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Abnormalities in serum osteocalcin values in children with chronic rheumatic diseases
Abnormalities in serum osteocalcin values in children with chronic rheumatic diseases A n n R e e d , MD, M a u r e e n H a u g e n , CPNP, Lauren M. P a c h m a n , MD, a n d C r a i g B. L a n g m a n , MD From the Department of Pediatrics, Northwestern University Medical School, and the Mineral Metabolism Laboratory, Division of Nephrology and Division of Immunology/Rheumatology, Children's Memorial Hospital, Chicago, Illinois We studied b o n e m i n e r a l m e t a b o l i s m p r o s p e c t i v e l y in t t 3 children with c h r o n i c r h e u m a t i c diseases ( j u v e n i l e arthritis, systemic lupus erythematosus, a n d juvenile d e r m a t o m y o s i t i s ) to d e t e r m i n e the r e l a t i o n s h i p of serum levels of o s t e o c a l cin to r h e u m a t i c d i s e a s e a c t i v i t y a n d c o r t i c o s t e r o l d usage, a n d to determine, in part, the cause of o s t e o p e n i a in this p o p u l a t i o n . Disease a c t i v i t y was quant i t a t e d b y historical, c l i n i c a l , and s e r o l o g i c means a n d an a c t i v i t y score d e r i v e d . The 113 children were d i v i d e d a c c o r d i n g to the expression of their disease, which was a c t i v e (group I: m e a n score 3.42, m e a n e r y t h r o c y t e sedimentation rate 28 m m / h r ) or i n a c t i v e (group 2: score 1.7, e r y t h r o c y t e s e d i m e n t a t i o n rate 15 m m / h r ) (p <0.02 group 1 vs group 2 for e a c h v a l u e ) , or which remitted during the study ( g r o u p 3). We found that serum levels of o s t e o c a l c i n , but not those of ionized c a l c i u m , 2 5 - h y d r o x y v i t a m i n D, 1,25-dihydroxyvitamin D, and p a r a t h y r o i d hormone, were r e d u c e d in g r o u p I children e v e n b e f o r e corticosteroid t h e r a p y was e m p l o y e d . Children in both g r o u p 2 a n d group 3 h a d normal o s t e o c a l c i n levels d e s p i t e the use of corticosteroids. The r e d u c e d l e v e l s of ost e o c a l c i n were p r e d i c t i v e of a r e d u c t i o n in b o n e mass m e a s u r e d by photon abs o r p t i o m e t r y in 16 of 19 children so studied. We c o n c l u d e that skeletal abnorm a l i t i e s that result in a r e d u c e d b o n e mass o c c u r in the c l i n i c a l course of the m a j o r i t y of children with a c t i v e c h r o n i c r h e u m a t i c diseases, are a s s o c i a t e d with r e d u c e d o s t e o c a l c i n levels, and are not r e l a t e d to the use of corticosteroids. Serum o s t e o c a l c i n levels m a y be a sensitive marker for r e d u c e d o s t e o b l a s t activity a n d b o n e f o r m a t i o n in children with c h r o n i c r h e u m a t i c diseases. (J PEDIATR 1990;116:574-80)
Children with chronic rheumatic diseases, including juvenile rheumatoid arthritis, systemic lupus erythematosus, and juvenile dermatomyositis, have alterations in skeletal integrity] demonstrated by both the presence of periarticSupported in part by National Institutes of Health grants AR30692 and DK33949, by the Arthritis Foundation, and by the Otto Sprague Memorial Fund. Submitted for publication Dec. 27, t 988; accepted Oct. 26, 1989. Reprint requests: Craig B. Langman, MD, Mineral Metabolism Laboratory, Divisionof Nephrology, Box 37, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL 60614. 9/20/17740
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ular bony destruction 2, 3 and the occurrence of generalized osteopenia. 48 The precise mechanisms that produce each of the bony changes in children with CRD are not completely understood but may involve abnormalities of mineral metabolism9,10 Corticosteroid-induced osteoporosis, the most frequent form of secondary osteoporosis in adults] 1 has often been ascribed as the cause of osteopenia in children with rheumatic diseases, although few studies have been performed. Additionally, some investigators have implicated immobilization12 as the cause of the osteopenic bone disease. During an initial retrospective chart review, we identified
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BMC CRD ESR JDMS JRA 25-OHD 1,25-(OH)2D SLE
Serum osteocalcin and chronic rheumatic diseases
Bone mineral content Chronic rheumatic diseases Erythrocyte sedimentation rate Juvenile dermatomyositis Juvenile rheumatoid arthritis 25-HydroxyvitaminD 1,25-DihydroxyvitaminD Systemic lupus erythematosus
37 ambulatory children with CRD who were not receiving corticosteroid therapy but who had generalized bone demineralization on standard radiographs or unexplained, repeated fractures. To better understand the alteration in bone homeostasis in children with CRD, we prospectively studied several measures of mineral metabolism. We postulated that generalized osteopenia in children with CRD may be the result of reduced bone formation as reflected in reduced serum osteocalcin levels, in addition to enhanced bone resorption as reflected by hypercalciuria. We further postulated that corticosteroid use would have an adverse effect on measured osteocalcin levels in this group of children. METHODS We studied children followed in the Children's Memorial Hospital Immunology/Rheumatology Clinic during an 18month period from February 1987 to April 1988. The patients included those with JRA, JDMS, and SLE. 13All patients with JRA fulfilled the criteria established by the American College of Rheumatology.14 Patients with J DMS fulfilled established criteria. 15 Patients with JRA were categorized as those having polyarticular JRA (five joints or more), pauciarticular JRA (less than five joints), systemic onset JRA, or systemic-onset JRA that had developed into polyarticular JRA. Clinical activity was assessed in standard fashion by history and physical examination of each patient; assessment was performed by one physician. A disease activity score was given on the basis of the presence of joint swelling, warmth, redness, range of motion, muscle weakness, complaints of pain and morning stiffness, and the use of antiinflammatory medications. The score ranged from 0 to 5, with 0 representing no complaints or physical findings of active disease and no use of antiinflammatory medication, and 5 representing very active clinical disease and the use of medication. We defined a disease as active if the patient score was >--3 and inactive if --<2. After the initial visit, each patient was placed in one of two groups on the basis of their initial activity score: group 1 (clinically active CRD) or group 2 (inactive disease). After the study (April 1988), group 1 patients whose disease activity remitted during the course of this study (score -< 2)
575
T a b l e I. Clinical characteristics of children with chronic rheumatic disease Disease
Poly JRA Pauci JRA Syst JRA Syst-Poly JRA JDMS SLE TOTAL
n
M/F
37 21 12 13 13 I_.Z
4/33 6/15 3/9 6/7 9/4 3/14
1V2-20 2-12 3-18 3-17 4-19 11-21
31/82
11/2-21
113
A g e range (yr)
Poly, Polyarticular; Pauei, pauciarticular; Syst, systemic,
were placed in group 3 (disease remission) and analyzed separately. There were no patients from group 2 whose disease became active during this study. Patients entered into the study at various times after their disease began, ranging from onset to more than 10 years after onset. Studies performed at the first visit included measurement of a Westergren erythrocyte sedimentation rate; determination of levels of blood ionized calcium, serum osteocalcin, parathyroid hormone, 25-hydroxyvitamin D, and 1,25dihydroxyvitamin D; and measurement of a random urine calcium/creatinineratio. On subsequent patient visits, at an interval determined by the attending physician, blood was obtained only for measurement of osteocalcin levels. Blood ionized calcium was measured in duplicate by an ion-specific electrode (Radiometer America, Westlake, Ohio). The mean (_+ 2 SD) normal blood ionized calcium level is 1.21 _+ 0.13 mmol/L. Levels of 25-OHD and 1,25(OH)zD were measured in triplicate by specific radioreceptor assays previously described. 16 Normal values (95% confidence intervals) for 25-(OH)D are 15 to 40 ng/ml, and for 1,25-(OH)zD are 15 to 80 pg/ml. Serum parathyroid hormone levels were measured in duplicate by a specific radioimmunoassay that detected the mid-portion of the hormone molecule (amino acids 44-68) and that was previously validated for clinical measurements over a wide range of parathyroid hormone levels 16, 17; normal values (95% confidence intervals) are 15 to 80 pmol/L. The ESR was determined by standard laboratory procedure. Serum osteocalcin levels were measured by specific radioimmunoassay (lncstar Corp., Stillwater, Minn.) with the use of bovine osteocalcin standards. Least detectable values ranged from 0.2 to 0.3 ng/ml; 50% displacement values ranged from 12 to 15 ng/ml. Interassay and intraassay coefficients of variation were 6% and 2%, respectively, over an osteocalcin range of 0.3 to 40 ng/ml. Normal values (95% confidence intervals) established in our laboratory are 15 to 25 ng/ml in children younger than 13 years of age (n = 126) and 2 to 8 ng/ml in children older than 131/2years (n = 98). We also measured serum osteocalcin in children
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Table II. I n i t i a l b i o c h e m i c a l m e a s u r e m e n t s in c h i l d r e n w i t h c h r o n i c r h e u m a t i c d i s e a s e
CRD PolyJRA
Activity + -
Pauci JRA Syst JRA Syst-Poly JRA SLE JDMS
+ + + + + -
ICa (mmol/L)
PTH (pmol/L)
25-OHD (ng/ml)
1,25-(OH)aD (pg/ml)
U Ca/Cr ratio
osteocalcin
1.22_+05(13) 1.17 -+ 06 (14) 1.25 _+ 06 (6) 1.24 _+ 08 (3) 1.15 _+ 05 (4) 1.23 _+ 03 (3) 1.17_+ 04 (2) 1.16 _+ 01 (3) 1.23 _+ 04 (10) 1.23 -+ 06 (7) 1.20 • 07 (6) 1.19 _+ 13 (2)
47_+ 15(14) 47 • 10 (13) 41 _+ 22 (5) 52 -+ 13 (3) 51 _+ 7 (4) 4l _+ 6 (2) 47 _+ 8 (3) 45 _+ 3 (3) 48 _+ 15 (11) 47 -+ 17 (7) 40 _+ 24 (9) 38 _+ 24 (9)
20_+7(14) 22 _+ 9 ( 1 3 ) 22 -+ 8 (5) 20 _+ 3 (3) 19 _+ 4 (4) 20 _+ 3 (2) 14 _+ 1 (2) 17 _+ 6 (3) 20 _+ 12 (10) 18 _+ 9 (7) 18 _+ 6 (9) 25 _+ 14 (4)
41_+23(I4) 41 _+ 31 ( 1 3 ) 54 _+ 19 (4) 50 + 6 (2) 30 _+ 2 (3) t5 _+ 1 (2) 50 -+ 23 (2) 45 _+ 23 (3) 25 _+ 12 (10) 33 _+ 21 (6) 46 _+ 32 (9) 32 _+ 20 (4)
0.13-+0.12(14) _+ .08 (10) 0.08 _+ 0.06 (6) 0.03 _+ 0.01 (2) 0.18 (1) 0.06 (2) 0.19 (1) 0.03 (1) 0.t8 _+ 0.02 (8) 0.08 _+ 0.07 (7) 0.30 _+ 0.22 (8) 0.31 _+ 0.16 (4)
11/14 0/I3 14/15 0/3 5/6 0 7/8 0 9/10 0/6 8/10 1/6
0.10
Reduced
Values are mean + SD for children in group 1 (Activity +) and group 2 (Activity - ) except wherc fewer than three children were studied, when the average or sole value is presented. Number in parentheses after each measurement represents the number of children studied. Activity represents disease activity score >~3 (+) or its absence, score --<2(-), as defined in the Methods section. For all disease groups combined, there was a significantdifferencein the number of children with disease activity versus those with absence of activity among those with reduced ostcocalcin values (p <0.05). ICa, Ionized calcium (in blood); PTH, parathyroid hormone; U Ca/Cr, urinary calcium/creatinine (ratio); Poly. polyarticular; Pauci, pauciarticular; Syst, systemic.
with two other chronic, n o n r h e u m a t i c diseases: congenital heart disease and common variable hypogammaglobulinemia. Neither group had a history of joint involvement, aut o i m m u n e disease, or recent infectious illness. The subjects included 4 female and 11 male patients with an age range from 1 to 21 years at tile time of serum collection. O f 15 of these latter children, 13 had normal osteocalcin levels for their age. Bone mineral content was determined by photon absorptiometry during a 9-month period, from July 1987 to April 1988, in 16 patients with clinical disease activity (group 1) and low osteocalcin values, in addition to four patients with inactive disease (group 2) or improved disease (group 3) and normal osteocalcin values. The B M C was determined in the distal one third of the n o n d o m i n a n t radius by conventional single-beam photon absorptiometry ( L u n a r Radiation Corp., Madison Wis.). Repetitive m e a s u r e m e n t error was less than 3%. From bone width and bone mineral values, B M C was calculated by computer methods ( L u n a r ) and expressed as grams per square centimeter. N o r m a l values for B M C were previously determined in children in a similar geographic area and latitude (Madison, Wis). js Statistical analysis of our data was done by standard computer-assisted methods ( B M D P computer program, University of California Press, Berkeley). Chi-square analysis was performed for analysis of bivariate relationships. Analysis of variance and a t test t h a t did not assume equality of variances an d t h a t used the Bonferroni correction were employed for comparison of data when more than one group was compared.
RESULTS Our study population consisted of 31 male and 82 female subjects whose ages ranged from 19 months to 21 years (Table I). G r o u p 1 children had a m e a n E S R of 28 m m / h r and an activity score of 3.42, each of which differed significantly (p <0.02) from those of group 2 children, who had a mean E S R of 15 m m / h r and an activity score of 1.7; group 3 children had a mean E S R of 27 m m / h r and an activity score of 3.4 during times of clinical disease activity, and an E S R of 13 m m / h r and an activity score of 1.7 during times of inactivity (p <0.01, activity vs inactivity, for each value). The frequency of corticosteroid use in patients with active disease (group 1, n = 62) ranged from 6.7% of patients with pauciarticular J R A (n = 15) to 90% of the children with J D M S (n = 10); the frequency in children with inactive disease (group 2, n = 23) ranged from 33% of pauciarticular J R A (n = 3) to 100% of the patients with inactive J D M S (n = 1). In group 3 (n = 28), 12 children were receiving corticosteroids throughout; 64% of group 3 patients with J R A or S L E were not receiving steroid therapy during times of blood sampling. The patients receiving steroid therapy in group 3 had a dose range during clinical activity (score > 3 ) that ranged from 0 to 0.44 m g / k g / d a y (mean 0.16 m g / k g ) , and while clinically inactive (score < 2 ) ranged from 0.27 to 0.025 m g / k g / d a y ( m e a n 0.21 m g / k g ) ; active versus inactive dose (p value not significant). All group 3 patients with J D M S were receiving steroids; the m e a n doses with clinical activity and inactivity were not significantly different. Irrespective of disease activity, the majority of the 113
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S e r u m osteocalcin and chronic r h e u m a t i c diseases
Poly JRA
577
Pouol JRA
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Fig. t. Serum osteocalcin levels in children with juvenile rheumatoid arthritis whose disease improved during course of study (group 3, see Methods section). Subtypes of juvenile rheumatoid arthritis are listed and individual osteocalcin measurements, connected by lines, are shown for each child during disease activity and inactivity. Closed boxes represent mean _+_+2 SD of group of children with that disease subtype at each time point. Asterisks indicate that mean values during remission, are significantly greater than during activity (p <0.05).
children with C R D who were studied had normal values for blood ionized calcium, parathyroid hormone, 25-OHD, and 1,25-(OH)zD levels (Table II). One patient had a low ionized calcium concentration, three had elevated parathyroid hormone levels, and three had low 25-OHD and low 1,25(OH)2D levels. There were no significant differences in the mean values in any of these studies among disease groups, within disease groups on the basis of activity levet, or in comparison with normal levels. Hypercalciuria (urine calcium/creatinine ratios >0.2519) was found in 14 (22%) of 64 children with CRD. The frequency of reduced serum osteocalcin levels was greater in group 1 children with active C R D in comparison with group 2 children with inactive disease, irrespective of disease classification (Table'lI). However, because of the absence of children with inactive systemic JRA' and systemic to polyartieular J R A , and because there was only one patient with inactive J D M S , this observation could not be
evaluated separately for each disease classification. Serum osteocalcin levels did not correlate significantly with any of the biochemical measurements, with the duration of disease, or with corticosteroid dosage employed. We had the opportunity to measure serum osteocalcin levels 2 to 6 months after the initial specimens were obtained from 28 children in group 1 and from l I children in group 2. There were no significant changes in the reduced or normal osteocalcin levels, respectively, in the follow-up specimen (data not shown). However, all patients in group 3 except one, irrespective of disease type, had an increase in the mean value of osteocalcin when their disease remitted and their activity score declined (Fig. 1). In the one child with a decreasing osteocalcin value during the period of this study, the second value represented a normal level for the child's age. In 16 of 19 children with active C R D (group 1) and reduced osteoealcin levels, B M C for age was reduced >2
578
Reed et al.
The Journal of Pediatrics April 1990
r
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o
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~r
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, 6
, 10
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YEARS Fig. 2. Bone mineral density determined by single-beam photon absorptiometry in normal children and in 16 children with clinically active CRD and reduced osteocalcin levels (group 1, see Methods section). Normal (mean _+ 2 SD) bone mineral density (line) is shown for children aged 6 to 18 years, 18 Values for 16 children aged 3 tO 19 years, with active CRD and reduced osteocalcin levels, are represented by triangles.
SD below normal (Fig. 2); three patients had normal BMC. Three patients with disease inactivity (group 2) and one patient with disease remission (group 3) and normal osteocalcin levels for age had normal BMC (data not shown). DISCUSSION Our studies demonstrate that alterations in mineral metabolism as reflected by reduced levels of serum osteocalcin occur early in the clinical course of the majority of children with CRD, are not related to the use of corticosteroids, but are associated with indexes of disease activity. Two distinct types of bone disease, local and systemic, may arise in children with CRD. Local bone disease, represented by juxtaarticular erosions and bony destruction in patients with J R A or avascular necrosis in patients with S L E , occurs with a frequency ranging from 20% to 55%.4,20.21 Juxtaarticular demineralization is thought to be a response to the hyperemia associated with the inflammatory process and is an early finding. Later in the course of the disease, as the involved areas become less mobile, disuse osteoporosis is thought to play a role in the local osteopenia. Additionally, in this study, we demonstrated a reduction in serum osteocalcin levels, which may account for some of the abnormalities in systemic bone disease present in children with CRD. Osteocalcin (bone Gla protein) is a vitamin K-dependent bone protein thought to be synthesized during the mineralization process in bone. It represents the most abundant noncollagenous protein in bone and constitutes 10% to 20% of the extracellular matrix. Osteocalcin is thought to be de-
rived from an osteoblast population 2225 and is present in serum in smaller amounts than in bone. Measurements of circulating levels of osteocalcin may serve as an index of bone formation. 26' 27 A reduction in the serum level correlates with dynamic histomorphometric evidence on bone biopsy of reduced bone formation rates.Z8, 29 Serum levels of osteocalcin are a sensitive marker of bone growth in normal children or in children with increased growth velocity3~ they are reduced in children with metabolic bone disease such as hypoparathyroidism and elevated in childre n with rickets being treated with vitamin D. 31 Reduced osteocalcin levels are specifically related to altered bone metabolism and not merely to chronic illness. 32 Osteocalcin that circulates in serum contains 7-carboxyglutamic acid. The process whereby the native molecule acquires Gla protein is vitamin K dependent. 33 We have previously documented that in a similar group of children with CRD, there was no evidence of vitamin K deficiency. 1~ It is unlikely that the inability to attach Gla protein to native osteocalcin is responsible for the reduction in serum levels of osteocalcin in our patients with CRD. In many previous studies of osteopenia in children and adults with CRD, eorticosteroids and physical immobilization have been implicated as the major factors in the pathogenesis of the osteopeniafl 1, 12 However, we measured a reduction in serum osteocalcin levels within the first year of the onset of disease in the majority of our patients, a time when corticosteroids were not used. Although administration of eorticosteroids can reduce the serum levels of osteocalcin, 34 we documented an increase in the levels de-
Volume 116 Number 4
spite the long-term use of corticosteroids. Total body immobilization produces changes in calcium homeostasis that may contribute to osteopenia, although relative immobilization, with decreased levels of physical activity, has not been studied. We had no evidence of such profound reduction in physical activity in our patient population that would account for the bony abnormalities. Therefore we can conclude that neither corticosteroid therapy nor significant immobilization is an important factor in the development of osteopenia in our patients with CRD. Previous studies of adults with rheumatoid arthritis have documented a reduction, 35' 36 an elevation, 37 or no change 38 in the circulating levels of osteocalcin; this variation may be more reflective of the therapy employed. However, a recent study employing transiliac bone biopsy and dynamic histomorphometry did demonstrate a reduction in bone formation rates in adults with rheumatoid arthritis before the use of corticosteroids. 39 The reductions in serum levels of osteocalcin seem to be associated with the extent of systemic inflammation, reflected by the elevated E S R and those criteria that defined C R D clinical activity. Several mediators of inflammation in children with C R D may profoundly affect bone metabolism. Elevated levels of interleukin-1 may be found in children with J R A . 4~ In vitro, interleukin- 1 inhibits both collagen synthesis and the production of osteocalcin in human osteoblast cell cultures. 41 Many other cytokines are produced during the acute inflammatory response of C R D , including tumor necrosis factor and interleukin-2. 42 The role of these mediators in the development of osteopenia remains speculative, although in vitro evidence suggests that they may reduce bone formation. 43 It is unlikely that the osteopenia is solely the result of reduced bone formation as reflected in the reduced osteocalcin levels. As in another study, 9 we found hypercalciuria in a high percentage of children with active C R D . The increased urinary calcium level is likely the result of increased bone resorption, not an increase in the fractional absorption of dietary calcium. W e found normal serum 1,25-(OH)2D levels in most children, unlike children with idiopathic hypercalciuria whose increased urine calcium level is associated with an increased serum 1,25-(OH)2D level and enhanced calcium absorption. 44 The long-term consequences of the disturbed mineral metabolism and resultant osteopenia that occur during the acute phase of the disease remain to be evaluated, but it is encouraging to us that osteocalcin levels improved or became normal in most patients when disease activity abated. Further studies in progress ,will allow us to determine whether the reduction in bone formation is repaired later in the course of CRD, when disease inactivity persists and serum osteocalcin levels are normal.
S e r u m osteocalcin and chronic rheumatic diseases
5 "7 9
We gratefully acknowledge the technical expertise of Ms. Melody Chapman-Hale and the proficient editorial assistance of Ms. Nancy Oliver and Ms. Janet Bojan. REFERENCES
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20. Cassidy JT, Martel W. Juvenile rheumatoid arthritis: clinicoradiologic correlations. Arthritis Rheum 1977;20:207-11. 21. Jacobs JC. Pediatric rheumatology for the practitioner. New York: Springer-Verlag, 1982:361-2. 22. Nishimoto SK, Price PA. Proof that the 3,-carboxyglutamic acid-containing bone protein is synthesized in calf bone. J Biol Chem 1979;254:437-41. 23. Price PA, Nishimoto SK. Radioimmunoassay for the vitamin K-dependent protein of bone and its discovery in plasma. Proc Natl Acad Sci (USA) 1980;77:2234-8. 24. Price PA, Williamson MK, Lothringer JW. Origin of the vitamin K-dependent bone protein found in plasma and its clearance by kidney and bone, J Biol Chem 1981;256: 12760;-6. 25. Hauschka PV, Frenkel J, DeMuth R, Gundberg CM. Presence of osteocalcin and related higher molecular weight 3,-carboxyglutamic acid-containing proteins in developing bone. J Biol Chem 1983;258:176-82. 26. Cecchettin M, Tarquini B, Manente P, Conte N, Albertini A. Serum osteocalcin assay and its clinical application. In: Cecchettin M, Segre G, eds. Calciotropic hormones and calcium metabolism. Amsterdam: Excerpta Medica, 1986:13-7. 27. Carmel R, Lau KHW, Baylink D J, Saxena S, Singer FR. Cobalamin and osteoblast-specific proteins. N Engl J Med 1988;319:70-4. 28. Delmas PD. Bone Gla-protein (osteocalcin): a specific marker for the study of metabolic bone diseases. In: Cecchettin M, Segre G, eds. Calciotropic hormones and calcium metabolism. Amsterdam: Excerpta Medica, 1986:19-28. 29. Garcia-Carrasco M, Gruson M, de Vernejoul C, Denne MA, Miravct L. Osteocalcin and bone morphometric parameters in adults without bone disease. Calcif Tissue Int 1988;42:13-7. 30. Johansen JS, Giwercman A, Hartwelt D, et al. Serum bone Gla-protein as a marker of bone growth in children and adolescents: correlation with age, height, serum insulin-like growth factor 1, and serum testosterone. J Clin Endocrinol Metab 1988;67:273-8. 31. Cote DEC, Carpenter TO, Gundberg CM. Serum osteocalcin concentrations in children with metabolic bone disease. J PEDIATR 1985;106:770-5. 32. Slovik DM, Gundberg CM, Neer RM, Lian JB. Clinical eval-
The Journal o f Pediatrics April 1990
uation of bone turnover by serum osteocalcin measurements in - a hospital setting. J Clin Endocrinol Metab 1984;59:228-30. 33. Hauschka PV, Reid ML. Vitamin K dependence of a calciumbinding protein containing ,y-carboxygtutamic acid in chicken bone. J Biol Chem 1978;253:9063-8. 34. Lukert BP, Higgins JC, Stoskopf MM. Serum osteocalcin is increased in patients with hyperthyroidism and decreased in patients receiving glucocorticoids. J Clin Endncrinol Metab 1986;62:1056-8. 35. Ekenstam EAF, Ljunghall S, Hallgren R. Serum osteocalcin in rheumatoid arthritis and other inflammatory arthritides: relation between inflammatory activity and the effect of glucocorticoids and remisslon-inducing drugs. Ann Rheum Dis 1986;45:484-90. 36. Franck H, van Valen F, Keck E, Kruskemper HL. Osteocalcin and bone metabolism in rheumatoid arthritis and osteoarthritis. Z Rheumatol 1986;45:241-6. 37. Gevers G, Devos P, De Roo M, Dequeker J. Increased levels of osteocalcin (serum bone Gla-protein) in rheumatoid arthritis. Br J Rheumatol 1986;25:260-2. 38. Hermann E, Aeschlimann A, Muller W. Serum osteocalcin in chronic polyarthritis in stage III. Z Rheumatol 1987;46:12931. 39. Compston JE, Vedi S, Mellish RW, Croucher P, O'Sullivan MM. Reduced bone formation in non-sterold-treated patients with rheumatoid arthritis. Ann Rheum Dis 1989;48:483-7. 40. Alpert SD, Koide J, Takada S, Engleman EG. T cell regulatory disturbances in the rheumatic diseases. Rheum Dis Clin North Am 1987;13:431-45. 41. Stashenko P, Dewhirst FE, Rooney ML, Desjardins LA, Heeley JD. Interleukin-l~ is a potent inhibitor of bone formation in vitro. J Bone Miner Res 1987;2:559-65. 42. Firestein GS, Tsai V, Zaifter N. Cellular immunity in the joints of patients with rheumatoid arthritis and other forms of chronic synovitis. Rheum Dis Clin North Am 1987;13:191213. 43. Canalis E. Effects of tumor necrosis factor on bone formation in vitro. Endocrinology 1987; 121 : 1596-604. 44. Stapelton FB, Langman CB, Bittle J, Miller LA. Increased serum concentrations of 1,25(OH)2 vitamin D in children with fasting hypercalciuria. J PED1ATR 1987;110:234-7.
Children with chronic rheumatic diseases, including juvenile rheumatoid arthritis, systemic lupus erythematosus, and juvenile dermatomyositis, have alterations in skeletal integrity] demonstrated by both the presence of periarticSupported in part by National Institutes of Health grants AR30692 and DK33949, by the Arthritis Foundation, and by the Otto Sprague Memorial Fund. Submitted for publication Dec. 27, t 988; accepted Oct. 26, 1989. Reprint requests: Craig B. Langman, MD, Mineral Metabolism Laboratory, Divisionof Nephrology, Box 37, Children's Memorial Hospital, 2300 Children's Plaza, Chicago, IL 60614. 9/20/17740
574
ular bony destruction 2, 3 and the occurrence of generalized osteopenia. 48 The precise mechanisms that produce each of the bony changes in children with CRD are not completely understood but may involve abnormalities of mineral metabolism9,10 Corticosteroid-induced osteoporosis, the most frequent form of secondary osteoporosis in adults] 1 has often been ascribed as the cause of osteopenia in children with rheumatic diseases, although few studies have been performed. Additionally, some investigators have implicated immobilization12 as the cause of the osteopenic bone disease. During an initial retrospective chart review, we identified
Volume l l 6 Number 4
BMC CRD ESR JDMS JRA 25-OHD 1,25-(OH)2D SLE
Serum osteocalcin and chronic rheumatic diseases
Bone mineral content Chronic rheumatic diseases Erythrocyte sedimentation rate Juvenile dermatomyositis Juvenile rheumatoid arthritis 25-HydroxyvitaminD 1,25-DihydroxyvitaminD Systemic lupus erythematosus
37 ambulatory children with CRD who were not receiving corticosteroid therapy but who had generalized bone demineralization on standard radiographs or unexplained, repeated fractures. To better understand the alteration in bone homeostasis in children with CRD, we prospectively studied several measures of mineral metabolism. We postulated that generalized osteopenia in children with CRD may be the result of reduced bone formation as reflected in reduced serum osteocalcin levels, in addition to enhanced bone resorption as reflected by hypercalciuria. We further postulated that corticosteroid use would have an adverse effect on measured osteocalcin levels in this group of children. METHODS We studied children followed in the Children's Memorial Hospital Immunology/Rheumatology Clinic during an 18month period from February 1987 to April 1988. The patients included those with JRA, JDMS, and SLE. 13All patients with JRA fulfilled the criteria established by the American College of Rheumatology.14 Patients with J DMS fulfilled established criteria. 15 Patients with JRA were categorized as those having polyarticular JRA (five joints or more), pauciarticular JRA (less than five joints), systemic onset JRA, or systemic-onset JRA that had developed into polyarticular JRA. Clinical activity was assessed in standard fashion by history and physical examination of each patient; assessment was performed by one physician. A disease activity score was given on the basis of the presence of joint swelling, warmth, redness, range of motion, muscle weakness, complaints of pain and morning stiffness, and the use of antiinflammatory medications. The score ranged from 0 to 5, with 0 representing no complaints or physical findings of active disease and no use of antiinflammatory medication, and 5 representing very active clinical disease and the use of medication. We defined a disease as active if the patient score was >--3 and inactive if --<2. After the initial visit, each patient was placed in one of two groups on the basis of their initial activity score: group 1 (clinically active CRD) or group 2 (inactive disease). After the study (April 1988), group 1 patients whose disease activity remitted during the course of this study (score -< 2)
575
T a b l e I. Clinical characteristics of children with chronic rheumatic disease Disease
Poly JRA Pauci JRA Syst JRA Syst-Poly JRA JDMS SLE TOTAL
n
M/F
37 21 12 13 13 I_.Z
4/33 6/15 3/9 6/7 9/4 3/14
1V2-20 2-12 3-18 3-17 4-19 11-21
31/82
11/2-21
113
A g e range (yr)
Poly, Polyarticular; Pauei, pauciarticular; Syst, systemic,
were placed in group 3 (disease remission) and analyzed separately. There were no patients from group 2 whose disease became active during this study. Patients entered into the study at various times after their disease began, ranging from onset to more than 10 years after onset. Studies performed at the first visit included measurement of a Westergren erythrocyte sedimentation rate; determination of levels of blood ionized calcium, serum osteocalcin, parathyroid hormone, 25-hydroxyvitamin D, and 1,25dihydroxyvitamin D; and measurement of a random urine calcium/creatinineratio. On subsequent patient visits, at an interval determined by the attending physician, blood was obtained only for measurement of osteocalcin levels. Blood ionized calcium was measured in duplicate by an ion-specific electrode (Radiometer America, Westlake, Ohio). The mean (_+ 2 SD) normal blood ionized calcium level is 1.21 _+ 0.13 mmol/L. Levels of 25-OHD and 1,25(OH)zD were measured in triplicate by specific radioreceptor assays previously described. 16 Normal values (95% confidence intervals) for 25-(OH)D are 15 to 40 ng/ml, and for 1,25-(OH)zD are 15 to 80 pg/ml. Serum parathyroid hormone levels were measured in duplicate by a specific radioimmunoassay that detected the mid-portion of the hormone molecule (amino acids 44-68) and that was previously validated for clinical measurements over a wide range of parathyroid hormone levels 16, 17; normal values (95% confidence intervals) are 15 to 80 pmol/L. The ESR was determined by standard laboratory procedure. Serum osteocalcin levels were measured by specific radioimmunoassay (lncstar Corp., Stillwater, Minn.) with the use of bovine osteocalcin standards. Least detectable values ranged from 0.2 to 0.3 ng/ml; 50% displacement values ranged from 12 to 15 ng/ml. Interassay and intraassay coefficients of variation were 6% and 2%, respectively, over an osteocalcin range of 0.3 to 40 ng/ml. Normal values (95% confidence intervals) established in our laboratory are 15 to 25 ng/ml in children younger than 13 years of age (n = 126) and 2 to 8 ng/ml in children older than 131/2years (n = 98). We also measured serum osteocalcin in children
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Table II. I n i t i a l b i o c h e m i c a l m e a s u r e m e n t s in c h i l d r e n w i t h c h r o n i c r h e u m a t i c d i s e a s e
CRD PolyJRA
Activity + -
Pauci JRA Syst JRA Syst-Poly JRA SLE JDMS
+ + + + + -
ICa (mmol/L)
PTH (pmol/L)
25-OHD (ng/ml)
1,25-(OH)aD (pg/ml)
U Ca/Cr ratio
osteocalcin
1.22_+05(13) 1.17 -+ 06 (14) 1.25 _+ 06 (6) 1.24 _+ 08 (3) 1.15 _+ 05 (4) 1.23 _+ 03 (3) 1.17_+ 04 (2) 1.16 _+ 01 (3) 1.23 _+ 04 (10) 1.23 -+ 06 (7) 1.20 • 07 (6) 1.19 _+ 13 (2)
47_+ 15(14) 47 • 10 (13) 41 _+ 22 (5) 52 -+ 13 (3) 51 _+ 7 (4) 4l _+ 6 (2) 47 _+ 8 (3) 45 _+ 3 (3) 48 _+ 15 (11) 47 -+ 17 (7) 40 _+ 24 (9) 38 _+ 24 (9)
20_+7(14) 22 _+ 9 ( 1 3 ) 22 -+ 8 (5) 20 _+ 3 (3) 19 _+ 4 (4) 20 _+ 3 (2) 14 _+ 1 (2) 17 _+ 6 (3) 20 _+ 12 (10) 18 _+ 9 (7) 18 _+ 6 (9) 25 _+ 14 (4)
41_+23(I4) 41 _+ 31 ( 1 3 ) 54 _+ 19 (4) 50 + 6 (2) 30 _+ 2 (3) t5 _+ 1 (2) 50 -+ 23 (2) 45 _+ 23 (3) 25 _+ 12 (10) 33 _+ 21 (6) 46 _+ 32 (9) 32 _+ 20 (4)
0.13-+0.12(14) _+ .08 (10) 0.08 _+ 0.06 (6) 0.03 _+ 0.01 (2) 0.18 (1) 0.06 (2) 0.19 (1) 0.03 (1) 0.t8 _+ 0.02 (8) 0.08 _+ 0.07 (7) 0.30 _+ 0.22 (8) 0.31 _+ 0.16 (4)
11/14 0/I3 14/15 0/3 5/6 0 7/8 0 9/10 0/6 8/10 1/6
0.10
Reduced
Values are mean + SD for children in group 1 (Activity +) and group 2 (Activity - ) except wherc fewer than three children were studied, when the average or sole value is presented. Number in parentheses after each measurement represents the number of children studied. Activity represents disease activity score >~3 (+) or its absence, score --<2(-), as defined in the Methods section. For all disease groups combined, there was a significantdifferencein the number of children with disease activity versus those with absence of activity among those with reduced ostcocalcin values (p <0.05). ICa, Ionized calcium (in blood); PTH, parathyroid hormone; U Ca/Cr, urinary calcium/creatinine (ratio); Poly. polyarticular; Pauci, pauciarticular; Syst, systemic.
with two other chronic, n o n r h e u m a t i c diseases: congenital heart disease and common variable hypogammaglobulinemia. Neither group had a history of joint involvement, aut o i m m u n e disease, or recent infectious illness. The subjects included 4 female and 11 male patients with an age range from 1 to 21 years at tile time of serum collection. O f 15 of these latter children, 13 had normal osteocalcin levels for their age. Bone mineral content was determined by photon absorptiometry during a 9-month period, from July 1987 to April 1988, in 16 patients with clinical disease activity (group 1) and low osteocalcin values, in addition to four patients with inactive disease (group 2) or improved disease (group 3) and normal osteocalcin values. The B M C was determined in the distal one third of the n o n d o m i n a n t radius by conventional single-beam photon absorptiometry ( L u n a r Radiation Corp., Madison Wis.). Repetitive m e a s u r e m e n t error was less than 3%. From bone width and bone mineral values, B M C was calculated by computer methods ( L u n a r ) and expressed as grams per square centimeter. N o r m a l values for B M C were previously determined in children in a similar geographic area and latitude (Madison, Wis). js Statistical analysis of our data was done by standard computer-assisted methods ( B M D P computer program, University of California Press, Berkeley). Chi-square analysis was performed for analysis of bivariate relationships. Analysis of variance and a t test t h a t did not assume equality of variances an d t h a t used the Bonferroni correction were employed for comparison of data when more than one group was compared.
RESULTS Our study population consisted of 31 male and 82 female subjects whose ages ranged from 19 months to 21 years (Table I). G r o u p 1 children had a m e a n E S R of 28 m m / h r and an activity score of 3.42, each of which differed significantly (p <0.02) from those of group 2 children, who had a mean E S R of 15 m m / h r and an activity score of 1.7; group 3 children had a mean E S R of 27 m m / h r and an activity score of 3.4 during times of clinical disease activity, and an E S R of 13 m m / h r and an activity score of 1.7 during times of inactivity (p <0.01, activity vs inactivity, for each value). The frequency of corticosteroid use in patients with active disease (group 1, n = 62) ranged from 6.7% of patients with pauciarticular J R A (n = 15) to 90% of the children with J D M S (n = 10); the frequency in children with inactive disease (group 2, n = 23) ranged from 33% of pauciarticular J R A (n = 3) to 100% of the patients with inactive J D M S (n = 1). In group 3 (n = 28), 12 children were receiving corticosteroids throughout; 64% of group 3 patients with J R A or S L E were not receiving steroid therapy during times of blood sampling. The patients receiving steroid therapy in group 3 had a dose range during clinical activity (score > 3 ) that ranged from 0 to 0.44 m g / k g / d a y (mean 0.16 m g / k g ) , and while clinically inactive (score < 2 ) ranged from 0.27 to 0.025 m g / k g / d a y ( m e a n 0.21 m g / k g ) ; active versus inactive dose (p value not significant). All group 3 patients with J D M S were receiving steroids; the m e a n doses with clinical activity and inactivity were not significantly different. Irrespective of disease activity, the majority of the 113
Volume 116 Number 4
S e r u m osteocalcin and chronic r h e u m a t i c diseases
Poly JRA
577
Pouol JRA
25 O
'!
,15
& 15
rt
0
/
Systemic JRA
Systemic to poly JRA
7is
i,o 0
ACTNI[
o
& is
o+ "
i ,o
i)~'TN[
0
ACTI~
IHACTI~
Fig. t. Serum osteocalcin levels in children with juvenile rheumatoid arthritis whose disease improved during course of study (group 3, see Methods section). Subtypes of juvenile rheumatoid arthritis are listed and individual osteocalcin measurements, connected by lines, are shown for each child during disease activity and inactivity. Closed boxes represent mean _+_+2 SD of group of children with that disease subtype at each time point. Asterisks indicate that mean values during remission, are significantly greater than during activity (p <0.05).
children with C R D who were studied had normal values for blood ionized calcium, parathyroid hormone, 25-OHD, and 1,25-(OH)zD levels (Table II). One patient had a low ionized calcium concentration, three had elevated parathyroid hormone levels, and three had low 25-OHD and low 1,25(OH)2D levels. There were no significant differences in the mean values in any of these studies among disease groups, within disease groups on the basis of activity levet, or in comparison with normal levels. Hypercalciuria (urine calcium/creatinine ratios >0.2519) was found in 14 (22%) of 64 children with CRD. The frequency of reduced serum osteocalcin levels was greater in group 1 children with active C R D in comparison with group 2 children with inactive disease, irrespective of disease classification (Table'lI). However, because of the absence of children with inactive systemic JRA' and systemic to polyartieular J R A , and because there was only one patient with inactive J D M S , this observation could not be
evaluated separately for each disease classification. Serum osteocalcin levels did not correlate significantly with any of the biochemical measurements, with the duration of disease, or with corticosteroid dosage employed. We had the opportunity to measure serum osteocalcin levels 2 to 6 months after the initial specimens were obtained from 28 children in group 1 and from l I children in group 2. There were no significant changes in the reduced or normal osteocalcin levels, respectively, in the follow-up specimen (data not shown). However, all patients in group 3 except one, irrespective of disease type, had an increase in the mean value of osteocalcin when their disease remitted and their activity score declined (Fig. 1). In the one child with a decreasing osteocalcin value during the period of this study, the second value represented a normal level for the child's age. In 16 of 19 children with active C R D (group 1) and reduced osteoealcin levels, B M C for age was reduced >2
578
Reed et al.
The Journal of Pediatrics April 1990
r
E
0.80
o
I.Z I,iJ I-Z
0.80
o
r
.-I
.
0.40
~r
i Ld Z
o m
0.20
T
, 6
, 10
, 14
!
18
YEARS Fig. 2. Bone mineral density determined by single-beam photon absorptiometry in normal children and in 16 children with clinically active CRD and reduced osteocalcin levels (group 1, see Methods section). Normal (mean _+ 2 SD) bone mineral density (line) is shown for children aged 6 to 18 years, 18 Values for 16 children aged 3 tO 19 years, with active CRD and reduced osteocalcin levels, are represented by triangles.
SD below normal (Fig. 2); three patients had normal BMC. Three patients with disease inactivity (group 2) and one patient with disease remission (group 3) and normal osteocalcin levels for age had normal BMC (data not shown). DISCUSSION Our studies demonstrate that alterations in mineral metabolism as reflected by reduced levels of serum osteocalcin occur early in the clinical course of the majority of children with CRD, are not related to the use of corticosteroids, but are associated with indexes of disease activity. Two distinct types of bone disease, local and systemic, may arise in children with CRD. Local bone disease, represented by juxtaarticular erosions and bony destruction in patients with J R A or avascular necrosis in patients with S L E , occurs with a frequency ranging from 20% to 55%.4,20.21 Juxtaarticular demineralization is thought to be a response to the hyperemia associated with the inflammatory process and is an early finding. Later in the course of the disease, as the involved areas become less mobile, disuse osteoporosis is thought to play a role in the local osteopenia. Additionally, in this study, we demonstrated a reduction in serum osteocalcin levels, which may account for some of the abnormalities in systemic bone disease present in children with CRD. Osteocalcin (bone Gla protein) is a vitamin K-dependent bone protein thought to be synthesized during the mineralization process in bone. It represents the most abundant noncollagenous protein in bone and constitutes 10% to 20% of the extracellular matrix. Osteocalcin is thought to be de-
rived from an osteoblast population 2225 and is present in serum in smaller amounts than in bone. Measurements of circulating levels of osteocalcin may serve as an index of bone formation. 26' 27 A reduction in the serum level correlates with dynamic histomorphometric evidence on bone biopsy of reduced bone formation rates.Z8, 29 Serum levels of osteocalcin are a sensitive marker of bone growth in normal children or in children with increased growth velocity3~ they are reduced in children with metabolic bone disease such as hypoparathyroidism and elevated in childre n with rickets being treated with vitamin D. 31 Reduced osteocalcin levels are specifically related to altered bone metabolism and not merely to chronic illness. 32 Osteocalcin that circulates in serum contains 7-carboxyglutamic acid. The process whereby the native molecule acquires Gla protein is vitamin K dependent. 33 We have previously documented that in a similar group of children with CRD, there was no evidence of vitamin K deficiency. 1~ It is unlikely that the inability to attach Gla protein to native osteocalcin is responsible for the reduction in serum levels of osteocalcin in our patients with CRD. In many previous studies of osteopenia in children and adults with CRD, eorticosteroids and physical immobilization have been implicated as the major factors in the pathogenesis of the osteopeniafl 1, 12 However, we measured a reduction in serum osteocalcin levels within the first year of the onset of disease in the majority of our patients, a time when corticosteroids were not used. Although administration of eorticosteroids can reduce the serum levels of osteocalcin, 34 we documented an increase in the levels de-
Volume 116 Number 4
spite the long-term use of corticosteroids. Total body immobilization produces changes in calcium homeostasis that may contribute to osteopenia, although relative immobilization, with decreased levels of physical activity, has not been studied. We had no evidence of such profound reduction in physical activity in our patient population that would account for the bony abnormalities. Therefore we can conclude that neither corticosteroid therapy nor significant immobilization is an important factor in the development of osteopenia in our patients with CRD. Previous studies of adults with rheumatoid arthritis have documented a reduction, 35' 36 an elevation, 37 or no change 38 in the circulating levels of osteocalcin; this variation may be more reflective of the therapy employed. However, a recent study employing transiliac bone biopsy and dynamic histomorphometry did demonstrate a reduction in bone formation rates in adults with rheumatoid arthritis before the use of corticosteroids. 39 The reductions in serum levels of osteocalcin seem to be associated with the extent of systemic inflammation, reflected by the elevated E S R and those criteria that defined C R D clinical activity. Several mediators of inflammation in children with C R D may profoundly affect bone metabolism. Elevated levels of interleukin-1 may be found in children with J R A . 4~ In vitro, interleukin- 1 inhibits both collagen synthesis and the production of osteocalcin in human osteoblast cell cultures. 41 Many other cytokines are produced during the acute inflammatory response of C R D , including tumor necrosis factor and interleukin-2. 42 The role of these mediators in the development of osteopenia remains speculative, although in vitro evidence suggests that they may reduce bone formation. 43 It is unlikely that the osteopenia is solely the result of reduced bone formation as reflected in the reduced osteocalcin levels. As in another study, 9 we found hypercalciuria in a high percentage of children with active C R D . The increased urinary calcium level is likely the result of increased bone resorption, not an increase in the fractional absorption of dietary calcium. W e found normal serum 1,25-(OH)2D levels in most children, unlike children with idiopathic hypercalciuria whose increased urine calcium level is associated with an increased serum 1,25-(OH)2D level and enhanced calcium absorption. 44 The long-term consequences of the disturbed mineral metabolism and resultant osteopenia that occur during the acute phase of the disease remain to be evaluated, but it is encouraging to us that osteocalcin levels improved or became normal in most patients when disease activity abated. Further studies in progress ,will allow us to determine whether the reduction in bone formation is repaired later in the course of CRD, when disease inactivity persists and serum osteocalcin levels are normal.
S e r u m osteocalcin and chronic rheumatic diseases
5 "7 9
We gratefully acknowledge the technical expertise of Ms. Melody Chapman-Hale and the proficient editorial assistance of Ms. Nancy Oliver and Ms. Janet Bojan. REFERENCES
1. Badley BWD, Ansell BM. Fractures in Still's disease. Ann Rheum Dis 1960;19:135-42. 2. Duncan H. Osteoporosis in rheumatoid arthritis and corticosteroid induced osteoporosis: symposium on metabolic bone disease. Orthop Clin North Am 1972;3:571-83. 3. Colton CL, Darby AJ. Giant granulomatous lesions of the femoral head and neck in rheumatoid arthritis. Ann Rheum Dis 1970;29:626-33. 4. Brewer E J, Giannini EH, Person DA. Juvenile rheumatoid arthritis. Major Probl Clin Pediatr 1970;6:60-71. 5. Bjelle AO, Nilsson BE. Osteoporosis in rheumatoid arthritis, Calcif Tissue Res 1970;5:327-32. 6. Maddison P J, Bacon PA. Vitamin D deficiency, spontaneous fractures, and osteopenia in rheumatoid arthritis. Br Med J 1974;4:433-5. 7. McConkey B, Fraser GM, Bligh AS. Osteoporosis and purpura in rheumatoid disease: prevalence and relation to treatment with corticosteroids. Q J Med 1962;55:419-27. 8. Taylor RT, Huskisson EC, Whitehouse GH, Hart FD. Spontaneous fractures of pelvis in rheumatoid arthritis. Br Med J 1971;4:663-4. 9. Stapelton FB, Hanissian AS, Miller LA. Hypercalciuria in children with juvenile rheumatoid arthritis: association with hematuria. J PEDIATR 1985;107:235-9. 10. Lian JB, Pachman LM, Gundberg CM, Partridge REH, Maryjowski MC. Gamma-carboxyglutamate excretion and calcinosis in juvenile dermatomyositis. Arthritis Rheum 1982; 25:1094-100. 11. Berg E, Moyle DD. Osteoporosis: an overview of causes, prevention, and therapy. J Musculoskeletal Med 1988;5: 64-81. 12. Ats OS, Gotfredsen A, Riis BJ, Christiansen C. Are disease duration and degree of functional impairment determinants of t3one loss in rheumatoid arthritis? Ann Rheum Dis 1985; 44:406-1 l. 13. Rodman GP, Schumacher HR, eds. Primer on the rheumatic diseases. 8th ed. Atlanta: Arthritis Foundation, i983:49-59. 14. In: Rodman GP, Sehumacher HR, eds. Primer on the rheumatic diseases. 8th ed. Atlanta: Arthritis Foundation, 1983:97103. 15. Bohan A, Peter JB. Polymyositis and dermatomyositis. N Engl J Med 1975;292:344-7; 403-7. 16. Gidding SS, Minciotti AL, Langman CB. Unmasking of hypoparathyroidism in tamilial partial DiGeorge syndrome by challenge with disodium edetate. N Engl J Med 1988;319: 1589-92. 17. Coe FL, Parks JH, Bushinsky DA, Langman CB, Favus MJ. Chlorthaiidone promotes mineral retention in patients with idiopathic hypercalciuria. Kidney Int 1988;33:1140-6. 18. Mazess RB, Cameron JR. Bone mineral content in normal U.S. whites. In: Mazess RB, ed. International Conference on Bone Mineral Measurement. Publication No. 75-683. Washington, D.C.: U.S. Department of Health, Education, and Welfare, 1974:228-38. 19. Kruse K, Kracht U, Kruse U. Reference values for urinary calcium excretion and screening for hypercalciuria in children and adolescents. Eur J Pediatr 1984;143:24-3E
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The Journal o f Pediatrics April 1990
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