron deficiency and xylose malabsorption, she was apparently able to absorb other fat-soluble vitamins because both the prothrombin time and the serum alpha tocopherol levels were normal. She had been taking 1.2 mg of vitamin D2 orally daily for 20 months prior to the present study. The fact that she responded within a matter of days to the small doses of orally-administered 25-OHDs after having failed to respond to large doses of dihydrotachysterol given over a prolonged period suggests that 25-OHDs should be the vitamin of choice in the treatment of osteomalacia due to intestinal malabsorption. The reasons for this patient’s failure to respond to dihydrotachysterol is unclear. If she had been able to absorb one thousandth of the daily dose of dihydrotachysterol she would have received her minimal daily requirements for the vitamin; it is difficult to see how she could have had total malabsorption of the vitamin in the presence of improved gastrointestinal absorption

AND CELIAC DISEASE-HEPNER

ET AL.

while on the gluten-free diet. Her failure to respond to 120 pg of vitamin Ds given parenterally is also puzzling. Failure of the liver to 25-hydroxylate vitamin Ds has been described in patients with cirrhosis 1181 and is presumably due to a decrease in hepatic microsomal 25-hydroxylating enzymes responsible for this biotransformation [ 191. Our patient had normal liver function and a normal liver biopsy, so that hepatic 25-hydroxylation was presumably normal. 25-OHD is the substrate for 1,25-(OH)*D, the renal metabolite thought to be the most biologically active form of vitamin D [ 20-241. There was no evidence of renal dysfunction in our patient so that we have no reason to believe that alterations in this metabolic pathway may have played a role in her failure to respond to dihydrotachysterol. A possible explanation of her failure to respond to the 120 pg dose of parenteral vitamin D3 is that she was vitamin D-depleted at the time of study and that the administered vitamin D was taken up by other vitamin D-deficient tissues, such as fat, and not available as a substrate for hepatic 25-hydroxylation. The myopathy seen in this patient is a common complication of osteomalacia. Muscle weakness does not seem to be correlated with serum calcium content since the level of serum calcium may be low, elevated or normal [25]. Moreover, our patient had progressive clinical and electrophysiologic improvement despite marked variation in serum calcium levels. Vitamin D deficiency or a disorder in its metabolism has been postulated as a cause of the weakness since repletion with vitamin D is often associated with marked improvement. Our patient vividly illustrates this clinically and electrophysiologically. However, the mechanism of action of vitamin D on muscle is not completely understood. Kodicek [26] has demonstrated that radioactive vitamin D is localized on muscle membranes in rachitic rats and suggested that the vitamin may have an important role in the transport of divalent cations across muscle membranes. Others have suggested that vitamin D is necessary for normal mitochondrial function [26, 271; the release of calcium from mitochrondria is influenced by vitamin D, and vitamin D deficiency may produce morphologic changes in mitochondria. This theory, however, is tenuous since human proximal muscle consists of equal amounts of both red and white fibers. Mallette et al. [28] have suggested that the weakness of osteomalacia is due to neurogenic disease rather than to a primary muscle disorder because of neurogenic atrophy and type II muscle fiber atrophy noted on muscle biopsy. These investigators also suggested that the electromyographic abnormalities and small polyphasic motor unit potentials represent neurogenic changes rather than myopathic abnormalities. This patient had no steatorrhea at the time that osteomalacia and vitamin D deficiency developed. The absence of steatorrhea in such patients is unusual, al-

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though a similar case in a patient also taking phenytoin was described by Moss et al. [5] and others, perhaps, by Hajjar et al. [ 171 and Munch [ 291. A more remarkable feature in this case was the development of osteomalacia and vitamin D deficiency at a time that the patient had responded to a gluten-free diet. Her response was incomplete, however, since apart from her vitamin D deficiency, she had iron deficiency and xylose malabsorption. Her case illustrates the point that response to gluten withdrawal in celiac patients may not be an all or none phenomenon, and that partial responsiveness to the diet may occur with residual defects that may cause severe nutritional deficits. Arnaud et al. [30] have reported that 25-hydroxyvitamin D3 has an enterohepatic circulation in man. They also found, in three patients with celiac disease, an

increased plasma turnover and fecal excretion of 25 OHD [31]. It is possible that this abnormality may contribute to the low serum 25OHD and osteomalacia in our patient, but further studies with radioactivelylabeled 25OHDs are required to investigate this matter. ACKNOWLEDGMENT We gratefully acknowledge the help of Dr. John Hinman from Upjohn Company, Kalamazoo, Michigan, who provided the 25-hydroxyvitamin Ds. Dr. Hector DeLuca also provided much invaluable advice concerning the evaluation of the patient, as well as the vitamin Ds for parenteral studies. D. Sara Arnaud provided invaluable insights and comments during the preparation of this manuscript.

REFERENCES Benson GD, Kowlessar OD, Sleisenger MHS: Adult celiac disease with emphasis upon response to gluten-free diet. Medicine (Baltimore) 43:1, 1964. 2. Brooks FP, Powell KC, Cerda JJ: Variable clinical course of adult celiac disease. Arch Intern Med 117: 789, 1966. 3. Melvin KEW. et al.: Calcium metabolism and bone pathology in adult celiac disease. Q J Med, 29: 83. 1970. 4. Juergens JL, Scholz DA, Wollaeger EE: Severe osteomalacia associated with occult steatorrhea due to nontropical sprue. Arch Intern Med 98: 774, 1956. 5. Moss AJ, Waterhouse C. Ten-y R: Gluten-sensitive enteropathy with osteomalacia but without steatorrhea. N Engl J Med 272: 825, 1965. 6. Mann 61, Brown WP, Kern F Jr.: The subtle and variable clinical expression of gluten-induced enteropathy (adult celiac disease, nontropical sprue). An analysis of twentyone consecutive cases. Am J Med 48: 357, 1970. 7. Hajjar ET, Vincenti F, Salti IS: Gluten-induced enteropathy. Osteomalacia as its principal manifestation. Arch Intern Med 134: 565, 1974. 8. Bordier P, Hepner GW, Matrajt H, et al.: L’effet du regimen sans gluten sur les lesions ossueuses de la maladie coeliaque. Biol Gastroenterol (Paris) 1: 75, 1969. 9. Thompson GR, Lewis Ft. Booth CC: Absorption of vitamin Ds-3H in control subjects and patients with malabsorption. J Clin Invest 45: 94, 1966. 10. Nassim JR, Saville PD, Cooke PB: Effects of vitamin D and gluten-free diet in iodiopathic steatorrhea. Q J Med 28: 141, 1959. 11. Belsey R, Deluca HF, Potts JT: Competitive binding assay for vitamin D and 25-DH vitamin D. J Clin Endocrinol Metab 33: 554,197l. 12. Rosen JF, Roginsky M, Nathenson G: 25-hydroxyvitamin D. Plasma levels in mothers and their premature infants with neonatal hypocalcemia. Am J Dis Child 127: 220, 1974. 13. Arnaud CD, Tsao HA, Littledike T: Radioimmunoassay of human parathyroid hormone in serum. J Clin Invest 50: 2 1, 1971. 14. Jowsey J: The Bone Biopsy, New York, Plenum Publishing Co., 1977. 15. Johnson KA, Riggs BL, Kelly PJ, et al.: Dsteoid tissue in normal and osteoporotic individuals. J Clin Endocrinol Metab 33: 745,197l. 16. Buchthal R: An introduction to Electromyography, Copenhagen, Gledendal, 1957. 17. Hepner GW, Vessel ES: Quantitative assessment of hepatic 1.

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19. 20. 21.

22.

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24.

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28.

29.

30.

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microsomal function by a breath analysis technique after oral administration of [ 14 C] aminopyrine. Ann Intern Med 83: 632, 1975. Hepner GW. Roginsky M, Moo HF: Abnormal vitamin D metabolism in patients with cirrhosis. Am J Dig Dis 21: 527, 1976. Parfitt MA: Hypophosphatemic vitamin D refractory rickets and osteomalacia. Ortho Clin North Am 3: 653, 1972. Ponchon G, DeLuca HF: The role of the liver in the metabolism of vitamin D. J Clin Invest 48: 1273, 1969. Fraser DR, Kodicek E: Unique biosynthesis by kidney of a biologically active vitamin D metabolite. Nature 228: 764, 1970. Haussler MR, Boyce DW, Littledike ET, et al.: A rapidly acting metabolite of vitamin D3. Proc Natl Acad Sci 68: 177. 1971. Holick MF, Garabedian M, DeLuca HF: 1,25-hydroxycholecalciferol: metabolite of vitamin D3 active on bone in anephric rats. Science 176: 1146, 1972. Wong RG, Myrtle JF, Normal AW: Studies on calciferol metabolism. C. The occurrence and biological activity of 1,25dihydroxyvitamin D3 in bone. J Biol Chem 247: 5728, 1972. Wolf SM, Lusk WL: Hypocalcemic myopathy. Bull Los Angeles Neurol Sot 37: 167. 1972. Kodicek E: Turnover and distribution of Vitamin D and its mode of action. The Transfer of Calcium and Strontium Across Biological Membranes (Wassermann RH, ed), New York, Academic Press, 1963, p 185. DeLuca HF. Sallis JD: Parathyroid hormone: its subcellular actions and its relationship to Vitamin D Parathyroid Glands (Gailland PJ, Talmage RV, Bundy AM, eds), Chicago, University of Chicago Press, 1965, p 181. Mallette LE, Patten BM, Engel WK: Neuromuscular disease in secondary hyperparathyroidism. Ann Intern Med 82: 474, 1975. Munch 0: Osteoporosis due to malabsorption of calcium responding favorably to large doses of vitamin D. Q J Med 33: 209, 1964. Arnaud SB, Newcomer AD, Hodgson SF, et al.: Serum 25hydroxyvitamin D (25-OH-D) and the pathogenesis of osteomalacia in patients with non-tropical sprue (NTS). Gastroenterology 72: 1025, 1977. Arnaud SB, Goldsmith RS, Lambert PW, et al: 25-hydroxyvitamin D3. Evidence of an enterohepatic circulation in man. Proc Sot Exp Bio Med 149: 570, 1975.