Histomorphometric profile and vitamin d status in patients with femoral neck fracture

Metab.

Bone Dis. & Rel. Res.4,85-93

0221-87481821020085-09$03.00/O Copyright 0 1982 Pergamon Press Ltd

(1982)

Printed in the USA. All rights reserved.

Histomorphometric ProfileandVitaminD Statusin Patientswith FemoralNeckFracture P. LIPS,” J.C. NETELENBOS,’ M.J.M. JONGEN,’ F.C. van GINKEL,’ A.L. ALTHUIS,‘C.L. W.J.F. van der VIJGH,’ J.P.W. VERMEIDEN’ and C. van der MEER.’

‘Academisch Ziekenhuis der Vrije Universiteit, ‘Onze Lieve Vrouwe Gasthuis, Amsterdam.

vanSCHAlK,*

Amsterdam.

Address for correspondence and reprints: P. Lips, M.D., Department Universiteit, P.B. 7057, 1007 MB Amsterdam, The Netherlands.

of Internal Medicine,

Key Words:

Abstract

Academlsch

Femoral Neck Metabolites - Osteomalacia Histomorphometry.

In order to detect metabolic bone disease vitamin D metabolites and other biochemical parameters of bone metabolism were measured in 125 patients with femoral neck fracture and 74 age-matched control subjects. Transilial bone biopsies from 89 patients, obtained within 1 week of the fracture, were evaluated by histomorphometric techniques. Trabecular bone volume (TBV) was significantly lower in trochanteric fractures in both sexes than in controls, whereas in cervical fractures TBV was not decreased. Other histomorphometric differences between both fracture types were not observed. Cortical porosity was higher in the patients than in autopsy controls. The osteoid surface was extended in the patients, but the osteoid seams were significantly thinner than in controls. The mean width of osteoid seams was not increased in any patient. On these grounds serious osteomalacia is unlikely. With remodeling parameters the biopsies were classified into three subgroups: “high turnover”(n=19), “uncoupling” between resorption and formation (n=9), and a “rest” group (n=61), in which bone turnover was normal or low. In the “high turnover” group mean cortical thickness was decreased significantly compared with the other groups. Mean serum concentrations of calcium (corrected for serum proteins), phosphate, creatinine, alkaline phosphatase, and parathyroid hormone were not different in patients and control subjects. Serum concentrations of the vitamin D metabolites 25(OH)D, 24,25(OH),D, and 1,25(OH),D were significantly lower in patients than in controls. The histomorphometric picture could not be predicted adequately by means of biochemical parameters. An increased osteoid volume (r5%), and/or surface (r25%), observed mainly in the “high turnover” group, was always associated with a serum 25(OH)D concentration below 30 nmolll. The “high turnover” may follow vitamin D deficiency. The latter, however, does not cause a clear histological reaction in most cases.

Ziekenhuis

der Vrl]e

Fractures - Vitamin D - Osteoporosis - Bone

Introduction The increased incidence of hip fractures in the female population over age 50 has been partly explained by the development of osteopenia in this age period (NewtonJohn and Morgan, 1970). With histomorphometric methods, in one third of the patients a more severe osteopenia was observed than would be expected for their age (Aaron et al., 1974). In another study, however, differences in bone volume between fracture patients and controls of the same age were not significant (Faccini et al., 1976). The dispersion of bone volume data in both series was very large, and in many individual patients osteopenia could not account for the fracture. In the last decade reports have been published, mainly in Britain, drawing attention to osteomalacia as a cause of femoral neck fractures. Chalmers et al. (1969) found histological signs of osteomalacia in 20% of patients with femoral neck fracture, and subsequent investigations demonstrated histological osteomalacia in up to 37% of the patients (Jenkins et al., 1973; Aaron et al., 1974; Faccini et al., 1976). However, in other series from Britain and elsewhere osteomalacia did not play an important part (Hodkinson, 1971; Rapin et al., 1979; Wootton et al., 1979). Aged people are less exposed to sunshine (Hodkinson et al., 1973), often have a low vitamin D intake (Chalmers et al., 1969; Panel on Nutrition of the Elderly, 1972), and show lower serum concentrations of 25 (OH) vitamin D than do younger subjects (Preece et al., 1975). In patients with femoral neck fractures, either serum 25 (OH) vitamin D was lower than in controls of the same age (Baker et al., 1979), or a difference was not observed (Lund et al., 1975; Wootton et al., 1979). Serum concen-

a5

P. LIPS et al

86

trations of the most important vitamin D metabolite, 1,25(0H),vitamin D, in patientswith hipfractures have not yet been reported. Elucidation of the role of osteomalacia and vitamin D deficiency in the pathogenesis of femoral neck fractures is important in considering prevention. In the Netherlands, vitamin D is added only to margarine (3 IU/gm). Consequently, this investigation was undertaken to establish the incidence of metabolic bone disease in patients with a fracture of the femoral neck with particular emphasis on osteomalacia and vitamin D deficiency.

Subjects and Methods The study includes all patients (125) who were admitted during a period of 15 months to the Onze Lieve Vrouwe Gasthuis in Amsterdam. Of these, 41 were male and 84 were female patients. The mean age r SD was 75.9 + 11 .O years. Of the fractures, 55 were cervical and 70 were in the trochanteric region. The patients were not selected and varied from active self-supporting aged persons to debilitated geriatric patients, Control subjects were healthy volunteers living in an apartment house for aged people. They lived independently and had to prepare their own meals. Controls with a history of femoral neck fracture were excluded, but other selection criteria were not used. The control group consisted of 20 men and 54 women with a mean age of 75.6 f 4.2 years. Histomorphometry Transilial bone biopsies were taken from the patients usually during operation for the hip fracture using an 8 mm (ID) trephine. From a total of 119 biopsies, 14 specimens were severely damaged and not suitable for histomorphometry. From the 105 biopsies that could be measured, 89 were taken within 1 week of the fracture(prefracturestate). Theother 16 biopsies, takenfromgto 82 days after the fracture, were regarded separately. The biopsy specimens were embedded undecalcified in methylmethacrylate, sectioned (5 pm) with a Jung microtome and stained with Goldner’strichromeafter removal of the plastic. Sections of some biopsies were also stained with solochrome cyanine R. Histomorphometry was performed with Zeiss integrating eyepieces I and II (volume and surface measurements), an eyepiece micrometer, calibrated to a stage micrometer (cortical thickness, width of osteoid seams), and with a drawing tube, cursor, digitizer, and computer (cortical porosity). The following cortical parameters were measured: Mean cortical thickness, i.e., the mean thickness of both cortices (pm): the thickness of each cortex was measured at four equidistant places, This parameter was not measured if the internal cortex was absent. Cortical porosity, i.e., the volume of canals and resorption spaces as a percentage of total cortical volume: this parameter was measured in the thicker of both cortices in a layer of 400 pm thickness on the periosteal side in order to avoid confusion with marrow spaces on the endosteal side (Meunier et al., 1972). When both cortical plates were thinner than 400 pm, porosity was not measured, and measurements were not corrected for obliquity of the section plane, as was discussed by Melsen (1978). Six sections were measured per biopsy. The following parameterswere measured in trabecular bone: i.e., the volume occupied by Trabecular bone volume (TN), trabecular bone and osteoid tissue, expressed as a percentage of the total volume between both cortices (Meunier et al., 1973). Relative osteoid volume (ROV), i.e., the osteoid volume, expressed as a percentage of trabecular bone volume (Meunier et al., 1977). Relative osteoid surface (ROS), i.e., the trabecular surface covered by osteoid seams, expressed as a percentage of total trabecular surface (Meunier et al., 1977).

: Histomorphometry

and vitamin D In hip fractures.

calculated according to Thickness index of the osteoidseams, Meunier et al. (1977) as 100 x ROVIROS, which isan estimate of osteoid thickness. Mean width of osteoid seams, i.e., the mean thickness of all trabecular osteoid seams in 1 or 2 sections, expressed in pm. Every seam was measured at four equidistant places and at least 30 seams were measured (Melsen, 1978). This parameter was only measured in biopsies with higher amounts of osteoid. Relative resorption surface (RRS), i.e, the trabecular surface occupied by resorption lacunae with or without osteoclasts, expressed as a percentage of total trabecular surface (Courpron, 1979., Osteoc/asrs/mm , I.e., the osteoclast count per mm’ of trabecular space. Osteoclasts on trabecular surfaces were counted in 4 sections with magnification x 100, or if in doubt, x 400 (Bordier and Tun Chot, 1972). All parameters were measured in sections stained with Goldner’s trichrome. Osteord parameters (ROV and ROS) were also measured in solochrome-stained sections of 42 biopsies, as the Goldner stain underestimates these parameters (Meunier et al., 1975). As reference value for cortical porosity, measurementswereusedfrom 18 bonesamplesoftheiliaccrestobtained at autopsy from subjects who had died suddenly and did not suffer from metabolic bone disease (mean age + SD, 70.7 + 12.6 years). For the other parameters, reference values of the same sex and age groups were taken from the control series of Meunier’s group (Courpron, 1972; Giroux et al., 1975; Meunier et al., 1977), from Melsen et al. (1978), and from Bordier et al. (1972). Biochemical

methods

Fasting morning blood samples were taken from the patients usually 1 or 2 days after admission. The examination of control subjects was distributed over the year with respect to seasonal variation. Creatinine was measured by calorimetry after complex formation with picrate (Jaffe reaction). Protein fractions (albumin globulins) were derived by densitometry from an electrophoretogram using cellulose acetate as support material and Ponceau S as dye. Calcium was measured by fluorimetric titration with EGTA. using calcein as fluorescence indicator (coefficient of variation 1.3%). Phosphate was determined by calorimetry according to Gomori. Alkaline phosphatase was determined by a modified Bessey technique. Serum PTH was determined in the endocrinologic laboratory of the Bergwegziekenhuis at Rotterdam. Serum PTH concentrationswere measured by radioimmunoassay after extraction from the serum samples by adsorption to and elution from Quso-32, a microcrystalline silicate. The antibody used was a cavia antiserum against bovine PTH (AS-31 1). It reacts with the complete PTH molecule and with the C-regional fragment. The interassay coefficient of variation is 10.3% (Lequin et al., 1970; Schopman et al., 1970). The vitamin D metabolites (25(OH)D, 24,25(OH),D, and 1,25(OH),D) were extracted from serum samples and purified by gradient HPLC. Concentrations of 25(OH)D and 24,25(OH),D were measured by a competitive protein-binding assay using rat serum as binding protein. Interassay coefficients of variation are 3.9 and 8.0% respectively. Concentrations of 1,251(0H)~D were measured by a competitive protein-binding assay using chicken duodenal mucosa receptor as binding protein. The interassay coefficient of variation is 4.8% (Jongen et al., 1981 a, b).

Statistical

methods

Differences in histomorphometric parameters between patients and controls were evaluated with Student’s T-test (two-sided). Histomorphometric subgroups were compared using analysis of variance (ANOVA) with age and sex as covariates. Biochemical parameters of patients and control subjects were evaluated using the same method. In the case of calcium, serum protein fractions were also included as covariates in order to correct for changes caused by the fracture. Serum concentrations of

P. LIPS et al:

Hrstomorphometry

Table 1. Histomorphometric

a7

and vrtamrn D In hip fractures.

data in patients with hip fracture compared with controls from the literature and autopstes Patients

Controls

Significant P

Source of controls n

mean f SD

791 * 37actm 656 f 290pm 7.7 + 3.8%

4.6 3.6 1.9 1.2 7.5 6.3

37 26 56 28 61 28 61

7.8 13.3 13.5 2.5 2.6 17.4 16.1

108

la.8 f 4.7

a9

15.6 + 6.3

Melsen et al., 1976

56

9.1 + 2.8

33

Courpron, 1972 Bordier et al., 1972

130

Courpron,

Cortical porosity

Autopsies

1972

(m) (f)

7 9

Trabecular bone volume

Giroux et al., 1975

Relative osteoid volume

Meunier et al., 1977

Relative osteoid surface

Meunier et al., 1977

comparison

Meunier et al., 1977

not valid, since this parameter

966 + 264 702 + 292

(m) ia

‘Staktical

mean ? SD

24 47 20

Mean cortical thickness

Thickness index osteord seams Mean width osteord seams Relative resorption surface Osteoclastslmm*

n

(f) (m) (f) (m) (f) (m) (f)

27 ;3 ;; 42

Results Histomorphometry Biopsies taken within a week of the fracture will be considered first. The histomorphometric parameters are compared with control values in Table I. The average value of cortical porosity was significantly increased in female patients compared to autopsy controls. When both sexes were combined, the difference in results from the autopsies was significant. Individual values of cortical porosity were increased (> 10.2%) in 11 patients. The TBV was lower only in male patients compared with controls of the same age. The dispersion of the values isvery large, as is shown for the female patients in Figure 1. When arranged according to fracture type the TBV in trochanteric fractures (n = 53) was significantly decreased in both sexes compared with control values (females: 11.9 f 3.9% vs 14.4 f 3.6%, P < 0.01; males: 12.6 f 5.0%~~ 17.6 & 4.80/o, P < 0.01). The TBV in cervical fractures (n = 36), however, was not significantly different from control values in either sex (females: 16.0 _+ 6.00/o, males: 14.3 f 5.7%). With respect to other histomorphometric parameters the two fracture types did not differ. Individual osteoid parameters, as measured in Goldner-stained sections, are compared with control values of Meunier et al. (1977) and Melsen et al. (1978) in Figure 2. The relative osteoid volume was moderately increased (between 5 and 9.9%) in nine patients. The relative osteoid surface exceeded 25% (which was taken as the upper limit) in 15 patients. The mean width of osteoid seams, which was measured in all biopsies with raised values for osteoid volume and/or surface, was not

0.05 <“p”<

0.10

5.8 f 2.2 17.6 14.4 3.1 2.0 15.9 10.7

r f f + f f

3.6 + 1.1 0.03 - 0.30(range)

was measured in particular

24,25(OH),D, showing a very skewed distribution, were evaluated also with Wilcoxon’s rank sum W test. Differences were considered significant if the P value was lower than 0.05. Correlations were calculated using Pearson’s product moment correlation coefficient. If necessary, partial correlations were calculated, controllmg for interfering variables. The histomorphometric subgroups were confronted with biochemical data by means of stepwise discriminant analysis.

ns

aa 46

+ + + c + f +

3.5% 5.4% 5.3% 1.7% 1.6% 12.4% 10.6%

7.8 + l.aFm 4.9 f 3.0% 0.42 f 0.35mm~2

P < 0.05 P < 0.01 ns 0.05 <“p” < 0.10 P :“0.01 P < 0.001 P < 0.05 P < 0.001 ??

In biopsies with increased resorption surface

increased in any biopsy. Comparison of the Goldner sections with results obtained from solochrome-stained sections yielded a high correlation between both stains, but values obtained with solochrome were 15% higher (ROV: r = 0.94, P < 0.001, y = 1 .I 5x - 0.33; ROS: r = 0.93, P < 0.001, y = 1.15x - 0.26). When values in Table I were corrected, the ROV in female patients became significantly greater than in controls. The thickness index of osteoid seams was not influenced by the correction (solochrome: mean + SD, 14.3 + 4.5). Individual data of relative resorption surface are compared with control values of Courpron (1972) and Melsen et al. (1978) in Figure 3. This parameter was increased (~6%) in 26 biopsies. The number of osteoclasts/mm* was counted in 46 biopsies, including all biopsies with increased resorption surface. A significant correlation (r = 0.77, P < 0.001) existed between the number of osteoclasts and the relative resorption surface. A positive correlation also existed between relative resorption and osteoid surface (r = 0.37, P < 0.001).

Fig. 1. Trabecular

bone volume in female patients with femoral neck fracture, compared with control values of Giroux et al. (1975) (mean f SD).

88

P. LIPS et al.: Histomorphometry

relative

osteold volume

%

r

relative o&mid

%

surface

t

50-

.

40-

: t i

-Et f L

COIlhIS

Mwniar

femoral neck fracture5 n-59

Meken

mean width of osteoid seam8

thickmass index of osteotd seams w

15.

t lo-$; .5-

wntrols Meunief

femoral neck fractures n-89

COn(rOlS Metsen

femoral neck fractums Il.33

and vitamin D in hip fractures.

SD 10.3 f 2.8% vs 13.5 + 5.3%, P < 0.05) and a decreased osteoid volume (1 .O f 0.6% vs 2.5 f 1.80/o, P < 0.01). Decreases in osteoid surface and thickness index of osteoid seams had borderline significance (0.05 < P < 0.10). The resorption was unchanged. Biochemistry Biochemical data from patients and controls are represented in Table Ill. Slight to moderate increments of serum creatinine (> 1 IO ~molll, max. value 184 cl.molll) werefound in 22 patients and 4 control subjects. Mild elevations of serum alkaline phosphatase (~3 Bessey U, max. value 5.1 Bessey U) were observed in, 12 patients and 4 control subjects. Fractionation by electrophoresis yielded only a liver and sometimes a gall fraction in these cases except in 1 hypercalcaemic patient, in which a skeletal fraction was observed and hyperparathyroidism was diagnosed. In the patients the serum calcium concentration correlated with all protein fractions, in particular with albumin and P-globulin (r = 0.70 and 0.38, respectively, P < O.OOl), whereas in the control subjects, only a correlation with albumin was observed (r = 0.29, P < 0.05). When serum proteins were included as covariates, there was no difference in serum calcium levels between patients and controls. The individual serum concentrations of 25(OH)D, 24,25(OH),D, and 1,25(OH),D are represented in Figure 4. Low values were common in the patients, many being close to the detection limit, or below in the case of 24,25(OH),D. In the patients, 25(OH)D correlated not only with 24,25(OH),D but also with 1,25(OH),D (r = 0.83 and 0.49, respectively, P < 0.0001). In the patient group, the vitamin D metabolites correlated negatively with age (in the above order: r = -0.25, -0.34, and -0.30, respectively, P < 0.005). Correlations with serum albu-

15 %

relative resorption surface .

Fig. 2. Osteoid parameters in patients with femoral neck fracture, compared with control values of Meunier et al., (1977) and Melsen et al., (1978) (mean f 2 SD).

: lo-

An attempt was made to classify the biopsies in subgroups based on the remodeling parameters, “High turnover” was characterized by an increased resorption surface (2 6%) and a normal to increased osteoid surface (> 18%). Two biopsies with normal resorption surface, but increased osteoclast count and increased osteoid surface were also included in this group. The “uncoupling” subgroup showed increased resorption surface (z 6%) but a normal or low osteoid surface (< 18%). In the “rest” group, resorption and osteoid surfaces were normal or low. Qualitatively judged active cuboidal osteoblasts were usually present in the “high turnover” group but absent in the “uncoupling” group. Remodeling and bone mass parameters in the three subgroups are summarized in Table II. Mean cortical thickness was significantly decreased in the “high turnover” group compared to the other groups. In 16 patients, biopsies were obtained from 9 to 82 days (mean 28 days) after the fracture. Compared with biopsies obtained within a week the histomorphometric analysis was characterized by a decreased TBV (mean +

T

t I

c.

5J

controls

Meunier hklsen fractures n.88

Fig. 3. Relative resorption surface in patients with femoral neck fracture compared with control values of Meunier’s group and Melsen et al., (1978) (mean f 2 SD).

P. LIPS et al.. Hrstomorphometry

Table II. Histomorphometnc

89

and vitamrn D rn hrp fractures.

data in three subgroups,

classified accordrng to remodeling parameters

“High turnover”

“Rest”

“Uncoupling”

Significant P n Mean cortical thrckness Cortrcal porosity Trabecular bone volume Relative osteoid volume Relative osteoid surface Thrckness index osteord seams Relative resorption surface Osteoclasts/mm*

n

mean f SD

14 15 18 19 19

507 7.5 15.1 4.8 31.1

19 19 19

+ + f + +

138 2.4 6.0 2.0 12.4

n

mean f SD r + + r +

mean ? SD

9 9 9 9 9

798 7.9 14.6 1.5 11.4

333 4.5 5.5 0.8 5.5

48 32 54 60 60

740 7.7 12.7 1.9 12.7

+ f f f +

353pm 3.9% 5.0% 1 10/o 7.2%

15.7 c 4.4

9

13.6 f 4.7

60

15 6 * 6.7

8.0 + 2.7 0.60 + 0.41

9 7

9.1 + 2.6 0.49 f 0.26

59 20

3.3 & 1.3% 0.22 r O.l8mm-’

c 0.05* ns ns < 0.001* < 0.001* ns < 0.001+ < 0.01+

*“High turnover” srgnrficantly different from “uncouplrng” and “rest” groups ‘“High turnover” and “uncoupling” slgnificantiy different from “rest” group

min were observed (r = 0.38, 0.26, and 0.32, respectively, P < 0.005). In the patients, serum 1,25(OH),D correlated with cr,-globulin (r = 0.27, P < 0.005). In the control subjects, a correlation was seen only between 1,25(OH),D and albumin (r = 0.24, P < 0.05). A negative correlation was observed in the patients between 1,25(OH),D and creatinine (r = - 0.30, P < O.OOi), which was independent of age (partial r = - 0.23, P < 0.05). When grouped according to serum creatinine concentrations, the patients with impaired renal function (serum creatinine > 110 pmolll) showed a significantly lower serum 1,25(OH),D than the patients with normal renal function (mean + SD, 52 f 33 pmolll vs 84 + 46 pmol/l, P < 0.01). The latter patients still had a significantly lower serum 1,25(OH),D than did the control subjects. Impairment of renal function did not cause significant changes in serum 24,25(OH),D.

Confrontation

of histomorphometry

with biochemical

0.31, P < 0.05, respectively). The relative osteoid surface correlated with serum PTH (r = 0.25, P < 0.05). The osteoid parameters did not correlate with the vitamin D metabolites. It was observed, however, that above a serum 25(OH)D concentration of 30 nmolll, osteoid volume and surface were not increased in any case (Fig. 5). With stepwise discriminant analysis on the three histomorphometric subgroups by means of biochemical parameters, only 56% of the biopsies could be classified correctly. The most discriminating variable was serum 1,25(OH),D which was significantly lower in the “uncoupling” subgroup than in the other groups (mean + SD 44 f 34~~84 + 46pmol/l, P < 0.05). Inthe”highturnover” and “uncoupling” subgroups, serum PTH was higher than in the “rest” group, but the difference was not quite significant (mean of: SD, 0.13 + 0.04 vs 0.11 + 0.05 Fgaeq bPTH/I, 0.05 < P < 0.10). Differences in other biochemical parameters between the three groups were not observed.

data Comparison of all biochemical and histomorphometric data yielded only minor correlations. Both relative resorption surface and the osteoclast number correlated with serum alkaline phosphatase (r = 0.30, P < 0.01 and r =

Discussion The heterogeneity of the group of patients with femoral neck fracture is apparent from the wide dispersion of his-

Table III. Biochemical values in patients with femoral neck fracture and control subjects. Control subjects

Patients Significant P

n Creatinine Total protein Albumin a,-Globulrn cr,-Globulin /3-Globulin y-Globulin Calcium Phosphate Alkaline phosphatase PTH 25(OH)D 24,25(OH)2D 1,25(OH),D

mean f SD

n

mean f. SD

units

74 74 74 74 74 74 74 74 74

85 73.5 41.9 2.4 8.2 9.0 12.1 2.35 1.09

f f k + f + + + +

16 3.8 2.8 0.4 1.1 1.2 2.7 0.12 0.15

125 125 125 125 125 125 125 125 125

86 61.6 32.5 3.4 8.0 7.5 10.3 2.13 1.11

f 26 f 7.5 + 4.8 f 0.8 of: 1.6 f 2.0 & 3.3 f 0.16 r 0.26

fimolll gmll gmll gmll gmll gmll gmll mmolll mmolll

ns < 0.001 < 0.001 < 0.001 ns < 0.001 < 0.001 < 0.001 ns

74 74 74 74 73+

2.1 0.12 32.9 2.3 105

f + f + +

0.5 0.05 13.6 1.9 31

125 125 124’ 124* 124’

2.0 0.11 18.5 1.0 79

f + + f f

Bessey U pgaeq bPTH/I nmolll nmol/l pmol/l

ns ns < 0.001 < 0.001 < 0.001

0.7 0.05 10.6 1.4 46

‘One patlent was excluded from statistical analysis because she had been treated with vitamin D 100.000 U/week for 4 years in addition to sodlum fluoride ‘One estimation failed.

90

P. LIPS et al.: Histomorphometry

25 (OH) vitamin D

24,25(OH)pitamin ?? 11.6

ntiol/l_

0123

:. . ” .

”

and vitamin D in hip fractures.

1,25(0H)pitamin

D

D

PmoVl-

. .

6-

15D-

. f

100 -

5D-

contK4ls

controls n.74

femoral neck fractures n-125

n.74

Fig. 4. Concentrations of vitamin D metabolites dotted lines mark the detection limits of the assays.

. . .

% . 40

??

t

.

.

. . 1

??

(

I

.*-

1

20

40 25(0H)vitamin

controls n=73

femoral neck fractures n.125

in patients with femoral neck fracture and control subjects of the same age. The

relative oateoid volume

relative osteoid suec&’

femoral neck fractures n-125

60 D

nnlcll/l

Fig. 5. Relationship between osteoid parameters and the serum 25(OH)D concentration in patients with femoral neckfracture. Dotted lines mark the upper reference limit of osteoid volume (W/o) and osteoid surface (25%) and the threshold concentration of 25(OH)D (30 nmol/l).

tomorphometric parameters. The mean cortical thickness tended to be lower in the male patients, but differences were not significant, due to the large variance and the small number of controls. Cervical fractures did not display a disproportionate loss of cortical thickness, in contrast with results of Nordin et al. (1980). The values of cortical porosity in our autopsy controls are equal to those reported by Melsen et al. (1978). The increase in cortical porosity in the patients may be an additional cause for the hip fracture. The data on trabecular bone volume that were obtained in female patients reflect the general population. However, the variance is larger, and thus more patients have a very low TBV than in the general population, which is in agreement with results of Faccini et al. (1976). An increased trabecular bone loss was observed in trochanteric fractures, whereas cervical fractures in neither sex showed a lower TBV than controls. The difference in TBV, also observed by Nordin et al. (1980), was the only histomorphometric distinction between the two fracture types. In patients with hip fracture, the osteoid surface is extended, but the seams are thinner than usual, as both parameters for osteoid thickness are decreased. The mean width of osteoid seams was not increased in any biopsy. It is the more reliable parameter since it is directly measured and not influenced by trabecular thickness (Melsen, 1978). On these grounds serious osteomalacia is unlikely, but borderline cases cannot be excluded, because the mineralization rate was not measured with tetracycline markers (Frost, 1969), as the biopsies had to be obtained soon after the fracture. This result is in agreement with Hodkinson (1971) and Wootton et al. (1979), who also used osteoid thickness as criterion for osteomalacia. In other studies, different criteria were used, such as osteoid volume (Chalmers et al., 1969) or osteoid surface and mineralization front (Aaron et al., 1974), leading to a higher frequency of osteomalacia.

P. LIPS et al:

Hrstomorphometry

and vitamrn D In hip fractures.

The increase in bone resorption, observed in many biopsies, was mainly active resorption, since the resorption surface and the number of osteoclasts correlated well. The large dispersion in remodeling parameters tempted us to a classification, as was described before in osteoporotic patients (Avioli et al., 1979; Meunier et al., 1979, 1981). Our classification is arbitrary, because appositional rate measurements are lacking. The “high turnover” group may contain cases with borderline osteomalacia, as this group comprises the highest osteoid values. “High turnover” leads to increased bone loss (Parfitt, 1979), which was visible in our patients as cortical thinning, which may havecaused the hipfracture. In the “uncoupling” group, the expected increase of bone loss (Baron et al., 1981) is not observed, which indicates that the defective coupling originated recently, perhaps only after immobilization. In the biopsies obtained later than a week after thefracture, the histological picture partly resembles data found in disuse osteoporosis (Minaire et al., 1974). However, in several patients the operation was postponed because of poor condition, which also may have caused the histological changes. In morphometric studies such as this, it appears important to obtain bone biopsies as soon as possible after the fracture in order to minimize changes induced by immobilization. When inspecting biochemical data of patients it should be borne in mind that blood samples were taken after the fracture and sometimes after operation. The fall in total protein is mainly due to the fall in serum albumin, which is reported to be about 25% on the first day after a closed fracture (Lal et al., 1976). The decrease of the serum calcium level was of the same magnitude as reported by Nilsson and Westlin (1972) following femoral neck fractures. It can be explained by the concomitant fall in serum albumin which was confirmed by disappearance of the difference between patients and controls after correction. Serum phosphate concentrations and alkaline phosphatase activity are reported to rise after a fracture, but they only do so after a week (Nilsson and Westlin, 1972; Lal et al., 1976; Hosking, 1978). The serum PTH concentration may increase 1 day after bone trauma (Hulth and Johnell, 1979). However, it is hazardous to compare different assays for PTH. In general with respect to the above parameters, patients with femoral neck fracture do not differ from the aged population as a whole, taking into account changes caused by the trauma. The difference in serum 25(OH)D between patients and controls confirms the results of Baker et al. (1979). It cannot be explained by seasonal variation, as both groups were distributed equally over the year. The effect of bone trauma on serum concentrations of vitamin D metabolites is not known. In the patients a correlation was observed between the vitamin D metabolites, albumin, and cY,-globulin. At physiological concentrations vitamin D binding protein, an cr,-globulin, binds almost all 25(OH)D and 62% of 1,25(OH),D, whereas the latter is bound to albumin for 23% (Silver and Fainaru, 1979). Vitamin D binding protein and albumin correlate well (Imawari et al., 1979; Brown et al., 1980), and in normal controls, serum 1,25(OH),D correlates also with vitamin D binding protein (Bouillon et al., 1981). Therefore, some decrease in concentration of serum vitamin D metabolites after a hip fracture is likely to occur, along with the fall in serum proteins. A correction will be postponed until vitamin D binding protein concentrations have been mea-

91

sured. Different results in serum 25(OH)D in studies of patients with hip fracture may be explained by the selection of controls, for example, orthopaedic patients (Wootton et al., 1979). The high serum 25(OH)D levels observed by Lund et al. (1975) in Denmark are caused by a high dietary vitamin D intake and the use of supplements. The low concentrations of 1,25(OH),D in our patients are explained by the higher prevalence of very old subjects in the patient group and by impairment of renal function, which caused a significantly decreased concentration in 22 patients. As seen from the positive correlation in the patients, a low serum 25(OH)D also may be associated with a low 1,25(OH),D. As shown by low correlations and by discriminant analysis, biochemical parameters cannot predict adequately the remodeling state that is observed in the bone biopsy. A similar negative result was obtained by other investigators (Kleerekoper et al., 1979; Teitelbaum et al., 1981). We were puzzled by the low serum 1,25(OH),D in the “uncoupling” group. The possibility that with a low serum 1,25(OH),D the coupling message is diminished remains entirely hypothetical. The relation between vitamin D deficiency and “high turnover” (Parfitt et al., 1981) is not supported by a lower serum 25(OH)D in this group than in the other groups, whereas the increase in serum PTH is only borderline significant. The latter situation may be due to insensitivity of the PTH assay. In general the serum 25(OH)D concentration was low in the patients, and increased osteoid values were not observed when serum 25(OH)D was higher than 30 nmolll. Therefore, at least in some of the cases “high turnover” may be considered a precursor of osteomalacia. The question remains why below a serum 25(OH)D level of 30nmolll osteoid parameters are increased in some patients only, while in most patients no histological reaction is observed. The average age of the patients was above 75 years, and in many of them the vitality of osteoblasts is decreased, as is suggested also by a decrease in mean wall thickness with advancing age (Lips et al., 1978). One may suppose that only a “vital” bone cell population can react to vitamin D deficiency. Cortical porosity appears to be a contributing factor to hip fractures. Trabecular bone loss is an important cause of trochanteric fractures. Serious osteomalacia does not occur in hip fractures in the Netherlands. Serum concentrations of the most important vitamin D metabolites are decreased in patients with hip fracture compared to controls of the same age. Vitamin D deficiency may lead to high turnover, which causes cortical thinning, but in most cases a histological reaction to low vitamin D levels is not observed. A causal relationship between vitamin D deficiency and femoral neck fractures remains hypothetical, Only a prospective study may demonstrate whether the frequency of hip fractures will decline when the vitamin D status in aged people is improved.

Acknowledgement: This study was supported by the Praeventiefonds, The Hague. The authors thank Dr. B. Veraart, Dr.,J. Borggreve, colleagues, and nurses of the orthopaedicand surgical departments (OLVG), without whom this study would not have been possible. They are indebted also to Dr. H. Kruyswijk (Clinical Chemistry Laboratory, OLVG), Dr. W. Hackeng (Laboratory, Bergwegziekenhuis), Mr. A. Kuizenga, and Mr. H. Willems (Research Laboratory, AZVU) for numerous biochemical estimations. Mrs. J. van der Hooft and Mrs. M. Stevens provided

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excellent organizational assistance. Dr. Alltree English text, and Mrs. A. Wiggers gave secretarial

P. Lips et al.:

reviewed

the

assistance.

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Received: September IO, 798 1 Revised: December 29, 1981 Accepted: February 1, 1982

RESUME Afin de dbceler une oatbopathie m~tabolique, lea auteura ont doaC lea mbtabolitea de la vitamine D et d’autrea parametrea biochimiquea reflbtant le m6taboliame oaaeux chez 125 patients ayant une fracture du col du famur, et 74 timoina adultea d’lge comparable. Ont 416 Btudika parallOlement chez 89 maladea, par dea techniques hiatomorphombtriquea, lea biopsies oaaeuaea pr6lev6aa dana la aemaine auivant la fracture. Le volume trabkulaire (TBV) eat aignificativement abaiaab par rapport aux tbmoina chez les maladea dea deux sexes ayant une fracture du trochanter alora qu’il eat normal chez lea maladea ayant une fracture tranacervicale. L’hiatomorphomBtrie ne montre pea d’autrea diffkencea entre les deux types de fractures. La poroait6 corticale eat augment&e chez lea maladea par rapport aux temoina autopalba. Lea surfaces oat&oidea aont plus Mendues maia lea bordurea oat6oidea aignlflcativement plus mlncea que chez lea tbmoina. Cher aucun malade, on ne trouve d’augmentation de I’~paiaseur dea bordurea oat&oidea, ce qui (carte toute oatbomalacie caracMria6e. Lea biopaiea oaaeuaea ont bt6 claaa~ea en troia groupea aelon lea param&trea du remodelage: (a) un premier groupe d haut nlveau de remodelage (n = 19), (b) un deuxi*me groupe avec un dkouplage entre la rhaorption et la formation (n = 9), (c) un troiaiame groupe dana lequel Ie remodelage eat normal ou baa (n = 61). Dana le premier groupe g haut nlveau de remodelage, I’ipaiaaeur moyenne dea corticalea eat nettement diminuk par rapport aux autrea groupes. Lea concentratlona abriquea en calcium (corrlgha en fonction du taux dea prot&inea akiquea), phosphora, crbtinine, phoaphataaea alcalinea, et hormone parathyroidlenne ne aont pas dtffbrentea cher lea maledea par rapport aux t6molna. Lea concentratlona aklquea dea m6tabolitea de la vitamine D: PJ(OH)D, 24-25(OH),D et l-25(OH)rD aont aigniflcatlvement diminuba chez lea maladea. La profll histomorphom~trique ne peut pas atre pr6dit valablement g partlr de la biochlmie. Une augmentation du tlaau oatioide (2 5%) etlou une extension dea surfaces osteoidea (t 25%) prlncipalement observ~ea dana Ie groupe B haut nlveau de remodelage eat toujoura aaaocibe g une diminution de la concentration abrique du 25(OH)D (lnfkieure g 30 nmolll). Le “haut nlveau de remodelage” peut relever dune dbflclence en vltamine D. Cette dernike, cependant, n’induit pas la plupart du tempa une rbponae hiatologique univoque.