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HYPERCALCAEMIC OSTEOMALACIA DUE TO ALUMINIUM TOXICITY
1009
HYPERCALCAEMIC OSTEOMALACIA DUE TO ALUMINIUM TOXICITY
G.S. FELL†
B. F. BOYCE* H. Y. ELDER‡ H. L. ELLIOT¶ I. FOGELMAN∥
B. J. JUNOR§ G. BEASTALL† I. T.
BOYLE∥ University Department of *Pathological, †Pathological Biochemistry, and Medicine, Royal Infirmary, Glasgow; and University Departments of ‡Physiology and ¶Materia Medica and §Renal Unit Western Infirmary, Glasgow In 16 patients with chronic renal failure and osteomalacia resistant to vitamin-D therapy, aluminium was demonstrated in bone biopsy specimens at the interface between thickened osteoid and calcified bone by means of both X-ray microanalysis and a specific histochemical stain. 14 patients also had hypercalcaemia. It is suggested that this is due to the blocking by aluminium of calcium uptake into bone coupled with the availability of additional calcium from dialysis fluid and vitamin-D therapy. This study provides more aetiological evidence linking aluminium and the development of osteomalacia in chronic renal failure. Further, if hypercalcaemia develops in such patients it is important that aluminium toxicity be excluded as the cause to prevent unnecessary parathyroidectomy.
Summary
Introduction ALUMINIUM has been implicated in the pathogenesis of a form of osteomalacia occurring in patients with chronic renal failure on regular haemodialysis.I-3 Aluminium-related osteomalacia differs from classical vitamin-D-deficiency osteomalacia in that patients are resistant to treatment with even large doses of vitamin D, have an increased incidence of bone fractures, and are particularly likely to experience bone
3. UCLA Bone Marrow Transplant Team: Bone marrow transplantation in acute leukemia. Lancet 1977; ii: 1197-200. 4. Herzig GP, Bull MI, Decter J, et al. Bone marrow transplantation in leukemia and aplastic anemia: NCI experience with four grafting regimens. Transplant Proc 1975;
pain.4,s The associated biochemical features are normal (or only slightly elevated) serum levels of alkaline phosphatase and parathyroid hormone and a tendency for hypercalcaemia to develop.6 Such patients have high aluminium concentrations in both serum and bone,1,8,9 where it accumulates predominantly at the calcification front between osteoid and calcified matrix.8,9 The source of the aluminium is usually the water-supply used to prepare the dialysis fluid10,11 but in some cases it is related to ingestion of
aluminium-based phosphate-binding drugs. 7,12 In this paper we describe the clinical, biochemical, and bone-histomorphometric findings in 16 patients with aluminium-related osteomalacia and discuss the mechanism which may underlie the development of hypercalcaemia. Patients and Methods
Patients 16 patients (9 males, 7 females, aged 12 to 60 years) with advanced chronic renal failure of various aetiologies were studied. All patients had received regular haemodialysis at home or in hospital for between 3 months and 8 years and had been treated with aluminiumcontaining phosphate-binding drugs. All had histological evidence ’of osteomalacia, and 13 had been unsuccessfully treated with large doses of vitamin D (la-hydroxycholecalciferol [1&agr;-OH D3] in most cases). Of the remaining 3 patients not treated with vitamin D before bone biopsy 2 (nos.4 and 6) were subsequently treated with 1 a-OH D3 without improvement. Thus, 15 patients were considered to have aluminium-related osteomalacia resistant to vitamin D. The remaining patient (no. 13) was never treated with vitamin D, and two subsequent bone biopsies, after withdrawal from exposure to aluminium, showed improvement and eventual resolution of the mineralisation defect. Patients were divided into three groups: Group A. -These 6 patients had the dialysis encephalopathy syndrome.All had received regular haemodialysis with water containing high aluminium concentrations. Group B. -These 6 patients were dialysed with aluminiumcontaminated water, pretreated by reverse osmosis to reduce the
MM, Gale RP, Kay HEM, Rimm AA. Factors associated with interstitial pneumonitis following bone marrow transplantation for acute leukaemia. Lancet
20. Bortin
1981;
5.
leukemia in first remission. N Engl J Med 1979; 301: 597-99. Morgenstern G, Clink HM, et al. The place of bone-marrow transplantation in acute myelogenous leukaemia. Lancet 1980; i: 1047-50. Gale RP. Clinical trials of bone marrow transplantation in leukemia. In: Gale RP, Fox CF (eds). Biology of bone marrow transplantation. New York: Academic Press,
nonlymphoblastic
8. Powles RL, 9
bone
33.
Bortin MM, Gale RP, Kay HEM, Rimm AA. Bone marrow transplantation for acute
34.
19
myelogenous
leukemia: factors associated with
early mortality. JAMA (in press).
press). Lumley HS,
RL, Morgenstern GR, Clink HM. Matched allogeneic sibling transplantation for acute myeloid leukaemia in first remission. Exp Hematol 1982; 10 (suppl 10): 70-71. 23. Zwaan FE, Hermans J. Bone marrow transplantation for leukaemia-European results in 264 patients. Exp Hematol 1982; 10 (suppl 10): 64-69. 24. Gale RP: A prospective controlled trial of bone marrow transplantation vs. chemotherapy in acute myelogenous leukemia. Blood 1981; 58 (suppl 1): 173a (abstract). 25. Zwaan FE, Jansen J, Colpin GGD, Simonis RFA. Marrow grafting for acute leukemia during remission-results in 23 patients. Exp Hematol 1982; 10 (suppl 10): 87 (abstract). 26. Forman SJ, Blume KG, Spruce WE, et al Bone marrow ablation and marrow transplantation in acute leukemia: influence of hematological pre-treatment status. Clin Res 1981; 29: 333a (abstract). 27. Dinsmore R, Shank B, Kapoor N, et al. A randomized trial of marrow transplantation (BMT) versus chemotherapy (CT) maintenance for acute myelogenous leukemia in first remission. Preliminary results. Exp Hematol 1981; 9 (suppl 9): 125 (abstract). 28. Speck B, Gratwohl A, Nissen C, et al. Further experience with cyclosporin-A in allogeneic bone marrow transplantation. Exp Hematol 1981; 9 (suppl 9) 124 (abstract). 29. Armitage J, Klassen L, Kugler J, et al. Allogeneic bone marrow transplantation (BMT) for the treatment of acute leukemia Clin Res 1981; 29: 842a (abstract). 30. Santos GW, Tutschka PJ, Beschorner WE. Marrow transplantation in acute nonlymphocytic leukemia (ANL) following busulfan (BU) and cyclophosphamide (CY). Blood 1981; 58 (suppl 1): 176a (abstract). 31. Mannoni P, Vernant JP, Rodet M et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission. Blut 1980; 41: 220-25. 32. Beutler E, McMillan R, Spruce W. The role of bone marrow transplantation in the 22.
1980: 11-27. KG, Beutler E, Bross KJ, et al. Bone-marrow ablation and allogeneic marrow transplantation in acute leukemia. N Engl J Med 1980; 302: 1041-46. 11. Okuma T, Rosner F, Levy RN, et al. Treatment of adult leukemia with 1-asparaginase (NCS-109229). Cancer Chemother Rep 1971; 55: 269-75. 12. Karnofsky DA, Abelman WH, Craver LF, et al. The use of nitrogen mustard in the palliative treatment of carcinoma. Cancer 1948; 1: 634-656. 13. Report from the ACS/NIH bone marrow transplant registry. Exp Hematol 1975; 3: 149-55. 14 Cutler SJ, Ederer F. Maximum utilization of the life table in analyzing survival J Chron Dis 1958; 8: 699-712. 15 Lee E, Desu M. A computer program for comparing K samples with right-censored data. Comput Programs Biomed 1972; 2: 315-21. 16 Jennrich RI, Moore RH. Maximum likelihood estimation by means of non-linear least squares. In: Proceedings of the statistical section of the American Statistical Association. Washington, D.C., 1975: 57-65. 17 Cox JR. The analysis of binary data. London: Methuen, 1970. 18. Bennett JM, Catovsky D, Daniel M-T, et al. Proposals for the classification of acute leukemias. Br J Haematol 1976; 33: 451-58. 10. Blume
437-39.
ED, Clift RA, Buckner CD. Marrow transplantation for patients with acute nonlymphoblastic leukemia who achieve a first remission. Cancer Treat Rep (in
7: 817-21.
Tutschka PF, Elfenbein GJ, Sensenbrenner LL, et al. Preparative regimens for marrow transplantation in acute leukemia and aplastic anemia. Am J Ped Hematol Oncol 1980; 2: 363-70. 6. Speck B, Cornu P, Nissen D, et al. The Basel experience with total body irradiation for conditioning patients with acute leukemia for allogeneic bone marrow transplantation. Pathol Biol (Paris) 1979; 27: 353-55. 7. Thomas ED, Buckner CD, Clift RA, et al. Marrow transplantation for acute
i:
21. Thomas
Powles
marrow
treatment of acute leukemia
in remission.
Blood 1982; 59: 1115-17.
Ramsay NKC, Kersey JH, Robison LL, et al. A randomized study of the prevention of acute graft-versus-host disease N Engl J Med 1982; 306: 392-97. Bortin MM, Gale RP, Rimm AA. Allogeneic bone marrow transplantation for 144 patients with severe aplastic anemia. JAMA 1981, 245: 1132-39.
1010 aluminium content. However, they became exposed to high concentrations of aluminium after deterioration and malfunction of the reverse-osmosis membranes. Group C. - These 4 patients were dialysed with water containing low concentrations of aluminium (<40 pg/1) but were exposed to aluminium-containing phosphate-binding drugs. An estimate of the total exposure to aluminium from this source had been calculated for all 16 patients from the dose and duration of therapy.
Biochemistry Serum calcium, inorganic phosphate, and alkaline phosphatase were measured with standard laboratory techniques. Immunoreactive parathyroid hormone (iPTH) was measured in plasma with a double-antibody radioimmunoassay which uses an antiserum (BW211/32) able to recognise both ends of the PTH molecule. Aluminium concentrations in serum and water were measured by means of electrothermal atomic-absorption
(AP)
spectrophotometry (ETA-AAS).
13
Bone Biopsies Transiliac bone biopsy specimens (8 mm diameter) were obtained from each patient and fixed in neutral phosphate-buffered formalin. The specimens were dehydrated in graded alcohols and embedded in methylmethacrylate. 10 µm serial sections were cut from each biopsy specimen on a Jung K sledge microtome. Histomorphometry.-Four representative sections from each biopsy specimen were stained (1% aqueous toluidine-blue) for quantitative histology, and the following variables were measured by point counting and line-intersect measurement with a Zeiss II eyepiece graticule: relative osteoid volume (OV),14 maximum number of birefringent lamellae (MNL) of osteoid15 (visualised under polarised light), total osteoid surface (OS),14 and extent of calcification fronts along osteoid seamsl6 were measured to give an indication of the severity of osteomalacia; and active osteoid surfaces14 and total14 and active14 resorption surfaces were measured to give an indication of the degree of secondary
hyperparathyroidism. a’bleasurement of bone aluminium content was measured with ETAAAS on particles of bone weighing 20 to 100 mg obtained from the methacrylate-embedded specimens.8 Localisation of aluminium in bone.-Two methods (electron-probe X-ray microanalysis and histochemical staining) were used to demonstrate the localisation of aluminium in the bone biopsy specimens. Electron-probe X-ray microanalysis17 is an established technique used for the localisation and measurement of elements
within tissue at the ultrastructural level. Ultrathin sections for electron microscopy were prepared from the bone biopsy specimens by means of a technique which we have described previously.s Various sites, including calcified matrix, osteoid, and osteocytes, were examined for the presence of aluminium. The microanalysis was carried out on a JEOL JEM 1 OOC electron microscope with an attached Link Systems 290 microanalysis system. The whole system had been modified for accurate ultrastructural analysis of hard tissues.18 An established histochemical staining method19 using ’Aluminon’ (BDH Chemicals, Poole, England) was used to detect aluminium in 10 µm sections cut as described above on the Jung K microtome.
Results
(<40 µg/1). Serum levels were elevated in all patients (62 - 700 µg/1) but lowest in group C (normal range20 3 - 35 µg/1). Those in group-B patients are the levels measured between 9 and 18 months after the start of home dialysis following the failure of their reverse-osmosis units. Bone aluminium levels were elevated in all cases, ranging from 51 to 275 µg/g dry weight (normal range8 3-23 µg/g, mean ±1SD 10 - .5±4. 2 µg/g). There was obvious overlap in values among the groups, and whereas those in group A appear higher, the numbers are too small and the range is too large for meaningful statistical comparison. The range of values in group B is much narrower than in the other groups (59-120 µg/g), perhaps indicating the shorter period of exposure to high aluminium levels during home dialysis. There is a positive correlation between bone and serum aluminium levels in the 16 patients (r=0’67, p<0.01). Estimated total oral aluminium consumption ranged from 1 -3to 10 -5kg aluminium and correlated significantly with the duration of dialysis (r = 0.86, p<0. 01, n= 16). were
CLINICAL AND BIOCHEMICAL DETAILS OF PATIENTS
NA=not available. *Including hospital and home
(see Table)
Aluminium Data Considerable variation was found from time to time in aluminium levels in water and serum in patients in groups A and B, reflecting the intermittent addition of aluminium salt to clarify water in local reservoirs. The values listed in the table are the highest recorded around the time of bone biopsy. Water aluminium levels were similar in groups A and B (110-1005 µg/1) and were much higher than those in group C
dialysis. Measured before start of dialysis.
1011
Discussion
Other Biochemical Data Serum calcium levels were at or above the upper limit of the normal range in 14 patients and normal or low in 2. These 2 patients had histological evidence of mild-to-moderate secondary hyperparathyroidism, although iPTH levels were elevated in only 1 (patient 11). iPTH levels were increased in 7 out of 13 patients in whom it was measured (610-1500 ng/1), and in some of these parathyroidectomy was contemplated for possible tertiary hyperparathyroidism but not carried out (see Discussion). Alkaline phosphatase was normal or only slightly elevated in most patients. Hyperphosphataemia was present in 11patients and serum levels correlated inversely with total oral aluminium -
consumption (r = -0’ 48, p = 0 - 05, n = 16). Localisation of Aluminium in Bone
in patients with chronic renal failure is "autonomous" to function of one or more of ascribed usually
Hypercalcaemia
parathyroid glands-so-called tertiary hyperparathyroidism.21,22 Bone biopsy in such patients usually reveals greatly increased bone resorption and formation with normal bone mineralisation.22 Circulating iPTH and AP levels are greatly increased.23 In the present study of 16 patients with chronic renal the
.
Aluminium was demonstrated with a specific stain as a bright-red line along most of the length of the osteoid/ calcified-bone interface. Toluidine-blue staining of adjacent sections failed to demonstrate calcification fronts at sites where aluminium was present. A typical example of the staining is seen in fig. 1, which depicts the severe osteomalacia present in patient 16. This localisation was confirmed ultrastructurally in all cases by electron-probe X-ray microanalysis. Linear deposition of aluminium was also noted in stained sections deep within some calcified trabeculae at reversal lines.
failure and histologically proven osteomalacia unrelated to vitamin-D deficiency, 14 had hypercalcaemia at the time of bone biopsy. In all cases, aluminium was demonstrated at the calcification fronts in bone both by histochemistry and by electron-probe X-ray microanalysis and was considered the cause of the mineralisation defect. There was only minimal histological evidence of secondary hyperparathyroidism and, in contrast to the biochemical features of tertiary
Histomorphometry Fig.2 displays the individual values for OV, MNL, OS, and extent
of calcification fronts. Calcification fronts extended
along less than 60% of the total osteoid surface in all cases (range 0-52%), indicating osteomalacia. The variation in
severity of the mineralisation defect is highlighted by the variation in OV and MNL. Whereas some patients had severe osteomalacia (OV >30%, MNL>10) others had mild disease with OV around 100/o and MNL normal (up to 4) or only slightly increased (up to 6). In the majority of cases the thickened osteoid seams had a characteristically focal distribution along bone trabeculae, intervening bone surfaces being fully mineralised or else covered by thin osteoid. Fig.3 shows the active osteoid surfaces and the total and active resorption surfaces in all cases. Most values were within or only slightly above the normal range and as such are indicative of minimal or absent hyperparathyroidism.
Fig. 1-Undecalcified section
of bone from
patient
Fig. in
2-Bone
histomorphometry severity of osteomalacia.
in 16
patients, indicating variation
Bars indicate the upper limit of the normal range for relative osteoid volume, ’ maximum number of lamellae of osteoid (M.N.L.), and total osteoid surface. The lower limit of the normal range for the extent of calcification fronts along total osteoid surfaces is indicated by the bar in the third column.
16 stained for
aluminium. Aluminium is seen as a bright-red line (arrowed) between osteoid (0) and calcified bone (CB). (Reduced by half from x 280.)
Fig. 3-Bone histomorphometry in 16 patients, indicating the degree of secondary hyperparathyroidism. Bars indicate the upper limit of the normal ranges.
1012
hyperparathyroidism, circulating iPTH and AP levels were either normal or only moderately elevated. The association between hypercalcaemia and osteomalacia,6,24,25 without osteitis fibrosa, although uncommon, has previously been reported in chronic renal failure, but the development of hypercalcaemia has not been adequately explained. We believe that hypercalcaemia is due to a combination of factors in which aluminium toxicity has a central role. Initially, patients with chronic renal failure tend to be hypocalcaemic,26 and secondary hyperparathyroidism ensues if an attempt is made to correct the serum calcium by means of the well-recognised effects of PTH on bone and kidney.16 Medical therapy, such as the addition of calcium to the dialysis fluid 21 and the use of vitamin-D metabolites,25 is usually instigated to complement this physiological response. If, however, mineralisation of osteoid is inhibited by the direct toxic effect of aluminium accumulating at the calcification front, available calcium cannot be taken up by bone and hypercalcaemia may develop. A subsequent fall in biologically active PTH secretion in response to this rise in calcium may then result in reduced liberation of calcium from bone, but unless the concentration of calcium in the dialysis bath is lowered and vitamin-D therapy discontinued, hypercalcaemia is likely to persist. Such a mechanism could explain the hypercalcaemia and osteomalacia reported by Hodsman et al.24 in a population of dialysis patients very similar to our own. These authors considered aluminium toxicity unlikely but surprisingly had taken few steps to exclude it. In their study, bone aluminium levels were measured in only 6 of the 19 patients and were found to be elevated, but bone sections were not stained for aluminium and no data on serum or water aluminium concentrations were reported. The failure of parathyroidectomy to reduce the hypercalcaemia in 7 of 9 patients in that study further supports our proposed mechanism. The distinction between tertiary hyperparathyroidism and aluminium-induced osteomalacia is clearly of considerable clinical importance because parathyroidectomy is indicated in the former whereas withdrawal of both aluminium and vitamin-D therapy and lowering of dialysis-bath calcium are more rational treatments of the latter. There have been several reports3,27,28 of osteomalacia developing after parathyroidectomy for what was presumably considered to be tertiary hyperparathyroidism. In most cases appropriate investigations for aluminium toxicity were not carried out. The differentiation between aluminium-induced hypercalcaemia and tertiary hyperparathyroidism is not always straightforward. 4 of our patients, for example, had iPTH levels above 1000 ng/1, and 3 of these were hypercalcaemic. In the presence of hypercalcaemia these iPTH levels are inappropriately high, and in the absence of osteitis fibrosa tertiary hyperparathyroidism seems unlikely. However, the possibility that the two conditions co-exist and that aluminium directly inhibits parathyroid function cannot be excluded. Whatever the explanation, the diagnosis of aluminium-induced osteomalacia will not be clear unless bone biopsy with appropriate analysis for aluminium is carried out. The histological appearances of aluminium-induced osteomalacia may allow differentiation from vitamin-Ddeficiency osteomalacia in many cases. The most striking feature is a tendency for the osteoid to be focally distributed along bone trabeculae, the intervening surfaces being fully mineralised, with little evidence of osteitis fibrosa. The osteoid also tends to vary considerably in thickness. Thus, in some parts of a biopsy specimen osteoid is of normal or only
serum
increased thickness, whereas in other parts greatly thickened seams are present. Nevertheless, specific staining for aluminium should be carried out in all cases of osteomalacia in chronic renal failure before vitamin-D therapy is started. Although contamination of dialysis water remains the most important potential source of aluminium, the prevalence of osteomalacia related causally to oral aluminium therapy remains to be established, since systematic studies of bone biopsies carried out after a minimum of 2-3 years’ therapy have not been reported. Several "new" syndromes, such as vitamin-D-resistant osteomalacia in patients without secondary hyperparathyroidism24 and osteomalacia following parathyroidectomy,27 have been reported and may, in our opinion, be related to aluminium toxicity. This may also explain the poor response to vitamin-D metabolites in the treatment of renal osteomalacia.2s,29 We think that aluminium-induced osteomalacia may be more widespread than has hitherto been recognised, but at the present time facilities for full evaluation of such cases will be limited to relatively few centres. Nevertheless, aluminium toxicity should be excluded in any patient with chronic renal failure in whom either vitamin-D-resistant osteomalacia or hypercalcaemia develops, particularly if parathyroidectomy is being considered.
slightly
We thank Mr J. Byars for excellent technical assistance; Mrs I. Main for typing the manuscript; and Dr I. A. MacDougall, Dr Anna Murphy, and Dr M. J. Boulton-Jones for information relating to their patients. This work was partly supported by grants from Greater Glasgow Health Board Research Support Group and the Scottish Hospital Endowments Research Trust. Correspondence should be addressed to B. F. B. REFERENCES 1. Ellis HA, McCarthy JH, Herrington J. Bone aluminium in haemodialysis patients and in rats injected with aluminium chloride: relationship to impaired bone mineralisation. J Clin Pathol 1979; 32: 832-44. 2. Drueke T. Dialysis osteomalacia and aluminium intoxication Nephron 1980; 26: 207-10. 3. Hodsman AB, Sherrard DJ, Alfrey AC, et al. Bone aluminium and histomorphometric features of renal osteodystrophy. J Clin Endocr Metab 1982; 54: 539-46. 4 Parkinson IS, Ward MK, Feest TG, Fawcett RWP, Kerr DNS. Fracturing dialysis osteodystrophy and dialysis encephalopathy. Lancet 1979; i: 406-09. 5. Pierides AM. Dialysis dementia, osteomalacic fractures and myopathy: a syndrome due to chronic aluminium intoxication. Int J Artif Organs 1978; 1: 841-44. 6. Parkinson IS, Ward MK, Kerr DNS. Dialysis encephalopathy, bone disease and anaemia: the aluminium intoxication syndrome during regular haemodialysis. J Clin Pathol 1981; 34: 1285-94. 7. Ward MK, Feest TG, Ellis HA, Parkinson IS. Osteomalacic dialysis osteodystrophy Evidence for water borne aetiological agent, probably aluminium Lancet 1978, r 841-45. 8. Boyce BF, Elder HY, Fell GS, et al. Quantitation and localisation of aluminium in human cancellous bone in renal osteodystrophy. Scanning Electron Microsc 1981, III: 329-37. 9. Cournot-Witmer G, ZingraffJ, Plachot JJ, et al. Aluminium localisation in bone from haemodialysed patients: Relationship to matrix mineralisation. Kidney Int 1981; 20: 375-85. 10. Platts MM, Goode GC, Hislop JS. Composition of the domestic water supply and the incidence of fractures and encephalopathy in patients on home dialysis Br Med J 1977; ii: 657-60. 11. Elliott HL, Dryburgh F, Fell GS, Sabet S, MacDougall AI. Aluminium toxicity during regular haemodialysis. Br Med J 1978; i: 1101-03 12. Rottembourge J, Jaudon MC, LeGrain M, Galli A. Les gels d’alumine chez les insuffisants renaux chroniques. Un risque potentiel d’encéphalopathie et d’ostéopathie. Ann Méd Intern 1980; 131: 71. 13. Gardiner PHE, Ottaway JM, Fell GS, Halls DJ. Determination of aluminium in blood plasma or serum by electrothermal atomic absorption spectrophotometry Analyt Chim Acta 1981; 128: 57-66. 14. Whyte MP, Bergfeld MA, Murphy WA, Avioli LV, Teitelbaum SL. Postmenopausal osteoporosis. A heterogeneous disorder as assessed by histomorphometric analysis of iliac crest bone from untreated patients. Am J Med 1982, 72: 193-202. 15. Woods CG, Morgan DB, Patterson CR, Grossman HH Measurement of ostesid in bone biopsy. J Pathol Bacteriol 1968; 95: 441-47. 16. Rasmussen H, Bordier P. The physiological and cellular basis of metabolic bone disease Baltimore: Williams & Wilkins Co, 1974; 62-63. 17. Chandler JA. X-ray microanalysis in the electron microscope Amsterdam North Holland Publishing Co, 1977. 18 Nicholson WAP, Gray CC, Chapman JN, Robertson BW Optimising thin film X-ray spectra for quantitative analysis. J Microsc 1982, 125: 25-40.
1013 a stable biochemical state and had not received blood transfusions or any drug known to affect platelet function in the month before the study. Their clinical condition was judged to be good, except for 4 patients who had PCV lower than 20%.
all in
URAEMIC BLEEDING: ROLE OF ANAEMIA AND BENEFICIAL EFFECT OF RED CELL TRANSFUSIONS MANUELA LIVIO DONATELLA MARCHESI GIUSEPPE REMUZZI
(b) 2 uraemic patients with symptom-producing anaemia and very6 long BT were repeatedly transfused with washed packed red cells.
ELIANA GOTTI GIULIANO MECCA GIOVANNI DE GAETANO
PCV and BT were measured in these patients after each transfusion.
(c)
Laboratory of Cardiovascular Clinical Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea, 62-20157 Milan, Italy, and Division of Nephrology, Ospedali Riuniti, 24100 Bergamo, Italy
Bleeding time, altered in most uraemic patients, may be influenced by packed cell volume (PCV). Uraemic patients are often anaemic, so the influence of PCV on bleeding time was assessed in several groups of patients with chronic uraemia. The findings were that bleeding times in uraemic patients are profoundly influenced by anaemia, and that the platelet-mediated haemorrhagic tendency in uraemia may be successfully managed by raising PCV values to above 30%. URAEMIC patients have a poorly understood bleeding tendency, and complex vascular and platelet abnormalities 1-3 have been reported in these patients. However, no correlation has been found between different abnormalities revealed by in-vitro tests and the ex-vivo variable believed to reflect the patient’s clinical state most closely, namely, bleeding time (BT). This test has been said to be the best marker for
haemorrhagic risk.4 An observation made by Hellem et a1.5 in the early sixties which has not been paid much attention suggested that BT was prolonged in anaemic patients and could be restored to normal by transfusion of washed red cells. Since uraemic patients are often anaemic, we undertook the present study to establish: (1) the influence of packed cell volume (PCV) on BT in’uraemic patients; (2) the relationship between BT and PCV in uraemic and anaemic patients with similar PCV values; and (3) the possible "haemostatic effect" of red-cell transfusion in uraemic bleeding. Patients and Methods
were
infused with
30%.
Summary
Introduction
6 additional patients with PCV of 16-21%
washed, filtered red cells.6 BT, platelet count, platelet retention on glass beads, prothrombin consumption, platelet cyclic-adenosinemonophosphate (cAMP), and serum thromboxane B2 (TxB2) were measured before transfusion and again when PCV values rose above Control
Groups
(a) 65 normal subjects matched for age and uraemic patients.
sex
with the group of
(b) 15 patients with anaemias unrelated to the uraemia (11with hypochromic anaemia and 4 with normochromic anaemia) were compared with 15 patients randomly selected from the 65 chronic uraemic patients and matched for age, sex, and PCV values.
Laboratory Tests PCV was measured by a routine micromethod. Platelets were counted by phase-contrast microscopy. BT was determined in duplicate by a template-like semiautomatic device.44 Platelet adhesion to glass beads was measured in native blood by a modification of Hellem’s methodwith standardised glass-beadfilled columns and a constant rate (1 ml blood/ 15s) infusion pump (Adeplate’s and Adeplate’s Pump System, Mascia Brunelli, Milan,
Italy).44
Prothrombin consumption was determined as a measure of platelet factor 3 availability by Quick’s method.8 Serum TxB2 production was measured by radioimmunoassay99 after blood clotting in vitro. 10 Intraplatelet CAMP determinations were done on I ml platelet-rich plasma to which 1 ml 5% trichloroacetic acid was added before the mixture was immediately frozen in liquid nitrogen. After being thawed at room temperature, platelets were shaken at 4°C for 30 min. The protein precipitate was removed by centrifugation at 2500 g for 30 min. The supernatants were extracted with water-saturated ether, and the residual ether in the aqueous extract was evaporated under nitrogen at 50°C. cAMP was determined by radioimmunoassayll with a commercially available kit (Becton-Dickinson, Novate Milanese, Italy).
Uraemic Patients
Statistical Analysis
(a) 65 chronic uraemic patients, aged 20 - 65 years, had their BT and PCV measured immediately before a routine haemodialysis,-i.e., 68 h after the end of the last dialysis sessions.These patients had been undergoing 12 m2 hours per week haemodialysis in thriceweekly sessions for from 1 month to 8 years. The dialyser used was the coil kidney with ’Cuprophane’ membrane. Blood flow was more than 300 ml/min and water flow was 500 ml/min. The patients were
Data were analysed by the following statistical regression analysis, X2 test, paired Student’st test. 12
Fig. 1 shows the correlation between BT and PCV in 65 chronic uraemic patients investigated before haemodialysis.
19 Lillie
25. Piendes
20
21 22
23 24
RD, Fullmer HM. Histopathologic technique and practical histochemistry. New York: McGraw-Hill, 1976: 534-35. Fleming LW, Stewart WK, Fell GS, Halls DJ. The effect of oral aluminium therapy in patients with chronic renal failure in an area with low water aluminium. Clin Nephrol 1982; 17: 222-27. Stanbury SW. Azotaemic renal osteodystrophy. In: MacIntyre I, ed. Clinics in endocrinology and metabolism. Philadelphia: WB Saunders, 1972: 267-304. Stanbury SW. Vitamin D and the syndromes of azotaemic osteodystrophy. In: Berlyne GM, Giovannetti S, Thomas S, eds. Contributions to nephrology, vol 13. Basel: Karger, 1978: 132-46. Arnaud CD. Hyperparathyroidism in renal failure. Kidney Int 1972; 6: 89-95. Hodsman AB, Sherrard DJ, Wong EGC, et al. Vitamin D-resistant osteomalacia in haemodialysis patients lacking secondary hyperparathyroidism. Ann Intern Med 1981; 94: 629-37.
tests:
linear
Results
26. 27.
28.
29.
AM,
Ellis
HA, Simpson W, Dewar JH,
Ward
MK, Kerr DNS. Variable
response to long term 1-&agr;-hydroxycholecalciferol in haemodialysis osteodystrophy. Lancet 1976; i: 1092-95. Bricker NS, Slatopolsky E, Reiss E, Avioli LV. Calcium, phosphorus, and bone in renal disease and transplantation. Arch Intern Med 1969; 123: 543-53. Felsenfeld AJ, Haurelson JM, Gutman RA, Wells SA, Drezner MK. Osteomalacia after parathyroidectomy in patients with uraemia. Ann Intern Med 1982; 96: 34-39. Teitelbaum SL, Bergfeld MA, Freitag J, Hruska KA, Slatopolsky E. Do parathyroid hormone and 1, 25-dihydroxyvitamin D modulate bone formation in uraemia? J Clin Endocr Metab 1980; 51: 247-51. Frost HM, Griffiths DL, Jee WSS, Kimmel DB, McCandlis RP, Teitelbaum SL. Histomorphometric changes in trabecular bone of renal failure patients treated with calcifediol. Metab Bone Dis Rel Res 1981; 2: 285-95.
HYPERCALCAEMIC OSTEOMALACIA DUE TO ALUMINIUM TOXICITY
G.S. FELL†
B. F. BOYCE* H. Y. ELDER‡ H. L. ELLIOT¶ I. FOGELMAN∥
B. J. JUNOR§ G. BEASTALL† I. T.
BOYLE∥ University Department of *Pathological, †Pathological Biochemistry, and Medicine, Royal Infirmary, Glasgow; and University Departments of ‡Physiology and ¶Materia Medica and §Renal Unit Western Infirmary, Glasgow In 16 patients with chronic renal failure and osteomalacia resistant to vitamin-D therapy, aluminium was demonstrated in bone biopsy specimens at the interface between thickened osteoid and calcified bone by means of both X-ray microanalysis and a specific histochemical stain. 14 patients also had hypercalcaemia. It is suggested that this is due to the blocking by aluminium of calcium uptake into bone coupled with the availability of additional calcium from dialysis fluid and vitamin-D therapy. This study provides more aetiological evidence linking aluminium and the development of osteomalacia in chronic renal failure. Further, if hypercalcaemia develops in such patients it is important that aluminium toxicity be excluded as the cause to prevent unnecessary parathyroidectomy.
Summary
Introduction ALUMINIUM has been implicated in the pathogenesis of a form of osteomalacia occurring in patients with chronic renal failure on regular haemodialysis.I-3 Aluminium-related osteomalacia differs from classical vitamin-D-deficiency osteomalacia in that patients are resistant to treatment with even large doses of vitamin D, have an increased incidence of bone fractures, and are particularly likely to experience bone
3. UCLA Bone Marrow Transplant Team: Bone marrow transplantation in acute leukemia. Lancet 1977; ii: 1197-200. 4. Herzig GP, Bull MI, Decter J, et al. Bone marrow transplantation in leukemia and aplastic anemia: NCI experience with four grafting regimens. Transplant Proc 1975;
pain.4,s The associated biochemical features are normal (or only slightly elevated) serum levels of alkaline phosphatase and parathyroid hormone and a tendency for hypercalcaemia to develop.6 Such patients have high aluminium concentrations in both serum and bone,1,8,9 where it accumulates predominantly at the calcification front between osteoid and calcified matrix.8,9 The source of the aluminium is usually the water-supply used to prepare the dialysis fluid10,11 but in some cases it is related to ingestion of
aluminium-based phosphate-binding drugs. 7,12 In this paper we describe the clinical, biochemical, and bone-histomorphometric findings in 16 patients with aluminium-related osteomalacia and discuss the mechanism which may underlie the development of hypercalcaemia. Patients and Methods
Patients 16 patients (9 males, 7 females, aged 12 to 60 years) with advanced chronic renal failure of various aetiologies were studied. All patients had received regular haemodialysis at home or in hospital for between 3 months and 8 years and had been treated with aluminiumcontaining phosphate-binding drugs. All had histological evidence ’of osteomalacia, and 13 had been unsuccessfully treated with large doses of vitamin D (la-hydroxycholecalciferol [1&agr;-OH D3] in most cases). Of the remaining 3 patients not treated with vitamin D before bone biopsy 2 (nos.4 and 6) were subsequently treated with 1 a-OH D3 without improvement. Thus, 15 patients were considered to have aluminium-related osteomalacia resistant to vitamin D. The remaining patient (no. 13) was never treated with vitamin D, and two subsequent bone biopsies, after withdrawal from exposure to aluminium, showed improvement and eventual resolution of the mineralisation defect. Patients were divided into three groups: Group A. -These 6 patients had the dialysis encephalopathy syndrome.All had received regular haemodialysis with water containing high aluminium concentrations. Group B. -These 6 patients were dialysed with aluminiumcontaminated water, pretreated by reverse osmosis to reduce the
MM, Gale RP, Kay HEM, Rimm AA. Factors associated with interstitial pneumonitis following bone marrow transplantation for acute leukaemia. Lancet
20. Bortin
1981;
5.
leukemia in first remission. N Engl J Med 1979; 301: 597-99. Morgenstern G, Clink HM, et al. The place of bone-marrow transplantation in acute myelogenous leukaemia. Lancet 1980; i: 1047-50. Gale RP. Clinical trials of bone marrow transplantation in leukemia. In: Gale RP, Fox CF (eds). Biology of bone marrow transplantation. New York: Academic Press,
nonlymphoblastic
8. Powles RL, 9
bone
33.
Bortin MM, Gale RP, Kay HEM, Rimm AA. Bone marrow transplantation for acute
34.
19
myelogenous
leukemia: factors associated with
early mortality. JAMA (in press).
press). Lumley HS,
RL, Morgenstern GR, Clink HM. Matched allogeneic sibling transplantation for acute myeloid leukaemia in first remission. Exp Hematol 1982; 10 (suppl 10): 70-71. 23. Zwaan FE, Hermans J. Bone marrow transplantation for leukaemia-European results in 264 patients. Exp Hematol 1982; 10 (suppl 10): 64-69. 24. Gale RP: A prospective controlled trial of bone marrow transplantation vs. chemotherapy in acute myelogenous leukemia. Blood 1981; 58 (suppl 1): 173a (abstract). 25. Zwaan FE, Jansen J, Colpin GGD, Simonis RFA. Marrow grafting for acute leukemia during remission-results in 23 patients. Exp Hematol 1982; 10 (suppl 10): 87 (abstract). 26. Forman SJ, Blume KG, Spruce WE, et al Bone marrow ablation and marrow transplantation in acute leukemia: influence of hematological pre-treatment status. Clin Res 1981; 29: 333a (abstract). 27. Dinsmore R, Shank B, Kapoor N, et al. A randomized trial of marrow transplantation (BMT) versus chemotherapy (CT) maintenance for acute myelogenous leukemia in first remission. Preliminary results. Exp Hematol 1981; 9 (suppl 9): 125 (abstract). 28. Speck B, Gratwohl A, Nissen C, et al. Further experience with cyclosporin-A in allogeneic bone marrow transplantation. Exp Hematol 1981; 9 (suppl 9) 124 (abstract). 29. Armitage J, Klassen L, Kugler J, et al. Allogeneic bone marrow transplantation (BMT) for the treatment of acute leukemia Clin Res 1981; 29: 842a (abstract). 30. Santos GW, Tutschka PJ, Beschorner WE. Marrow transplantation in acute nonlymphocytic leukemia (ANL) following busulfan (BU) and cyclophosphamide (CY). Blood 1981; 58 (suppl 1): 176a (abstract). 31. Mannoni P, Vernant JP, Rodet M et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission. Blut 1980; 41: 220-25. 32. Beutler E, McMillan R, Spruce W. The role of bone marrow transplantation in the 22.
1980: 11-27. KG, Beutler E, Bross KJ, et al. Bone-marrow ablation and allogeneic marrow transplantation in acute leukemia. N Engl J Med 1980; 302: 1041-46. 11. Okuma T, Rosner F, Levy RN, et al. Treatment of adult leukemia with 1-asparaginase (NCS-109229). Cancer Chemother Rep 1971; 55: 269-75. 12. Karnofsky DA, Abelman WH, Craver LF, et al. The use of nitrogen mustard in the palliative treatment of carcinoma. Cancer 1948; 1: 634-656. 13. Report from the ACS/NIH bone marrow transplant registry. Exp Hematol 1975; 3: 149-55. 14 Cutler SJ, Ederer F. Maximum utilization of the life table in analyzing survival J Chron Dis 1958; 8: 699-712. 15 Lee E, Desu M. A computer program for comparing K samples with right-censored data. Comput Programs Biomed 1972; 2: 315-21. 16 Jennrich RI, Moore RH. Maximum likelihood estimation by means of non-linear least squares. In: Proceedings of the statistical section of the American Statistical Association. Washington, D.C., 1975: 57-65. 17 Cox JR. The analysis of binary data. London: Methuen, 1970. 18. Bennett JM, Catovsky D, Daniel M-T, et al. Proposals for the classification of acute leukemias. Br J Haematol 1976; 33: 451-58. 10. Blume
437-39.
ED, Clift RA, Buckner CD. Marrow transplantation for patients with acute nonlymphoblastic leukemia who achieve a first remission. Cancer Treat Rep (in
7: 817-21.
Tutschka PF, Elfenbein GJ, Sensenbrenner LL, et al. Preparative regimens for marrow transplantation in acute leukemia and aplastic anemia. Am J Ped Hematol Oncol 1980; 2: 363-70. 6. Speck B, Cornu P, Nissen D, et al. The Basel experience with total body irradiation for conditioning patients with acute leukemia for allogeneic bone marrow transplantation. Pathol Biol (Paris) 1979; 27: 353-55. 7. Thomas ED, Buckner CD, Clift RA, et al. Marrow transplantation for acute
i:
21. Thomas
Powles
marrow
treatment of acute leukemia
in remission.
Blood 1982; 59: 1115-17.
Ramsay NKC, Kersey JH, Robison LL, et al. A randomized study of the prevention of acute graft-versus-host disease N Engl J Med 1982; 306: 392-97. Bortin MM, Gale RP, Rimm AA. Allogeneic bone marrow transplantation for 144 patients with severe aplastic anemia. JAMA 1981, 245: 1132-39.
1010 aluminium content. However, they became exposed to high concentrations of aluminium after deterioration and malfunction of the reverse-osmosis membranes. Group C. - These 4 patients were dialysed with water containing low concentrations of aluminium (<40 pg/1) but were exposed to aluminium-containing phosphate-binding drugs. An estimate of the total exposure to aluminium from this source had been calculated for all 16 patients from the dose and duration of therapy.
Biochemistry Serum calcium, inorganic phosphate, and alkaline phosphatase were measured with standard laboratory techniques. Immunoreactive parathyroid hormone (iPTH) was measured in plasma with a double-antibody radioimmunoassay which uses an antiserum (BW211/32) able to recognise both ends of the PTH molecule. Aluminium concentrations in serum and water were measured by means of electrothermal atomic-absorption
(AP)
spectrophotometry (ETA-AAS).
13
Bone Biopsies Transiliac bone biopsy specimens (8 mm diameter) were obtained from each patient and fixed in neutral phosphate-buffered formalin. The specimens were dehydrated in graded alcohols and embedded in methylmethacrylate. 10 µm serial sections were cut from each biopsy specimen on a Jung K sledge microtome. Histomorphometry.-Four representative sections from each biopsy specimen were stained (1% aqueous toluidine-blue) for quantitative histology, and the following variables were measured by point counting and line-intersect measurement with a Zeiss II eyepiece graticule: relative osteoid volume (OV),14 maximum number of birefringent lamellae (MNL) of osteoid15 (visualised under polarised light), total osteoid surface (OS),14 and extent of calcification fronts along osteoid seamsl6 were measured to give an indication of the severity of osteomalacia; and active osteoid surfaces14 and total14 and active14 resorption surfaces were measured to give an indication of the degree of secondary
hyperparathyroidism. a’bleasurement of bone aluminium content was measured with ETAAAS on particles of bone weighing 20 to 100 mg obtained from the methacrylate-embedded specimens.8 Localisation of aluminium in bone.-Two methods (electron-probe X-ray microanalysis and histochemical staining) were used to demonstrate the localisation of aluminium in the bone biopsy specimens. Electron-probe X-ray microanalysis17 is an established technique used for the localisation and measurement of elements
within tissue at the ultrastructural level. Ultrathin sections for electron microscopy were prepared from the bone biopsy specimens by means of a technique which we have described previously.s Various sites, including calcified matrix, osteoid, and osteocytes, were examined for the presence of aluminium. The microanalysis was carried out on a JEOL JEM 1 OOC electron microscope with an attached Link Systems 290 microanalysis system. The whole system had been modified for accurate ultrastructural analysis of hard tissues.18 An established histochemical staining method19 using ’Aluminon’ (BDH Chemicals, Poole, England) was used to detect aluminium in 10 µm sections cut as described above on the Jung K microtome.
Results
(<40 µg/1). Serum levels were elevated in all patients (62 - 700 µg/1) but lowest in group C (normal range20 3 - 35 µg/1). Those in group-B patients are the levels measured between 9 and 18 months after the start of home dialysis following the failure of their reverse-osmosis units. Bone aluminium levels were elevated in all cases, ranging from 51 to 275 µg/g dry weight (normal range8 3-23 µg/g, mean ±1SD 10 - .5±4. 2 µg/g). There was obvious overlap in values among the groups, and whereas those in group A appear higher, the numbers are too small and the range is too large for meaningful statistical comparison. The range of values in group B is much narrower than in the other groups (59-120 µg/g), perhaps indicating the shorter period of exposure to high aluminium levels during home dialysis. There is a positive correlation between bone and serum aluminium levels in the 16 patients (r=0’67, p<0.01). Estimated total oral aluminium consumption ranged from 1 -3to 10 -5kg aluminium and correlated significantly with the duration of dialysis (r = 0.86, p<0. 01, n= 16). were
CLINICAL AND BIOCHEMICAL DETAILS OF PATIENTS
NA=not available. *Including hospital and home
(see Table)
Aluminium Data Considerable variation was found from time to time in aluminium levels in water and serum in patients in groups A and B, reflecting the intermittent addition of aluminium salt to clarify water in local reservoirs. The values listed in the table are the highest recorded around the time of bone biopsy. Water aluminium levels were similar in groups A and B (110-1005 µg/1) and were much higher than those in group C
dialysis. Measured before start of dialysis.
1011
Discussion
Other Biochemical Data Serum calcium levels were at or above the upper limit of the normal range in 14 patients and normal or low in 2. These 2 patients had histological evidence of mild-to-moderate secondary hyperparathyroidism, although iPTH levels were elevated in only 1 (patient 11). iPTH levels were increased in 7 out of 13 patients in whom it was measured (610-1500 ng/1), and in some of these parathyroidectomy was contemplated for possible tertiary hyperparathyroidism but not carried out (see Discussion). Alkaline phosphatase was normal or only slightly elevated in most patients. Hyperphosphataemia was present in 11patients and serum levels correlated inversely with total oral aluminium -
consumption (r = -0’ 48, p = 0 - 05, n = 16). Localisation of Aluminium in Bone
in patients with chronic renal failure is "autonomous" to function of one or more of ascribed usually
Hypercalcaemia
parathyroid glands-so-called tertiary hyperparathyroidism.21,22 Bone biopsy in such patients usually reveals greatly increased bone resorption and formation with normal bone mineralisation.22 Circulating iPTH and AP levels are greatly increased.23 In the present study of 16 patients with chronic renal the
.
Aluminium was demonstrated with a specific stain as a bright-red line along most of the length of the osteoid/ calcified-bone interface. Toluidine-blue staining of adjacent sections failed to demonstrate calcification fronts at sites where aluminium was present. A typical example of the staining is seen in fig. 1, which depicts the severe osteomalacia present in patient 16. This localisation was confirmed ultrastructurally in all cases by electron-probe X-ray microanalysis. Linear deposition of aluminium was also noted in stained sections deep within some calcified trabeculae at reversal lines.
failure and histologically proven osteomalacia unrelated to vitamin-D deficiency, 14 had hypercalcaemia at the time of bone biopsy. In all cases, aluminium was demonstrated at the calcification fronts in bone both by histochemistry and by electron-probe X-ray microanalysis and was considered the cause of the mineralisation defect. There was only minimal histological evidence of secondary hyperparathyroidism and, in contrast to the biochemical features of tertiary
Histomorphometry Fig.2 displays the individual values for OV, MNL, OS, and extent
of calcification fronts. Calcification fronts extended
along less than 60% of the total osteoid surface in all cases (range 0-52%), indicating osteomalacia. The variation in
severity of the mineralisation defect is highlighted by the variation in OV and MNL. Whereas some patients had severe osteomalacia (OV >30%, MNL>10) others had mild disease with OV around 100/o and MNL normal (up to 4) or only slightly increased (up to 6). In the majority of cases the thickened osteoid seams had a characteristically focal distribution along bone trabeculae, intervening bone surfaces being fully mineralised or else covered by thin osteoid. Fig.3 shows the active osteoid surfaces and the total and active resorption surfaces in all cases. Most values were within or only slightly above the normal range and as such are indicative of minimal or absent hyperparathyroidism.
Fig. 1-Undecalcified section
of bone from
patient
Fig. in
2-Bone
histomorphometry severity of osteomalacia.
in 16
patients, indicating variation
Bars indicate the upper limit of the normal range for relative osteoid volume, ’ maximum number of lamellae of osteoid (M.N.L.), and total osteoid surface. The lower limit of the normal range for the extent of calcification fronts along total osteoid surfaces is indicated by the bar in the third column.
16 stained for
aluminium. Aluminium is seen as a bright-red line (arrowed) between osteoid (0) and calcified bone (CB). (Reduced by half from x 280.)
Fig. 3-Bone histomorphometry in 16 patients, indicating the degree of secondary hyperparathyroidism. Bars indicate the upper limit of the normal ranges.
1012
hyperparathyroidism, circulating iPTH and AP levels were either normal or only moderately elevated. The association between hypercalcaemia and osteomalacia,6,24,25 without osteitis fibrosa, although uncommon, has previously been reported in chronic renal failure, but the development of hypercalcaemia has not been adequately explained. We believe that hypercalcaemia is due to a combination of factors in which aluminium toxicity has a central role. Initially, patients with chronic renal failure tend to be hypocalcaemic,26 and secondary hyperparathyroidism ensues if an attempt is made to correct the serum calcium by means of the well-recognised effects of PTH on bone and kidney.16 Medical therapy, such as the addition of calcium to the dialysis fluid 21 and the use of vitamin-D metabolites,25 is usually instigated to complement this physiological response. If, however, mineralisation of osteoid is inhibited by the direct toxic effect of aluminium accumulating at the calcification front, available calcium cannot be taken up by bone and hypercalcaemia may develop. A subsequent fall in biologically active PTH secretion in response to this rise in calcium may then result in reduced liberation of calcium from bone, but unless the concentration of calcium in the dialysis bath is lowered and vitamin-D therapy discontinued, hypercalcaemia is likely to persist. Such a mechanism could explain the hypercalcaemia and osteomalacia reported by Hodsman et al.24 in a population of dialysis patients very similar to our own. These authors considered aluminium toxicity unlikely but surprisingly had taken few steps to exclude it. In their study, bone aluminium levels were measured in only 6 of the 19 patients and were found to be elevated, but bone sections were not stained for aluminium and no data on serum or water aluminium concentrations were reported. The failure of parathyroidectomy to reduce the hypercalcaemia in 7 of 9 patients in that study further supports our proposed mechanism. The distinction between tertiary hyperparathyroidism and aluminium-induced osteomalacia is clearly of considerable clinical importance because parathyroidectomy is indicated in the former whereas withdrawal of both aluminium and vitamin-D therapy and lowering of dialysis-bath calcium are more rational treatments of the latter. There have been several reports3,27,28 of osteomalacia developing after parathyroidectomy for what was presumably considered to be tertiary hyperparathyroidism. In most cases appropriate investigations for aluminium toxicity were not carried out. The differentiation between aluminium-induced hypercalcaemia and tertiary hyperparathyroidism is not always straightforward. 4 of our patients, for example, had iPTH levels above 1000 ng/1, and 3 of these were hypercalcaemic. In the presence of hypercalcaemia these iPTH levels are inappropriately high, and in the absence of osteitis fibrosa tertiary hyperparathyroidism seems unlikely. However, the possibility that the two conditions co-exist and that aluminium directly inhibits parathyroid function cannot be excluded. Whatever the explanation, the diagnosis of aluminium-induced osteomalacia will not be clear unless bone biopsy with appropriate analysis for aluminium is carried out. The histological appearances of aluminium-induced osteomalacia may allow differentiation from vitamin-Ddeficiency osteomalacia in many cases. The most striking feature is a tendency for the osteoid to be focally distributed along bone trabeculae, the intervening surfaces being fully mineralised, with little evidence of osteitis fibrosa. The osteoid also tends to vary considerably in thickness. Thus, in some parts of a biopsy specimen osteoid is of normal or only
serum
increased thickness, whereas in other parts greatly thickened seams are present. Nevertheless, specific staining for aluminium should be carried out in all cases of osteomalacia in chronic renal failure before vitamin-D therapy is started. Although contamination of dialysis water remains the most important potential source of aluminium, the prevalence of osteomalacia related causally to oral aluminium therapy remains to be established, since systematic studies of bone biopsies carried out after a minimum of 2-3 years’ therapy have not been reported. Several "new" syndromes, such as vitamin-D-resistant osteomalacia in patients without secondary hyperparathyroidism24 and osteomalacia following parathyroidectomy,27 have been reported and may, in our opinion, be related to aluminium toxicity. This may also explain the poor response to vitamin-D metabolites in the treatment of renal osteomalacia.2s,29 We think that aluminium-induced osteomalacia may be more widespread than has hitherto been recognised, but at the present time facilities for full evaluation of such cases will be limited to relatively few centres. Nevertheless, aluminium toxicity should be excluded in any patient with chronic renal failure in whom either vitamin-D-resistant osteomalacia or hypercalcaemia develops, particularly if parathyroidectomy is being considered.
slightly
We thank Mr J. Byars for excellent technical assistance; Mrs I. Main for typing the manuscript; and Dr I. A. MacDougall, Dr Anna Murphy, and Dr M. J. Boulton-Jones for information relating to their patients. This work was partly supported by grants from Greater Glasgow Health Board Research Support Group and the Scottish Hospital Endowments Research Trust. Correspondence should be addressed to B. F. B. REFERENCES 1. Ellis HA, McCarthy JH, Herrington J. Bone aluminium in haemodialysis patients and in rats injected with aluminium chloride: relationship to impaired bone mineralisation. J Clin Pathol 1979; 32: 832-44. 2. Drueke T. Dialysis osteomalacia and aluminium intoxication Nephron 1980; 26: 207-10. 3. Hodsman AB, Sherrard DJ, Alfrey AC, et al. Bone aluminium and histomorphometric features of renal osteodystrophy. J Clin Endocr Metab 1982; 54: 539-46. 4 Parkinson IS, Ward MK, Feest TG, Fawcett RWP, Kerr DNS. Fracturing dialysis osteodystrophy and dialysis encephalopathy. Lancet 1979; i: 406-09. 5. Pierides AM. Dialysis dementia, osteomalacic fractures and myopathy: a syndrome due to chronic aluminium intoxication. Int J Artif Organs 1978; 1: 841-44. 6. Parkinson IS, Ward MK, Kerr DNS. Dialysis encephalopathy, bone disease and anaemia: the aluminium intoxication syndrome during regular haemodialysis. J Clin Pathol 1981; 34: 1285-94. 7. Ward MK, Feest TG, Ellis HA, Parkinson IS. Osteomalacic dialysis osteodystrophy Evidence for water borne aetiological agent, probably aluminium Lancet 1978, r 841-45. 8. Boyce BF, Elder HY, Fell GS, et al. Quantitation and localisation of aluminium in human cancellous bone in renal osteodystrophy. Scanning Electron Microsc 1981, III: 329-37. 9. Cournot-Witmer G, ZingraffJ, Plachot JJ, et al. Aluminium localisation in bone from haemodialysed patients: Relationship to matrix mineralisation. Kidney Int 1981; 20: 375-85. 10. Platts MM, Goode GC, Hislop JS. Composition of the domestic water supply and the incidence of fractures and encephalopathy in patients on home dialysis Br Med J 1977; ii: 657-60. 11. Elliott HL, Dryburgh F, Fell GS, Sabet S, MacDougall AI. Aluminium toxicity during regular haemodialysis. Br Med J 1978; i: 1101-03 12. Rottembourge J, Jaudon MC, LeGrain M, Galli A. Les gels d’alumine chez les insuffisants renaux chroniques. Un risque potentiel d’encéphalopathie et d’ostéopathie. Ann Méd Intern 1980; 131: 71. 13. Gardiner PHE, Ottaway JM, Fell GS, Halls DJ. Determination of aluminium in blood plasma or serum by electrothermal atomic absorption spectrophotometry Analyt Chim Acta 1981; 128: 57-66. 14. Whyte MP, Bergfeld MA, Murphy WA, Avioli LV, Teitelbaum SL. Postmenopausal osteoporosis. A heterogeneous disorder as assessed by histomorphometric analysis of iliac crest bone from untreated patients. Am J Med 1982, 72: 193-202. 15. Woods CG, Morgan DB, Patterson CR, Grossman HH Measurement of ostesid in bone biopsy. J Pathol Bacteriol 1968; 95: 441-47. 16. Rasmussen H, Bordier P. The physiological and cellular basis of metabolic bone disease Baltimore: Williams & Wilkins Co, 1974; 62-63. 17. Chandler JA. X-ray microanalysis in the electron microscope Amsterdam North Holland Publishing Co, 1977. 18 Nicholson WAP, Gray CC, Chapman JN, Robertson BW Optimising thin film X-ray spectra for quantitative analysis. J Microsc 1982, 125: 25-40.
1013 a stable biochemical state and had not received blood transfusions or any drug known to affect platelet function in the month before the study. Their clinical condition was judged to be good, except for 4 patients who had PCV lower than 20%.
all in
URAEMIC BLEEDING: ROLE OF ANAEMIA AND BENEFICIAL EFFECT OF RED CELL TRANSFUSIONS MANUELA LIVIO DONATELLA MARCHESI GIUSEPPE REMUZZI
(b) 2 uraemic patients with symptom-producing anaemia and very6 long BT were repeatedly transfused with washed packed red cells.
ELIANA GOTTI GIULIANO MECCA GIOVANNI DE GAETANO
PCV and BT were measured in these patients after each transfusion.
(c)
Laboratory of Cardiovascular Clinical Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea, 62-20157 Milan, Italy, and Division of Nephrology, Ospedali Riuniti, 24100 Bergamo, Italy
Bleeding time, altered in most uraemic patients, may be influenced by packed cell volume (PCV). Uraemic patients are often anaemic, so the influence of PCV on bleeding time was assessed in several groups of patients with chronic uraemia. The findings were that bleeding times in uraemic patients are profoundly influenced by anaemia, and that the platelet-mediated haemorrhagic tendency in uraemia may be successfully managed by raising PCV values to above 30%. URAEMIC patients have a poorly understood bleeding tendency, and complex vascular and platelet abnormalities 1-3 have been reported in these patients. However, no correlation has been found between different abnormalities revealed by in-vitro tests and the ex-vivo variable believed to reflect the patient’s clinical state most closely, namely, bleeding time (BT). This test has been said to be the best marker for
haemorrhagic risk.4 An observation made by Hellem et a1.5 in the early sixties which has not been paid much attention suggested that BT was prolonged in anaemic patients and could be restored to normal by transfusion of washed red cells. Since uraemic patients are often anaemic, we undertook the present study to establish: (1) the influence of packed cell volume (PCV) on BT in’uraemic patients; (2) the relationship between BT and PCV in uraemic and anaemic patients with similar PCV values; and (3) the possible "haemostatic effect" of red-cell transfusion in uraemic bleeding. Patients and Methods
were
infused with
30%.
Summary
Introduction
6 additional patients with PCV of 16-21%
washed, filtered red cells.6 BT, platelet count, platelet retention on glass beads, prothrombin consumption, platelet cyclic-adenosinemonophosphate (cAMP), and serum thromboxane B2 (TxB2) were measured before transfusion and again when PCV values rose above Control
Groups
(a) 65 normal subjects matched for age and uraemic patients.
sex
with the group of
(b) 15 patients with anaemias unrelated to the uraemia (11with hypochromic anaemia and 4 with normochromic anaemia) were compared with 15 patients randomly selected from the 65 chronic uraemic patients and matched for age, sex, and PCV values.
Laboratory Tests PCV was measured by a routine micromethod. Platelets were counted by phase-contrast microscopy. BT was determined in duplicate by a template-like semiautomatic device.44 Platelet adhesion to glass beads was measured in native blood by a modification of Hellem’s methodwith standardised glass-beadfilled columns and a constant rate (1 ml blood/ 15s) infusion pump (Adeplate’s and Adeplate’s Pump System, Mascia Brunelli, Milan,
Italy).44
Prothrombin consumption was determined as a measure of platelet factor 3 availability by Quick’s method.8 Serum TxB2 production was measured by radioimmunoassay99 after blood clotting in vitro. 10 Intraplatelet CAMP determinations were done on I ml platelet-rich plasma to which 1 ml 5% trichloroacetic acid was added before the mixture was immediately frozen in liquid nitrogen. After being thawed at room temperature, platelets were shaken at 4°C for 30 min. The protein precipitate was removed by centrifugation at 2500 g for 30 min. The supernatants were extracted with water-saturated ether, and the residual ether in the aqueous extract was evaporated under nitrogen at 50°C. cAMP was determined by radioimmunoassayll with a commercially available kit (Becton-Dickinson, Novate Milanese, Italy).
Uraemic Patients
Statistical Analysis
(a) 65 chronic uraemic patients, aged 20 - 65 years, had their BT and PCV measured immediately before a routine haemodialysis,-i.e., 68 h after the end of the last dialysis sessions.These patients had been undergoing 12 m2 hours per week haemodialysis in thriceweekly sessions for from 1 month to 8 years. The dialyser used was the coil kidney with ’Cuprophane’ membrane. Blood flow was more than 300 ml/min and water flow was 500 ml/min. The patients were
Data were analysed by the following statistical regression analysis, X2 test, paired Student’st test. 12
Fig. 1 shows the correlation between BT and PCV in 65 chronic uraemic patients investigated before haemodialysis.
19 Lillie
25. Piendes
20
21 22
23 24
RD, Fullmer HM. Histopathologic technique and practical histochemistry. New York: McGraw-Hill, 1976: 534-35. Fleming LW, Stewart WK, Fell GS, Halls DJ. The effect of oral aluminium therapy in patients with chronic renal failure in an area with low water aluminium. Clin Nephrol 1982; 17: 222-27. Stanbury SW. Azotaemic renal osteodystrophy. In: MacIntyre I, ed. Clinics in endocrinology and metabolism. Philadelphia: WB Saunders, 1972: 267-304. Stanbury SW. Vitamin D and the syndromes of azotaemic osteodystrophy. In: Berlyne GM, Giovannetti S, Thomas S, eds. Contributions to nephrology, vol 13. Basel: Karger, 1978: 132-46. Arnaud CD. Hyperparathyroidism in renal failure. Kidney Int 1972; 6: 89-95. Hodsman AB, Sherrard DJ, Wong EGC, et al. Vitamin D-resistant osteomalacia in haemodialysis patients lacking secondary hyperparathyroidism. Ann Intern Med 1981; 94: 629-37.
tests:
linear
Results
26. 27.
28.
29.
AM,
Ellis
HA, Simpson W, Dewar JH,
Ward
MK, Kerr DNS. Variable
response to long term 1-&agr;-hydroxycholecalciferol in haemodialysis osteodystrophy. Lancet 1976; i: 1092-95. Bricker NS, Slatopolsky E, Reiss E, Avioli LV. Calcium, phosphorus, and bone in renal disease and transplantation. Arch Intern Med 1969; 123: 543-53. Felsenfeld AJ, Haurelson JM, Gutman RA, Wells SA, Drezner MK. Osteomalacia after parathyroidectomy in patients with uraemia. Ann Intern Med 1982; 96: 34-39. Teitelbaum SL, Bergfeld MA, Freitag J, Hruska KA, Slatopolsky E. Do parathyroid hormone and 1, 25-dihydroxyvitamin D modulate bone formation in uraemia? J Clin Endocr Metab 1980; 51: 247-51. Frost HM, Griffiths DL, Jee WSS, Kimmel DB, McCandlis RP, Teitelbaum SL. Histomorphometric changes in trabecular bone of renal failure patients treated with calcifediol. Metab Bone Dis Rel Res 1981; 2: 285-95.