Boneandhfinerd, 8(1994)69-86 Elsevier

BAM M)243

69

Bone disease in children with homozygous @-thalassemia

Lilimar Rioja, Robert Girot, Michhle Garab6dian and Giulia Coumot-Witmer

CNRS URA.58.Wniwrsir~ PnLv V, oeparr~“rsrHcmororopy. HBpittddes Enfanrr.Mafades. 149 Rue de Pvm, 73743 Paris CPdex 15, France

(Received 27lanuary lYS9) (Awpted 22Seplember 1989)

The histological features of thalascmtc bone ere impwfectly known. end the rates of bone marrow hy- peractivity, iron overload or vitamin D deficiency in the pathogen& of the disease are not cleerly iden- tified. In this study we examined iliac crest biopsies from 17 transfusion-dependent children with home. zygous,%thakwemia and severe radiolo&el skeletel thalassemic changes. including widening of medul- lary spews end osteoporosis. Rachilic lesions were not observed. Serum ferritin cottcentretions were in- creased in all but one subject. Iron deposits were bistochemically detected in bone marrow, et the mer- row-bone interface, along cement liner and mia?ralhing perimeters. Minor changes were present in tn.. becular bone, and osleomalacia wes absent. By cootrest, cortical bane exhibited severe changer incled- ing ftisurer and focal mineralization de$cls. Plasma 2%hydroxyviwmin D (25(OH)D) concenrrations measured during the winter (December-May, 6.5 f 4.9 q/ml, meen f SD, n = 6) end duringthe sum. mer (June-November. 13.8 f 8.4 nplml. n = 9) did not differ from those of ege.matcbed children living in the same country. Seven patients had moderate hypocalccmis but no biolo&al signs suggestive of vi temin D deficiency: all had normal alkaline phosehetase ectivitv, normal or sliahtly elevated plasma phosphate, only t&o had low plarma 2S(OHiD c&wdions aed two others s~pr&mrmal &ss of piasma immenoreactive parethyroid hormone.

There resolts show that iron overload and vitamin D deticiency do not seem to play en important role in the pathogen&r of thalassemic tone disease, which is characterized hy cwticel lesions probably R- lated to marrow hypsractivity.

Key words: Tbalasemia; Iron; 25.Hydmxyeholecaldferol; Bone histomotphometry

Homozygous @thalassemia is an hereditary disorder due to the defective ability 10

synthesize the p-chain of adult hemoglobin. This defect leads to severe anemia for

Correspondence to: Dr Giulie Cownot, CNRS IJRASS3, Hbpital des EofeeV.-MeledeS. 149% me de S6vre.s. 75143 Paris C6dcx 15, Rena.

O~~S.~OS?J~~Q~~~.SO@ 19% Elsevier Science PublishersB.V. (Biomedicsi Division)

70

which regular blood transfusions are required. Radiological changes include diffuse osteoporosis with thin cortices and lack of

normal trabeculation, thickening of cranial bones and widened medullary cavities of the small tubular bones of the hands and the feet. Moreover skeletal maturation is delayed [l].

The bone changes are mainly attributed to mechanical pressure from the hyper- active erythroid marrow, but their histological aspect is not precisely known [2-51. Moreover, thalassemia is only one of the many causes of marrow stimulation pro- ducing such severe lesions. It has therefore been suggested :hat factors other than medullary expansion, such as iron overload [3] vitamin D deficiency [ci] or de- creased hepatic production of 25-hydroxyvitamin D [7] may contribute to the pathogenesisof the skeletal abnormalities.

The purpose of the present study was to examine iliac crest biopsies obtained dur-

ing splenectomy from a group of children with thalassemia, and to compare results of bone histology with clinical data and plasma biochemistry. In addition, in order to establish the influence of possible hepatic iron overload on the production of 25- hydroxyvitamin D, liver samples were examined.

Patients and Methods

Seventeen patients with homozygous @ltalassemia (16 patients, thalassemia ma- jor; one patient (patient 15). thalassemia intermedia). aged 3 years 9 months to 17 years 2 months, were investigated. The diagnosis was established by clinical exami- nation and hemoglobin (Hb) electrophoresis of the children and their families. All patients but one (patient 17) lived in Algeria and were foilowed as outpatients. They had received transfusions approximately once a month for several years, but complete data on the total amount of red blood cells transfused were not available. None of the patients had received regular iron chelation therapy or treatment by vi- tamin D or vitamin D derivatives. They were admitted at the HBpital des Enfants- Malades for splenectomy (except patient 17). Infortned consent was obtained from the patient’s parents for removal of spleen, bone and liver biopsies.

Radiology

Bone age [S] and the severity of skeletal lesions were evaluated on standard radio- graphs. Cortical diameter of the second metacarpal bone was obtained by subtract- ing the medullaty width from the total width measured at midshaft. Patient’s values were compared with the modified data from Gam et al. for normal subjects re- ported by F.N. Silverman [9].

Erythroblast percentage was evaluated on sternal bone marrow smears. Mean an- nual Hb concentration was calculated from the values measured every month dur- ing the year preceding splenectomy.

71

Calcium, phosphorus, albumin and protein concentrations were measured using an automatic autoanalyzer (SMA II, Technicon, Domont, France). Alkaline phospha- tase and glutamate oxaloacetate transaminase (SGOT) activities were determined according to a kinetic method [lo].

Serum ferritin and immunoreactive parathyroid hormone (IPTH) concentrations were measured using radioimmunoassays. For PTH an antiserum against the 53-84 fragment was used (LPTI-IoK CIS). This assay detects iPTH changes induced by 2% decrease in total plasma calcium following citrate perfusion [ll] (lower limit of detection, 20 pg/ml; normal adult range, 20-60 pglml). 25Hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D (1,25(OH),D) concentrations were mea- sured by competitive protein assays after extraction and chromatography of the plasma samples [12,13].

Liver biopsies were fixed in Bouin’s solution. Specimens were examined for sidero- sis and fibrosis.

Iliac crest biopsies obtained during surgery were embedded in methyl methacrvlate

blue (PI-~ 6.4) and by the Gomori method for the demonstration of iron. Before the histochemical reaction sections were briefly treated with HsO, in order to release iron molecules strongly bound to proteins [14]. Moreover, ‘the aurintricarboxylic acid method [lS] was applied to some sections, in order to investigate whether the presence of iron may interfere with the issue of this reaction.

The following histological variables were measured for tfabecular bone with a 100~point integrating eye piece (Carl Zeiss, FRG) and are given as two dimensional values: bone area, percentage of spongy bone arca composed of mineralized and unmineralixed matrix; osteoid area, percentage of bone area composed of osteoid; osteold perimeter, percentage of the total trabccular perimeter covered by an os- teoid seam; osteoblast perimeter, percentage of the total trabecular perimeter cov- ered by large osteoblasts; mineralizing perimeter, percentage of osteoid perimeter with fine granular aspect at the limit with the mineralized matrix on toluidine blue- stained sections [16]; osteoclast perimeter, percentage of the total trabecular pcr- imeter covered by osteoclasts; iron perimeter, percentage of total trabacular per- imeter covered by iron deposits.

Osteoid seam width was measured on toluidine blue-stained sections with a semi automatic technique (Morphomat 10, Zeiss, FRG) at a magnification of x.500.

Cortical width, cortical porosity and cortical osteoid area were measured with the same technique, at a magnification of x50. The values are given as the mean of the measurements performed on two non-consecutive sections. Porosity represents the percentage of cortical bone area occupied by resorption cavities and haversian channels, and cortical osteoid area the percentage occupied by osteoid tissue.

Since specimens from normal Algerian children were not available, we compared

72

data obtained in thalassemic children with those reported by Pierre Marie et al. [17] for mineralizing perimeter, and with those found in our laboratory for trahecular ]18] and cortical parameters.

Results

Clinical data, plasma biochemistry and treatment are shown in Table 1.

Growth failure was present in eight of the 17 patients (below -2 SD for height). Skeletal maturation was delayed in 15 subjects. As assessed by clinical examination and plasma testosterone concentrations (values not shown) puberty was delayed in the three older patients.

Plasma biochemistry The values for plasma calcium and phosphorus concentrations indicated on Table 1 represent the mean of two or three measurements performed during the week pre- ceding surgery. Values for caIcium arc adjusted for albumin or protein concentra- tions [19]_ Moderate hypocalcemia was present in seven patients. No decrease in plasma phosphorus concentration and/or elevated alkaline phosphatase activity was observed in any of these patients.

Plasma iPTH concentrations were normal (n = 12) or above normal (n = 3). They were not elevated in four of the hypocalccmic children.

For the patients studied during the winter period (December to May) (n = 6), the mean (+ SD) plasma 2S(OH)D concentration was65 f 4.9 @ml, while it was 13.8 + 8.4 q/ml for those studied during the summer (June to November) (n = 9). These values did not differ significantly from those in twenty age-matched controls living in Algeria with no clinical or radiological cvidencc of rickets, and normal cal- cemia and phosphatemia. Their mean ZS(OH)D concentration was 5.6 & 2.5 ng/ml (mean f SD) (n = 10) during the winter and 13.5 + 4.8 ng/ml (n = 10) during the summer. Eououi sebjects had extremeiy low piasma 2S(OH)D. T%s of them had hy- pocalcemia but none had increased alkatine phosphatase activities or increased iPTH concentrations. Plasma 1,25(OH)aD concentration was available for patients 2,s. 6 and 17. This concentration was found increased in patient 6 (165 pg/ml), and in the normal range in the others (47,Sg and 69 pg/ml, respectively).

Plasma SGOT activity was increased in all but patients 1,2 and 15. No correla- tion was observed between this activity and plasma 25(OH)D concentrations.

Serum f&tin was increased in all but patient 15 who had thalassemia interme- dia. Significant correlation was observed between ferritin concentration and SGOT activity (r = 0.567. P <&OS).

Liver hisrology

Abundant iron deposits were present in Kupffer cells and hepatocytes in all sub- jects. Severe portal and interportal fibrosis was observed in patients 5 and 6, fibrot- ic lesions were less severe in patients 4, 9, 12, 13 and 14, and small lesions were present in the other children. Liver biopsy was not available for patient 17. No rela-

Tab

le

1

Clin

ical

, pl

asm

a bi

oche

mis

try

and

trea

tmen

t da

ta o

f th

e th

alas

sem

ic

child

ren

Patients

No.

se

x A

ge

Hd-

(Y

ma)

IM

3 9

fl 3

2 F

3

11

-2

2

3 M

4

7 -4

3

4M

4 7

-1.5

5

5M

6 3

-2s

4

6M

6 3

-I

4

7 F

6

II

-1.5

4

RM

7

3 -1

.2

6

9F

7 4

-1.5

6

10

F

7 4

-3

5

11

M

7 5

-2

6

12

M

7 6

-1.j

4

13

F

7 10

-3

6

14

M

IL

5 -2

.1,

10

15

M

13

11

-3

11

16

M

1s

2 -6

11

17

M

17

2 --

4 12

6 2.

22

1.20

6 2.

45

-

6 2.

56

1.50

2.29

1.

15

2.L

6 1.

89

6 2.

37

L.8

3

6 2.

15

1.75

2.17

1.

70

2.06

1.

45

2.37

1.

50

2.23

1.

20

6 2.

34

1.55

2.15

1.

55

6 2.

16

1.52

2.33

1.

70

2.31

1.

85

2.15

I.

90

Nor

mal

val

ues

2.

20-2

.M)

1.1-

2

Pla

sma

bioc

bem

isfl

y T

ran

sfu

sion

s

25(O

H)D

A

:Wh

S

GO

T

F&

tin

M

ean

t S

tart

D

UW

iDll

(WI)

(n

@m

l)

An

nu

al

age

Y

Ino

Y

mo

- 45

17

18

0

31

8”

142

65

27

-

53

3 13

1

52

3’

281

50

3”

258

- -

94

102

6 21

0

80

19

128

29

24

-

_ _

243

59

9 I5

1

30

7 26

7

23

2w

164

56

w

110

70

12

129

48

SW

14

5

20-a

5.

6r2.

P

<3w

13.5

+4.

f?

14.4

57

0 6.

9 1

11

1 9

22

1850

7.

6 1

4 1

7

159.

3

86

77

32.5

87

71

7 -4

43

3200

6.

5 -

IO

3 9

3200

5.

4 1

10

4 5

2120

6.

1 1

10

4 5

575

6.8

3 3

1

6680

6.

3 -

S

6 7

66al

S

.2

I IO

5

6

7760

8.

1 5

1 2

5

4450

7

-47-

2480

9

136

3

3400

8.

8 3

6 4

4

1940

6.

5 -

8 10

9

220

6.2

7 6

1

9w

5.7

3 12

2

J@j

12.8

2

3 15

:

20-2

80

90

44

97

51

13

52

1W.6

8220

’ H

, h

eigh

t. e

xpre

ssed

as

SD

of

rbe

mea

n.

b A

dju

sted

acca

rdin

gtoa

lbu

min

or

prot

ein

co

nxm

trat

ion

~

1191

.

’ D

urb

~g

the

Yea

r pr

eced

ing

spkn

ecto

my.

w w

inte

r:

’ su

mm

er.

SG

OT

, ~

cru

m g

luta

mie

o~

aloa

ceti

r ac

id t

ran

sam

inas

e;

Alk

Ph

, al

kal

ine

phor

phat

are;

Y

. Yea

rs:

mo.

mon

ths.

74

tionship was observed between the severity of these lesions and plasma ZS(OH)D concentrations.

Radiology and bone histology All children had skeletal thalassemic radiographic changes. Metacarpal cortical diameters could be measured in 14 subjects and were found greatly reduced in all of them, whereas medullary spaces were widened (Table 2). Small cysts were ob- served in spongy and in cortical bone. Broadening of the diploic space of the skull was present in the majority of the patients. Rachitic lesions were not observed.

The iliac crest histomorphometric data are indicated in Table 3: minor changes were present in trabecular bone, whereas cortical bcne exhibited severe focal le- sions.

Eight patients had trabecular bone area below -2 SD from the nomud mean. Os- teomalacic lesions, i.e., increased osteoid area and osteoid seam width, together with reduced extent of the mineralizing perimeter, were not observed. However. in several patients osteoid area and osteoid perimeter were slightly increased. Os- teoid seam width was high in patients 14 and 15 and osteoblast perimeter was above normal in seven children. This latter parameter was positively correlated with os- teoid area (I = 0.58, P < 0.02) a.:d with osteoid perimeter (r = 0.82, P-c 0.01). No correlation was observed between osteoblast perimeter and serum ferritin concen- trations. Osteoid lamellae were present deep within the calcified matrix in patients

Table 2

Metacarpal co:tical diameter and medullary width in the thalassemic children

Metacarpal cortical diameter (mm)

Padents Age-marched controls’

Mcdullary width (2~)

Patients Ape-malchedcantrols’

1 I.0

2 1.0

3 2.0 4 1.0 5 - 6 1.0 7 0.5 B - 9 I.5

IO 1.5 11 1.0 I2 - 13 0.5 14 1.0 15 1.5 16 0.5 17 0.5

2.31 k 0.47 6.0 2.30 * 0.45 5.0 2.54 f 0.43 2.54 f 0.43

2.61 + 0.43 5.5 2.88 k 0.46 6.5

2.8B f 0.46 2.8B f 0.46 2.82 t 0.52 5.5

3.11 f0.54 6.5 3.47 + 0.57 6.0 4.31 5 0.84 4.5 4.75 f 0.77 5.5 4.92 + 0.71 9.5

3.0 4.5

5.0

5.0

3.10 * 0.63 2.91 f 0.6S 3.12 f 0.61 3.12 f 0.61

3.23 f 0.63 2.95 * 0.64

2.95 + 0.64 2.95 f 0.64 3.63 f 0.7B

2.96 f 0.67 3.63 k 0.78 3.81 * 0.82 3.92 f 0.81 3.92 f 0.81

* Ref. 9.

Tab

le 3

Ilia

c bo

ne b

isto

mor

phom

etry

of

the

thal

asse

mic

chi

ldre

n

1 2 3 4 5 6 7 8 9 10

11

f2

13

14

15

16

17

14.2

3.

7 19

.2

80

7 0.

8 13

.9

3.0

12.6

81

8.

1 7.

3 29

.2

2.5

24.1

76

.9

12

1.9

20

3 31

.6

50.6

6.

5 8.

5 17

5.

9 53

.9

69.3

10

.3

27.5

12

.4

3.2

44.1

67

.0

9.6

28.4

15

.2

2.8

11.5

62

.1

7 5.

4 20

.6

4.3

40.6

44

.6

9 4.

6 20

.7

2.1

29.1

61

.3

7 9.

5 26

.7

0.5

18.3

65

.3

10.8

1.

5 18

.4

6.4

34.4

23

.7

11.1

1.

6 15

2

28

29.3

14

.3

6.2

12.5

3.

6 40

.4

28.6

13

.5

17.6

4.

4 41

.5

65.6

16

.8

21.6

23

.4

7.3

63.3

63

.5

16.2

35

.7

13.9

6.

2 33

65

.3

12.4

23

.3

2:.5

5.

0 39

.3

78.4

14

.5

13.6

22f1

.9

4.2k

2.0

22k

7.6

65.4

S.2

9.

3i3.

7 8.

3f4.

4 1.

8f0.

7 -

0.77

f0.2

7 0.

!6kO

.2:

5.8t

2.:

____

(%)

Cm

) (%

) (%

‘o)

I%)

(mm

1 (%

I

0.7

52.8

0.

6:

0.5

4.8

1.6

60.1

0.

18

1.6

2.45

2.

24

35.6

0.

84

6.25

4.

25

2.23

25

.3

0.79

5.

3 !.9

3.

26

37.8

-

3.00

11

.9

0.38

0.

87

4.35

1.

7 19

.9

0.44

0.

29

4.75

2.

42

65.7

0.

62

0.75

1.

89

2.90

41

.4

0.67

7.

6 9.

05

4.8

18.9

0.

51

0.3

17.0

5 2

27.8

1.

09

1.7

3.65

1.

83

51.4

0.

59

1.5

3.5

2.41

35

.4

0.77

4.

2 11

2.

3 40

0.

88

2.45

0.

05

1.05

6.

9 0.

62

O/:5

1.

35

3.3

56.2

0.

58

4.8

1.2

1.4

41.4

-

76

4,5,6,13 and 14. All but three patients (patients5,lO and 16) had osteoclast perim- eter in the normal range. Marrow fibrosiswas absent in all subjects.

Cortical width was extremely reduced in patients 2, 6 and 7 but normal in the others. Cortical osteoid area was above normal in nine children (Table 3). This in- crease resulted naiaiy from the presence of osteoid focally distributed in one or both cortices, often localized deep within the mineralized matrix (Figs. 1, ZA, 3 and 4). These lesions were the most severe in patients 3,4,9,13 and 16. No such lesions were observed is patients 10 and 15. Conices from patients 5 and 17 had beeo dam- aged during biopsy removal. No relationship was observed between cortical osteoid and plasma 25(OH)D concentrations.

In eight subjects cortices were interrupted at one or several places. In the inter- space between the two cortical extremities, bone marrow, macrophage clusters or blood vessels were present. The extremities were partially covered by large and ir- regular areas of osteoid tissue, which extended under the periosteum or deep into the neighbowing mineralized matrix (Fig. 4A,B,C,D). Cortical porosity varied greatly, and was above normal in patients 10 and 13. No relationship was observed between cortical width, cortical osteoid and porosity, nor between cortical and spongy bone parameters.

Abundant iron deposits were present in the bone marrow of the 17 patients. Nu-

Pig. 1. Iliaccorricalbenefrom pa!icnt 12. Periorteum at the topof theimage. Alargeosteoidarea (open

arrow) is present within the mineraXvd matrix (toluidine blue, X !Yf;).

Fig. 2. Conicalbone frompadent4. Ostcoidtissue (openarrow)isobservedonthetoluidinestainedsec- don (A). Ontheserialwcrionslained bytheGomori method (B)irondeposiasrepnrentst the limitbe- twcen osteaid and the mineralized matrix (closed snows) in some zones, but absent in other regions

(open arrows) (X155).

Fig. 4. nisc cortical bone from patients 4 (A), 9 (It). 13 (C) and 14 03). Periosteum is locslired at the top ofeach micrograph. Cortiss arc interrupted (closed arrows) and osteoid tissue (open arrows) is present

under the periosteum and within the mineralized matrix (toltidine blue, X30).

mercnt~ large macrophages containing Gomori-positive material were arranged in clusters near the bone surfaces or in contact with the periosteum. In the bone matrix iron was detected at the interface between the marrow and the mineralized IX’ the

:

. . .

. .

Fig. 5. Bone trakcula from patient 8. km deporitsarepresent inlhe bone marrow. at the marrow bone

interface (closed arrow) and along cemcnl lines (open arrow) (Gomori, X155).

81

osteoid perimeters, and along cement lines (Fig. 5). Moreover the metal was pres- ent at the limit between osteoid and the mineralized tissue, and in these areas evi- dence for active mineralization was absent. However, at this site the distribution and the intensity of the histochemical reaction varied greatly along the same miner- alizing perimeter (Fig. 2B) and also between two different areas from the same sec- tion. In some patients Gomori-positive zones were present throughout the osteoid tissue (Fig. 3B).

Osteocytes and osteoclasts contained iron-loaded granules (Fig. 6). No iron was detected in osteoblasts.

On Prussian b!ue-stained sections thin osteoid seams are difficult to identify with precision. Therefore. the iron perimeter indicated in Table 2 represents the total extent of the iron-positive perimeters, i.e., marrow-bone interfaces, neutral and mineralizing perimeters. All measurements were done within 2 weeks of the histo- chemical reaction being performed, because Prussian blue may fade with time. No correlation was observed between iron perimeters. the parameters reflecting bone remodelling, and plasma bioche!nistry.

The aurinotricarboxylic arid method showed deep violet deposits on the intense Gomori-positive bone perimeters, but no stain was detected in the majority of the iron-loaded marrow cells. This violet color was easily distinguishable from the bright red which indicates the presence of aluminum.

Hemarology Mean marrow erythroblast count was 58.9 f 14.6% (range 32-81%) for the chil- dren for whom marrow aspirates were available (patients 3-7, 9-11, 13-E). No correlation was observed between this parameter and bone histomorphometric data.

This study describes the bone histology from a large group of transfusion-depen- dent children with homozygous p-thalassemia. In the iliac crest of these patients cortical bone exhibited severe lesions, includr.., ‘“n small fissures and focal osteoid areas, whereas minor changes were present in trabecu!ar bone.

All but one patient originated from Algeria and were followed in this coumry. They were regularly transfused. However, as indicated by their low mean annual hemoglobin concentrations, :he blood supply available appears to have been insuf- ficient for adequate treatment. The magnitude of iron overload was estimated from serum ferritin concentrations, as the total amount of blood received by each patient could not be exactly known. The ferritin concentrations were above normal in all patients with thalassemia major, and normal in the patient with thalassemia inter- media, who required less frequent transfusions. In some children, the ferritin in- cre.tse was probably disproportionate to that of the iron stores. As a mdtter of fact a positive correlation was observed between ferritin concentration and SGOT activi- ty, suggesting that part of the increase in ferritin levels was the consequence of liver

82

damage [SOJ.

Osteoporosis, as assessed on X-rays hy the cortical diameter of the second meta-

carpal, was observed in all children, and was the most severe in the oldest patients.

Osteoporosis was less evident in iliac crest biopsies, although in several children

cortical width and/or trahecular bone area were reduced. The origin of this osteope-

nia is unknown. In experimental animals acute iron overload has been shown to de-

crease osteohlast number and bone formation rate [Zl]. In the present stuay, osteo-

blast number was not reduced, and even increased in several children. However,

findings ot a normal plasma alkaline phosphatase activity in all of them, even in the

two hypocalcemic subjects with elevated plasma IPTH concentrations, suggest a

defective osteoblast response to PTH. An alternative hypothesis could be titat of a

direct effect of iron on alkaline phosphatase activity, since in vitro Fe” inhibita this

enzyme [22].

The pathogenesis of the small fissures and of the focal osteoid areas observed in

cortical hone remains unclear.

Microfractures could be involved. However, the surface of the cortical extremi-

ties was smooth. and the interspace between them was often relatively large and

contained numerous macrophages. These cells have been shown to synthesize sev-

eral factors which stitnulate the resorptive activity of osteociasts 1231. It is possible

that marrow macrophages, activated by the presence of abnormal protein mole-

cules. produce stimuli for cortical bone resorption. Moreover, medullary spaces

were greatly enlarged in all patients and this observation further suggests that hy

perplastic marrow may directly influence bone metabolism. This hypothesis is sup

ported by the observation of Pootrakul et al. that, in two thalassemic patients,

transfusion therapy reduced the rate c: erythropoiesis, stimulated bone turnover

and decreased osteoid seams width [4]. Moreover, radiological bone changes are

improwd by tran;fusions [24].

Osteoid remnants can he observed in the mineralized matrix (IF healing osteoma-

lacia [I&b], but large areas such as those observed in OUT patients have never been

described. It is not known whether such osteoid tissue is the consequence of the dis-

ease or would have also been found in the control children living in the same coun-

try. as bone of the control Algerian children could not he examined. The plasma

25(OH)D concentrations were low in thalassemic and control children, as com-

pared to the values reported for subjects living in other countries and especially

when Gompared to values found in France and measured in our laboratory [26].

Thus, although no evidence was found for radiological or biological vitamin D defi-

ciency in thalassemic patients, it cannot he excluded that cortical hone, which has a

lower turnover rate than trahecular bone, still contained osteoid tissue reflecting

previous osteomalacia, whereas bone mineralization had improved in trabecular

hone.

Iron deposits were observed in the bone of all the children of the present study.

The presence of iron has been histochemically demonstrated along the mineralizing

perimeter in some thalassemic patients [3-51, osteomalacic dialysis patients [27,28]

and in the bone of pigs who received dexiran iron [21]. It has been suggested that

iron may influence the mineralizing process because it belongs to the same group of

83

elements as aluminum, which induces low-rurnover osteomalacia [15,29]. Howev- er, gallium does not influence bone mineralization (301. Osteomalacic lesions have been reported in thalassemic patients, but the pathogenesis of these lesions cannot be ascertained in the absence of ZS(OH)D data [3-S]. In contrast, bone mineraliza- tion was not impaired in the iron overloaded animals [Zl]. Moreover, children with constitutional red-cell aplasia, who are submitted to a transfusion regimen similar to that for thalassemic patients, do not develop skeletal changes, in spite of severe iron overload [31]. In addition, mineralization rate was found increased in a 41. year-old woman who received over 400 transfusions for chmnic erythroid hyperpla- sia [32]. Similarly, none of the children of the present study displayed low-turnover osteomalacia, in spite of extensive iron surfaces. As suggested by the positive corre- lation between osteoblast surface and osteoid area the increase in osteoid area ob- served in some children appears to be the consequence of accelerated bone turn- over rather than to delayed mineralization. In cortical bone, iron deposits were not regularly found along the mineralizing perimeter, and their intensity was greatly variable, suggesting passive depos:rion a+ inactive surfaces.

The mechanism by which iron is fixed to the bone tissue is not known. In vitro this metal does not adsorb to calcium hydroxyapatite cristals in the same manner as ah:- minum [33]. Autoradiographic studies in mice have shown that, 3 h after the injec- tion of radioactive iron, extensive iron deposits are present in osteoid and bone, but that a rapid decrease occurs thereafter [34]. These observations suggest that iron incorporation within the bone tissue is unstable, and this could explain the great variability in the iron perimeters and of the intensity of the histochemical stain ob- served among the uatients of the present study. For all these reasons it is unlikely thai iron deposits aid affect the mineralization process.

Moderate hypocalcemia was present in seveli children. Hypoparathyroidism is a frequent compiication of transfusional iron overioad, and its incidence increaes with age [3S-371. Indeed, among the six hypocalccmic patients from whom iPTH concentrations were assayed, plasma iPTH was increased in two, whereas in the four othrr patients normal iPTH concentrations suggest an impairment of the para- thyroid response to hypocalcemia. For the older of these fox patients this hypothe- sis is further supported by the relatively elevated P&ma phosphorus concentra- tions. The impact of these biochemical changes on bone histology is dift%ult to anal- yse, as osteoclast perimeter was high in only one subject among the hyperparathp roid patients, and no significant decrease in osteoclasts was observed in the hypo- calcemic patients with relative hypoparathyroidism. Clearly the responses in indi- viduals are diverse and may depend on the age of the children. But detailed histo- morphometric data during normal development are not yet available.

Vitamin D-deficiency or a decrease in 25(OH)D production by the liver could be another cause of hypocalcemia. Several studies have reported that, in thalassemic patients, plasma Z(OH)D concentrations are reduced [S-7]. This has been attrib- uted to impaired hepatic hydroxylation of vitamin D resulting from hemosiderosis, because in four thalassemic patients an increase in plasma 2S(OH)D levels was ob- served in response to iron chelation therapy [7]. In hereditaq hemochromatosis, plasma 2S(OH)D concentrations are related to hepatic histological changes and

84

plasma SGOTactivity (381. In our patient group the mean plasma 25(OH)D concentrations in winter and in

summer were not reduced, as compared to control children and no relationship was found between 25(OH)D concentrations and liver injury, assessed by histoological examination and plasma SGOT activity. Both findings suggest that hepatic vitamin D hydroxylation was not disturbed in our patients. In addition, they did not exhibit pathological signs of vitamin D deficiency, even those children with the lowest pIas. ma 25(OH)D. There was no rachitic lesion shown by X-rays, no osteomalacic le- sions in the bone biopsies and no increase in plasma alkaline phosphatase activities and plasma iPTH concentrations.

In conclusion, this study shows that thalassemic bone disease in children is char- acterized by focal lesions of cortical bone, which include fissures and defective min- eralization. Thtve changes ate probably related to marrow hyperactivity, whereas iron overload. ! itamin D deficiency and defective hepatic vitamin D hydroxylation do not appear to play an important role.

AcknowIedgements

We wish to acknowledge Dr Esseghir, Dr &rdjoen, Dr Bemabadj and Dr Belhari for allowing us to study their patients. We thank Dr P. McKelvie for the analysis of liver biopsy specimens, Mrs C.L.. Thil for expert technical assistance and Mrs M. Poitou for preparing the manwript.

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