J Clin Pathol 1982;35:1283-1293

Localisation of aluminium by histochemical andelectron probe x-ray microanalytical techniques inbone tissue of cases of renal osteodystrophyPS SMITH, J McCLURE

From the Adelaide Bone and Joint Research Unit and the Division of Tissue Pathology, Institute ofMedical andVeterinary Science, Frome Road, Adelaide, South Australia

SUMMARY Histochemical studies and electron probe x-ray microanalysis for aluminium havebeen performed on 16 samples of undecalcified bone from cases of renal osteodystrophy associ-ated with a syndrome suggestive of dialysis encephalopathy (five cases), age and sex matchedcontrols for these and a group of patients with chronic renal failure (six cases) who have never

been on haemodialysis. Aluminium was detected only in the patients with a dialysisencephalopathy-like syndrome. This group had significant histological bone disease the features ofwhich were broadly consistent with the so-called atypical renal osteomalacia which is thought to bedue to a metal toxin. Aluminium was demonstrated at the interface between osteoid andmineralised tissue-that is, at the site of the calcification front, where it could interfere with themineralisation process. In the group of patients who had never been subjected to haemodialysisthere was also significant histological bone disease but no evidence of aluminium accumulation. Inthis group the bone disease was of a more typical pattern of osteomalacic changes coupled withthose of hyperparathyroidism.

Berlyne et all reported that hyperaluminaemiaoccurred in patients with advanced renal failure andsuggested that the sources of aluminium were thedialysis fluid and orally consumed aluminium contain-ing ion exchange resins and aluminium hydroxideused therapeutically to control hyperkalaemia andhyperphosphataemia respectively. More recentlyKaehny et at2 have demonstrated significant transfersof aluminium from dialysate to patient duringhaemodialysis. The importance of aluminium trans-fer to and accumulation in patients on haemodialysisis illustrated by the work of Alfrey et at3 who suggestthat aluminium intoxication is responsible for thedialysis encephalopathy syndrome.

It has also been demonstrated by Parsons et at4that bone samples from patients treated with inter-mittent haemodialysis have high aluminium concen-trations. Platts et at5 found that patients undergoinghome dialysis in the Trent region who developeddialysis encephalopathy and suffered spontaneousfractures were being dialysed against water whichhad a higher concentration of aluminium than those

Accepted for publication 16 March 1982

who did not develop these complications.Ward et at6 found that osteomalacia was present in

15% of patients dialysed against deionised water andin 70% of patients dialysed against softened waterfrom the same original source in the Newcastle-upon-Tyne area. The aluminium content of thissource was high. Serum aluminium concentrationswere higher in patients using softened water thanthose using deionised water and it was found thatdeionisation diminished the water aluminium contentto a greater extent than the softening process. A latersurvey by the Newcastle group of 1293 patientsbeing treated in 18 dialysis centres in Great Britainrevealed a highly significant rank correlation of theincidence of both fracturing osteomalacic dialysisosteodystrophy and dialysis encephalopathy with thealuminium content of the water used to prepare thedialysate.7

All this adds up to a substantial body of evidencesuggesting a link between aluminium accumulationand the development of dialysis osteodystrophy. Yetthe nature of this evidence is still circumstantial andthere is no proven causal relation between thedevelopment of osteomalacia and increased concen-trations of aluminium in blood or bone. Thus Ellis et

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al8 found no significant relation between the pres-ence or severity of osteomalacia and the bonealuminium content of 34 patients with chronic renalfailure. These workers were unable to demonstratethe precise localisation of aluminium in undecalcifiedbone blocks either by Stereoscan scanning electronmicroscopy or by histochemical means and theyattributed this failure to the low concentrations ofaluminium involved.

It is of course of prime importance to determinethe localisation of accumulated aluminium in thebones of renal failure patients on haemodialysis tosee if it is capable of exerting an inhibitory effect onthe mineralisation process. Towards this end unde-calcified bone tissue from a total of 16 patients wasstudied by a number of available histochemical tech-niques and by electron probe x-ray microanalysis todetermine the presence or absence of aluminium andits localisation if present.

Material and methods

The studied specimens were derived from threegroups of patients. The first group (of five indi-viduals) consisted of patients in chronic renal failurewho had been treated by haemodialysis and who haddeveloped a syndrome suggestive of dialysisencephalopathy. The second group (again of fiveindividuals) consisted of age and sex matched con-trols. These were patients in whom bone biopsieshad been performed and in whom there was no evi-dence of renal failure by clinical and biochemicalassessment. The third group (of six individuals) alsohad renal failure and had never been treated byhaemodialysis. Members of this group were not ageor sex matched against the first group. Clinical dataon these patients are given in Tables 1, 2, and 3.

In each instance cores of bone were taken fromthe left anterior iliac crest from just behind the

Table 1 Clinical and biochemical details ofpatients in group 1 who had chronic renal failure on haemodialysis and in whomthere was evidence ofdialysis encephalopathy

Case Age Sex Cause ofrenal failure Duration of Renal status at time ofbone biopsy Evidence ofencephalopathy(yr) haemodialysis

1 63 F Chronic 42 months Creatinine 0-92 mmol/l (NRO-05 -> 0.12) Hallucinations, decreasedglomerulonephritis. Urea 31-8 mmol/l (NR3.0 -> 8-0) consciousness, CT scanAnti-GBM antibodies Calcium 2.70 mmol/I (NR2.2 -> 2.55) suggested cerebral atrophy.

Phosphate 1-86 mmol/l (NRO-70 -> 1.25)Alkaline phosphatase 78 U/I (NR30-1 10)Albumin 37 g/l (NR34-> 45)

2 59 M Congenital absence of 24 months Creatinine 1-03 mmol/l (NRO-05 -> 0.12) "Shakes" afterright kidney. Urea 26.8 mmolIl (NR3.0.* 8.0) haemodialysis accompaniedPolycystic left kidney Calcium 2-48 mmolfl (NR2.2 -> 2.55) by slurring of speech and

Phosphate 1-31 mmol/l (NRO.70 - 1.25) short term memory loss.Alkaline phosphatase 50 U/l (NR30-110) Marked intention tremor.Albumin 40 g/l (NR34-> 45)

3 55 F Analgesic 20 months Creatinine 1-04 mmol/l(NRO-05 -> 0.12) Shaking of hands andnephropathy Urea 45-2 mmol/l (NR3-0-a 8-0) aphasia during

Calcium 2-38 mmol/l (NR2-20 2.55) haemodialysis. EEGPhosphate 2-48 mmol/l (NRO-70 -> 1.25) showed slow a waveAlkaline phosphatase 166 U/l (NR30-1 10) activity. Grand mal fits.Albumin 39 g/l (NR 34* 45)

4 52 F Reflux nephropathy 41 months Creatinine 1-02 mmol/l(NRO-05 -> 0.12) Grand mal epileptiform fits.Urea 33-3 mmol/l (NR3.0*. 8-0) EEG showed "generalisedCalcium 2.78 mmol/l (NR2.20 -> 2.55) abnormality". Unable toPhosphate 1-32 mmol/l (NRO-70 -> 1.25) follow complex commands.Alkaline phosphatase 161 U/1 (NR30-1 10) Expressive dysphasia afterAlbumin 43 g/l (NR34 -> 45) haemodialysLs. Memory

impairment. History ofmanic depressive psychosis.

5 48 M Chronic 48 months Creatinine 0-74 mmol/l (NRO-05 -> 0-12) Grand mal epileptiform fits.glomerulonephritis Urea 22-6 mmol/l (NR3-0 -> 8-0) EEG showed generalisedand malignant Calcium 2-4 mmol/l (NR2-20 -> 2.55) abnormality which washypertension Phosphate 1-97 mmol/l (NRO70 -> 1-25) more marked after

Alkaline phosphatase 205 U/1 (NR30-1 10) haemodialysis. Slurring ofAlbumin 36 g/l (NR34 -> 45) speech, memory

impairment and myoclonus.History of manic depressivepsychosis.

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Table 2 Clinical and biochemical details ofthe age and sex matched controlsfor group I

Case Age Sex Clinical history Assessment ofrenalfunction(yr)

1 63 F Six months previously she had a fall and sustained a Creatinine 0.10 mmol/l (NR0-05 -> 0-12)crush fracture of Ti 1, T12. Investigated for Urea 6-0 mmolfl (NR3.0-> 8-0)osteoporosis. Calcium 2-45 mmol/l (NR2.20 -> 2.55)PMH: nil of significance. Phosphate 0-68 mmol/l (NRO-7 -> 1-25)

Albumin 42 g/l (NR39 -> 48)

2 59 M Investigated for episodic mild hypercalcaemia. Creatinine 0-09 mmol/l (NRO05-OS 0-12)Thyroid function normal. Serum parathormone level Urea 4-70 mmol/l (NR3-0-> 8-0)normal. Calcium 2-33 mmol/l (NR2.20-> 2.55)PMH: three proven myocardial infarcts. Phosphate 0-86 mmol/l (NRO-7 -v 1.25)

Albumin 41 g/l (NR39-> 48)Creatinine clearance 101 ml/min (NR90-v 180)24-hour urine volume 2150 mlUrinary creatinine 13-1 mmol/24h (NR8-8 + 23.0)

3 55 F Fractured two right metacarpals after a fall. Creatinine 0.07 mmol/l (NRO-05 * 0-12)Investigated for osteoporosis. Urea 5-1 mmol/l (NR3-0 8-0)PMH: nil of significance. Calcium 2-3 mmol/l (NR2-20.* 2.55)

Phosphate 1*02 mmol/l (NR0-7-> 1.25)Albumin 40 g/l (NR39 -v 48)

4 52 F Right colles fracture after a fall. Investigated for Creatinine 0-06 mmol/l (NRO.05-O 0-12)osteoporosis. Urea 6-0 mmol/l (NR3.0-> 8-0)PMH: nil of significance. Calcium 2-40 mmol/l (NR2-20-> 2.55)

Phosphate 0-72 mmol/l (NRO-7 -v 1.25)Albumin 43 g/l (NR39 -> 48)

5 48 M Reformed alcoholic who fell and sustained a crush Creatinine 0-07 mmolfl (NRO.05-O 0-12)fracture of T12. Investigated for metabolic bone Urea 4-5 mmol/l (NR3-0-> 8-0)disease. Calcium 2-33 mmol/l (NR2.20-> 2.55)

Phosphate 1-2 mmol/l (NR0-7-> 1.25)Albumin 39 g/l (NR39-> 48)Creatinine 11-8 mmol/24 h (NR8.8-> 23-0)Serum 25 hydroxycholecalciferol 87 mmol/l (NR15 -> 90)

anterior superior spine and through the top of thecrest passing down between outer and inner cortices.The specimens were processed and embedded inaraldite. Thin (7 ,.m) sections were cut on a Jung Kmicrotome without prior decalcification. Multiplesections were cut and stained by the von Kossamethod with various counterstains includinghaematoxylin and eosin and the van Gieson method.Calcification fronts were stained with toluidine blueand with haematoxylin as suggested by Raina.9 Inthree cases (in group 3) tetracycline had beenadministered in vivo and the calcification fronts weredetermined by ultraviolet light fluorescence. Therewas a high degree of correlation between the resultsof this technique and staining with either toluidineblue or haematoxylin.

Quantitative assessments of several parameterswere made in each case. These assessments wererestricted to cancellous bone. The total cancellousbone volume, the osteoid volume and the percentageof the total bone which was mineralised were esti-mated from multiple sections (treated by the vonKossa technique and the van Gieson counterstain)using the Quantimet image analysis computer. Thepercentage of the trabecular surface covered byosteoid was estimated by a line-intersect technique

using an eyepiece graticule. Calcification fronts weredetermined by a similar technique and expressed asthe percentage of osteoid seams bearing a calcifica-tion front. An assessment of the thickness of theosteoid seams was made by inspecting the seams inpolarised light and determining the maximumnumber of birefringent lamellae present. Totalresorbing or crenated surfaces were expressed as thepercentage of the total trabecular surfaces occupiedby these surfaces. A distinction was also made be-tween active resorbing surfaces and inactive resorb-ing surfaces in that the former were occupied byosteoclasts and the latter not occupied by these cells.

Sections from each case were stained by threeseparate histochemical methods which may be usedto demonstrate the presence of aluminium salts.These methods were obtained from Pearse10 andwere (i) the ammonium aurintricarboxylate (alumi-non) method (ii) the solochrome azurine method(with and without alkali pretreatment) and (iii) theacid solochrome cyanine method. The latter twomethods were used to stain one section from eachcase.

After sections had been obtained for histologicaland histochemical studies the surface of each Aralditeblock was polished to within 1 ,um using diamond

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paste, coated with carbon and analysed with a Jeol733 Superprobe scanning electron microanalyser.This instrument had both energy dispersive andwavelength dispersive x-ray analytical capabilities.Initially studies were performed using the OrtecEEDS-II energy dispersive x-ray analysis system butbecause of negative results obtained with this system

the wavelength dispersive x-ray analysis system waseventually used to perform the complete analyticalstudy.

This involved examining selected areas at a stan-dard magnification and photographing the secondaryelectron images. These areas were analysed for cal-cium and aluminium x-ray emissions. Linear focusing

Table 3 Clinical and biochemical details ofpatients in group 3 who had never been treated by haemodialysis

Case Age Sex Cause(s) ofrenalfailure Renalfunction at the time ofbiopsy(yr)

1 53 F Hypertension. Duration of renal failure approximately Creatinine 0-70 mmol/l (NRO05 -- 0-12)10 yr Urea 32-2 mmolAl (NR3.0.* 7-6)

Calcium 2-53 mmolAl (NR2.10-> 2 60)Phosphate 1-63 mmolfl (NRO-7 - 1.30)Alkaline phosphatase 148 U/I (NR30 -v 110)Albumin 40 g/l (NR35 -+- 50)

2 49 M Nephrolithiasis. Gout. Hypertension. Duration of Creatinine 0.37 mmol (NRO-05 0-012)renal failure approximately 8 yr Urea 16-3 mmol/l (NR3.0-> 7-6)

Calcium 1-18 mmol/l (NR2-10- 2-60)Phosphate 1-38 mmol/l (NRO-7 v 1-30)Alkaline phosphatase 333 U/l (NR30O- 110)Albumin 37 g/l (NR35 -v 50)

3 50 M Polycystic kidneys. Duration of renal failure Creatinine 1.40 mmol/l (NRO.05-O 0-12)approximately 10 yr Urea 54-9 mmol/l (NR3-0 - 7-6)

Calcium 2-36 mmol/l (NR2.10- 2-60)Phosphate 1-79 mmol/l (NRO-7 -> 1.30)Alkaline phosphatase 60 U/l (NR30 -> 110)Albumin 40 g/l (NR35 -v 50)

4 15 M Reflux nephropathy, fashioning of ileal loop, recurrent Creatinine 0-72 mmol/l (NRO-05 -v 0-12)urinary tract infections. Duration of renal failure Urea 51-7 mmol/l (NR3.0 -> 7-6)approximately 15 yr Calcium 1-91 mmol/l (NR2.10- 2.60)

Phosphate 1 38 mmol/l (NRO 7 -v 1-30)Alkaline phosphatase 417 U/I (NR30-> 110)Albumin 37 g/l (NR35 -> 50)

5 30 F Reflux nephropathy, megaureters, scarred cotracted Creatinine 0-45 mmol/l (NRO-05 -> 0-12)kidney, recurrent urinary tract infections. Duration Urea 34-6 mmol/l (NR3.0 - 7-6)approximately 30 yr Calcium 2-56 mmolAl (NR2.10* 2.60)

Phosphate 2-03 mmol/l (NRO.7 v 1.30)Alkaline phosphatase 447 U/l (NR30 - 110)Albumin 29 g/l (NR35 -> 50)

6 53 F Analgesic nephropathy. Duration of renal failure Creatinine 0-66 mmolAl (NRO-05 -v 0.12)approximately 6 yr Urea 32-9 mmol/l (NR3-0 -> 7-6)

Calcium 1 99 mmolAl (NR2.10* 2 60)Phosphate 2-06 mmol (NRO-7-* 1.30)Alkaline phosphatase 228 U/l (NR30 - 110)Albumin 36 g/l (NR35 -> 50)

Table 4 Mean values and standard error of the mean (SEM) of histoquantitative parameters of each of the three study groups. ThUsing Student's t test a comparison has been made between the means of each group for each parameter. A significanc

TBV OV %M OS

Normal range <50 yr 18-28% <0-2% >99% <50 yr 5-20%350 yr 12-18% ¢50 yr 5-40%

Group 1 17-7 3-4 82-8 73-6(I) SEM= (1-8) (1-0) (4-8) (9-0)Group 2 13-9 0-2 98-4 20-8(II) (2-6) (0.1) (0-6) (8-0)Group 3 31-5 3-4 90-0 76-5(III) ( 1-4) ( 1-4) (4-4) (5 *7)Comparison of group means I v II-NS I > II (p < 0.006) I < II (p < 0.007) I > II (p < 0.002)

I < III (p < 0.0001) I v III-NS I v III-NS I v III-NSII < III (p < 0-0001) II < III (p < 0.04) II v III-NS II < III (p < 0.000"

TBV = total bone volume; OV = osteoid volume; %M = percentage mineralisation; OS = percentage of trabecular surface covered by osteoid; CF percentage of osteoid sealresorbing surtace.

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Bragg crystal spectrometers with PET and TAPcrystals were used for x-ray detection using calciumphosphate and aluminium oxide as standards. Thehigh beam currents of 120 nA at 15 kV used todemonstrate aluminium in the tissues severely dam-aged the softer components (osteoid and marrow tis-sues) of the analysed area, but left the mineralisedbone intact. Three areas from each patient were

analysed in this way and identical beam currentswere used for each analysis.

Results

The results of the histoquantitative assessments are

detailed in Table 4. Both in group 1 (Fig. 1) andgroup 3 (Fig. 2) there was significant bone disease. In

Fig. 1 Case 1, group 1. This undecalcifiedbone trabecular structure has a thickenedosteoid seam (0). This is observed in polarisedlight to demonstrate birefringent parallellamellae. von Kossa and haematoxylin andeosin x 200.

both groups there was an increased osteoid volume,diminution in the degree of mineralisation, an

increase in the extent of the trabecular surfacecovered by osteoid, increased osteoid seam thicknessand a decrease in the extent of the calcificationfronts. There were differences in the degree ofchange in these parameters. Although in two cases ofgroup 1 there was increased resorptive activity, mani-fest as increased surface resorption and trabecular"tunnelling" (Fig. 3), the mean total resorbing sur-

face extent for the group was not abnormally high.Resorptive activity was a much more prominent fea-ture of group 3. In the only case in which the totalresorbing surface was not raised the active resorbing

.% ....

Fig. 2 Undecalcifled bone section from case 2,

group 3. Thickened, extensive osteoid seams (0)

cover the black mineralised tissue. von Kossa and

haematoxylin and eosin x 200.

2ormal range is that usedfor the population ofthe Adelaide area.evel ofp < 0 05 is used and the results are included in the Table (NS = not significant)

'F MOL AR IR TR

*70% <4 <1-5% <15% <16-5%

0-5 8 2-4 12-0 14-47-0) (2-3) (1-1) (1.7) (2-7)2-3 1-6 0-5 9-4 9-9)-8) (0-2) (0-5) (3-8) (4-0)1-9 2-8 3-4 14-9 18-33-6) (0-4) (0-8) (3-3) (3-7)< II (p < 0-0003) I > II (p < 0-02) I > II (p < 0.05) I v II-NS I v II-NSv III-NS I > III (p < 0.02) I v III-NS I v II-NS IvIII-NSI > III (p < 0.006) II < III (p < 0-02) II < III (p < 0-01) II v III-NS II v III-NS

.earing a calcification front; MOL = maximum number of birefringent lamellae in osteoid seams; AR = active resorbing surface; IR = inactive resorbing surface; TR = total

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Fig. 4 In this samplefrom case 2, group 3 there is excessosteoid (0). There is a deep excavation (E) ofthe trabecularsubstance by osteoclasts. von Kossa and haematoxylin andeosin x 200.

Fig. 3 The trabecular surface is covered by osteoid(0) in this section from case 1, group 1. There istrabecular "tunnelling" (T) by osteoclasts. von Kossaand haematoxylin and eosin x 200.

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Localisation of aluminium by histochemical and electron probe x-ray microanalytical techniques

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Fig. 6 The margin ofa trabecular structure is shown. There is a thin osteoidseam (0) and a linear positive aluminon reaction (I). Mineralised tissue is markedM (case 1, group 1). Aluminon x 400.

surface value was at the limit of normality and histo-logically trabecular excavations were observed(Fig. 4).

Positive histochemical staining reactions wereobserved with the three techniques listed above in allsections studied from the cases in group I (Figs. 5and 6). The ammonium aurintricarboxylate methodwas found to be the most useful and numerous sec-tions were stained by this technique which has nowbeen adopted as a routine test. Negative stainingreactions were obtained with the three techniques inall sections studied from the cases of groups 2 and 3.

Positive reactions were manifest as a linearcolouration at the junction of osteoid andmineralised tissue-that is, in the region of the cal-cification front. These reactions were very extensivein all five cases and were present at the osteoid/mineralised tissue junctions of thick and thin osteoidseams. Positive reactions were not obtained at thesurface of the osteoid seams (marrow/osteoid junc-tions) or in the mineralised tissue. Examination ofthe sections in polarised light rendered the osteoidbirefringent and emphasised the linear positivealuminon reaction. Positive reactions were not con-fined to the cancellous bone being present in corticalbone at the osteoid/mineralised tissue interfacearound Haversian canals.The secondary electron images (Figs. 7a and 8a)

clearly distinguish mineralised tissue, osteoid andmarrow tissue and the junctional zones of these com-ponents. Mineralised tissue had a homogeneous lightgrey appearance and the osteoid was a darker greyusually with an array of parallel lamellae. The dis-tribution of calcium determined by x-ray analysis atthe same magnification precisely matched themineralised bone area thus confirming the reality of

the latter and by implication the reality of the osteoid(Figs. 7b and 8b). The aluminium x-ray distributionswere such that in group 1 cases there was an accumu-lation of this element at the osteoid/mineralised tis-sue junction and these distributions were thereforedisposed in a linear fashion (Figs. 7c and 8c). Linearx-ray distributions for aluminium were not found inany of the areas studied in any of the cases in groups2 and 3.

In group 1 cases a very faint aluminium x-ray dis-tribution was seen in both mineralised bone and themarrow tissue, the concentration appearing to behigher in the former.

Discussion

The histochemical reaction used to demonstratealuminium in the majority of the tissue sectionsstudied was the aluminon method which was origi-nally devised by Irwin."1 Aluminon is a dye(ammonium aurintricarboxylate) which forms a lakewith a number of metallic ions under appropriateconditions. The lake is a complex precipitate formedby the dye molecule reacting with the particularmetallic ion. The formation of lakes by aluminonwith the hydroxides or basic acetates of aluminium,beryllium, iron, thorium and the rare earths has beenreported by Middleton.12 Most of these lakes, withthe exception of those formed by aluminium andberyllium are dissolved or decolorised by a solutioncontaining ammonium ion buffered at a pH of 7-2.The beryllium lake is unstable in the absence ofexcess dye. Under the conditions of the Irwin tech-nique the lake formed, which has a cherry-red/purplecolour, is considered specific for the demonstration

1 289

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Smith, McClure

Fig. 7 Electron probe analysis ofcase 2, group 1.(a) secondary electron image demonstratingmarrow tissue (M), wide osteoid seams (0) andmineralised tissue (B) x 1200. (b) x-raydistribution ofcalcium (C) in the same area. Thisdistribution corresponds exactly to the mineralisedtissue x 1200. (c) x-ray distribution ofaluminium indicating the high abundances alongthe osteoid/mineralised tissue junction (arrows)x 1200.

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Localisation of aluminium by histochemical and electron probe x-ray microanalytical techniques

Fig. 8 Electron probe analysis ofcase 3,group 1. (a) secondary electron imagedemonstrating marrow tissue (M), wideosteoid seams (0) and mineralised tissue(B) x 1200. (b) x-ray distribution ofcalcium (C) in this area x 1200. (c) x-raydistribution ofaluminium again indicatinghigh abundances along theosteoid/mineralised tissue junction(arrows) x 1200.

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of the presence of aluminium.This specificity was tested by the use of electron

probe x-ray microanalysis. Initially studies were per-formed with the Ortec EEDS-II energy dispersivesystem but aluminium could not be demonstrated inhistochemically positive cases. Bearing in mind thatEllis et al8 were unable to demonstrate aluminium byStereoscan scanning electron microscopy it was con-cluded that the energy dispersive system lacked therequired sensitivity to demonstrate aluminium sincethis was probably present in low concentration.Assays for aluminium have not been performed onour material but in the cases studied by Ellis et al8 theconcentration in haemodialysed patients was of theorder of 150 parts per million. It was thereforedecided to use the more sensitive wavelength disper-sive system and by using high beam currents positiveresults were obtained and satisfactory photographsobtained by using prolonged exposure times.

Both histochemical and electron probe techniquesdemonstrated the localisation of aluminium at thejunction of osteoid and mineralised tissue-that is, atthe site of the calcification fronts. In the x-ray analyt-ical images very faint dispositions were also noted inmineralised tissue and marrow (the latter less thanthe former). These are regarded as backgroundemissions although the slightly higher emissionsnoted in the mineralised tissues might indicate thepresence of aluminium in very low concentrationsnot detectable by the Irwin technique. However, itmust be emphasised that the vast bulk of the x-rayemissions were at the osteoid/mineralised tissueinterface thereby confirming the selectivity of theIrwin technique and suggesting a very high degree ofsensitivity.

The patients in group 1 were chosen for studybecause they had a clinical syndrome suggestive ofdialysis encephalopathy. The evidence for this ispresented in Table 1. The diagnosis might be con-sidered doubtful in patients 4 and 5 in view of thepsychiatric history in these two cases. It will be seenfrom Table 4 that in group I there was significantbone disease characterised by hyperosteoidosis andreduced calcification fronts. Group 2 represent ageand sex matched controls for group 1. In this groupare patients who had been investigated for suspectedmetabolic bone disease. There was no evidence ofosteomalacia and clinical and biochemical studiesspecifically excluded renal dysfunction in these cases.These patients were used as controls in preference toavailable necropsy material because their renal func-tion status at the time of bone biopsy was accuratelyknown (Table 2). In addition an identical volume oftissue was taken from the same skeletal site and pro-cessed in an identical way in both groups (this alsoapplies to group 3 biopsies).

It was decided to examine the tissues of patientsincluded in group 3 because these patients (althoughsuffering from chronic renal failure) had never beentreated by haemodialysis or any form of dialysis. Theavailability of biopsy material from such cases isextremely limited and it was not therefore possible tomatch them precisely for age and sex with groups 1and 2. Their mean age of 41.6 yr is less than that ofgroups 1 and 2 (55.4 yr) and this due to the inclusionof a 15-year-old boy and a 30-year-old woman in thegroup. Their clinical data are presented in Table 3. Itis evident from Table 4 that these patients also hadsignificant histological bone disease. Their meanosteoid volume was the same as that of group 1. Thiswas a coincidence since the cases were not selectedto obtain this parity. The histological pattern of thebone disease was hyperosteoidosis with diminishedcalcification fronts and increased resorptive activity.In contrast to groups 1 and 2 the mean total bonevolume was higher in group 3. Their calcificationfronts were also diminished but not to the sameextent as group 1. A major difference was thatresorptive activity was greater in group 3 than group1.Aluminium was demonstrated only in group 1

cases and these patients had been treated by haemo-dialysis (mean duration = 35 months). The dialysateused contained water derived from the Adelaidedomestic water supply which, during the period oftreatment of patients in group 1, had an aluminiumcontent ranging from 1.23 mg/l to 1-00 mg/l (sincethat time active steps have been taken to reduce thealuminium content of the tap water and dialysate sothat the concentration in the latter is now 0*005 to0*01 mg/l).The fact that aluminium was not demonstrated in

group 3 (five of whose patients had been taking oralaluminium hydroxide as a phosphate-binding agent)indicates that the aluminium burden of group 1 isdictated by haemodialysis using a dialysate with ahigh aluminium content. The localisation ofaluminium at the calcification front could result in aninterference with the mineralisation process bymechanisms which at this stage must remain specula-tive but perhaps involving the preferential formationof aluminium phosphates. Aluminium was notdemonstrated in the tissues of patients in group 3who had never been treated by haemodialysis. Thesepatients exhibited significant histological bone dis-ease and the absence of aluminium indicates that thiselement does not play a role in the causation of theirrenal osteodystrophy.

Cochran13 has distinguished between typical (orclassical) and atypical renal osteomalacia and sug-gests that the latter is a consequence of aluminiumtoxicity. In typical renal osteomalacia plasma calcium

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Localisation of aluminium by histochemical and electron probe x-ray microanalytical techniques

is often low, phosphate concentration is normal orraised, alkaline phosphatase activity is raised, andhistological features include osteosclerosis andhyperparathyroidism. The majority of the patients ingroup 3 fit well into this category.

In atypical renal osteomalacia, plasma calcium isoften high, phosphate is normal or raised andalkaline phosphatase activity is normal. Spontaneousfractures occur and there is failure of response tovitamin D or its active metabolites. Histologicallythere is no evidence of secondary hyperparathyroid-ism. There is a strong association with dialysisencephalopathy. In group 1, cases 1 and 2 fit into thiscategory on biochemical grounds. Histologically,however, there is increased resorption in case 1.Cases 3, 4, and 5 have raised alkaline phosphataseactivities but not as marked as in group 3 and thehistological findings are more coflsistent with atypicalrenal osteomalacia except that case 3 has increasedresorptive activity. Case 1 was being treated with 1,25 hydroxyvitamin D3 at the time of biopsy andabout this time developed an avascular necrosis ofthe right head of femur. Cases 4 and 5 both sus-tained fractured necks of femur during an epilepti-form fit and case 5 had rib fractures which resultedin rib cage deformities. Both of these cases had beentreated with dihydrotachysterol.Therefore, it seems that the cases in group 1 fit

broadly into the category of atypical renal osteo-malacia as defined by Cochran.13 Case 3 fits least wellbut this case exhibits the least severe histologicalbone disease and had been the least time on haemo-dialysis. Therefore this category of renal osteo-malacia does indeed exhibit aluminium accumulationat a locus where the aluminium could interfere withthe mineralisation process and produce the observedhistological changes.

We wish to thank Mr W Mussard in the LapidaryLaboratory of the Department of Economic Geol-

ogy, University of Adelaide for help with the prep-aration of the blocks for electron probe analysis. Wewish to acknowledge the assistance of Mr B Griffin inthe performance of the electron probe analysis at theElectron Optical Centre, University of Adelaide. Wethank Mrs M Rowlands and Miss EA Goodwin fortyping the manuscript.

Referenoes

' Berlyne GM, Ben-Ari J, Pest D, et al. Hyperaluminaemia fromaluminium resin in renal failure. Lancet 1970;ii:494-6.

2Kaehny WD, Alfrey AC, Holman RE, Shorrn WJ. Aluminiumtransfer during haemodialysis. Kidney Int 1977;12:361-5.

'Alfrey AC, Le Gendre GR, Kaehny WD. The dialysisencephalopathy syndrome. Possible aluminium intoxication. NEngl J Med 1976;294:184-8.

4 Parsons V, Davies C, Goode C, Ogg C, Siddiqui J. Aluminium inbone from patients with renal failure. Br Med J 1971 ;iv:273-5.

Platts MM, Goode GC, Hislop JS. Composition of domestic watersupply and the incidence of fractures and encephalopathy inpatients on home dialysis. Br Med J 1977;ii:657-60.

6 Ward MD, Feest TG, Ellis HA, et al. Osteomalacic dialysis osteo-dystrophy: evidence of a water-borne aetiological agent, prob-ably aluminium. Lancet 1978;i:841-4.

7Parkinson IS, Ward MK, Feest TG, Fawcett RWP, Kerr DNS.Fracturing dialysis osteodystrophy and dialysis encephalopathy.Lancet 1979;i:406-9.

6 Ellis HA, McCarthy JH, Herrington J. Bone aluminium in haemo-dialysed patients and in rats injected with aluminium chloride:relationship to impaired bone mineralization. J Clin Pathol1979;32:832-44.

9 Raina V. Normal osteoid tissue. J Clin Pathol 1972;25:229-32.*'Pearse AGE. Histochemistry: theoretical and applied Edinburgh

and London: Churchill Livingstone, 1972.Irwin DA. The demonstration of aluminium in animal tissues. Arc-

hives of Industrial Health 1955;12:218-20.12 Middleton AR. Reaction of "Aluminon" with hydroxides of beryl-

lium, rare earths, zirconium and thorium. J Am Chem Soc1926;48:2125-6.

13 Cochran M. Aspects of renal bone disease. Aust NZ J Med1981;11 (suppl 1):33-7.

Requests for reprints to: Dr J McClure, IMVS, Box 14,Rundle Street PO, Adelaide, South Australia 5000

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