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Changes in serum aluminium, blood zinc, blood lead and erythrocyte δ-aminolaevulinic acid dehydratase activity during haemodialysis
Toxicology Letters, 4 (1979) 419-424 o Elsevier/North-Holland Biomedical Press
419
CHANGES IN SERUM ALUMINIUM, BLOOD ZINC, BLOOD LEAD AND ERYTHROCYTE 6-AMINOLAEVULINIC ACID DEHYDRATASE ACTIVITY DURING HAEMODIALYSIS
P.A. MEREDITH*,
H.L. ELLIOTT, B.C. CAMPBELL and M.R. MOORE**
Department of Materia Medica, University of Glasgow, Stobhill General Hospital, Glasgow, G21 3UW, and **Department of Medicine, University of Glasgow, Gardiner Institute, Western Infirmary, Glasgow, Gll (U.K.) (Received July 9th, 1979) (Accepted July 12th, 1979)
SUMMARY
In a study of 18 patients on haemodialysis, erythrocyte 6 -aminolaevulinic acid dehydratase (ALAD), serum aluminium, blood zinc and blood lead concentrations were increased significantly following haemodialysis. The increase in erythrocyte ALAD activity was found to be significantly linearly correlated with the increase in blood zinc concentrations. In comparison with control subjects, the patients had significantly lower activities of ALAD and significantly increased serum aluminium concentrations. When zinc and lead concentrations were corrected for packed cell volume they were found to be significantly higher in the patient group than in the control group.
INTRODUCTION
The activity of the second enzyme of the haem biosynthetic pathway, ALAD measured in erythrocytes is now widely accepted as an accurate bioanalytical measure of environmental lead exposure [l].This has been aided in Europe by the development of a standardised assay [2] for use as indicated in the Council Directives of the Commission of the European Communities [3]. Other metals, in particular aluminium and zinc, can alter the activity of ALAD [4, 51. Both metals have been shown to activate the enzyme, but only zinc has been shown to have a significant effect on its activity at physiological concentrations [6] and a greater effect following a poisoning episode [7]. There is evidence of active uptake of both zinc [8] and aluminium [9] during haemodialysis and the dialysis encephalopathy, which often includes an increasing anaemia, has been shown to be due to uptake of aluminium from the *Correspondence to Dr. P.A. Meredith. Abbreviation: ALAD, 6 -aminolaevulinic acid dehydratase.
420
water used in the preparation of the dialysis fluid [lo]. The present study was designed to measure any changes in erythrocyte ALA dehydratase activity, blood lead, blood zinc and serum aluminium concentrations associated with haemodialysis and to evaluate possible inter-relationships between the changes in these parameters. PATIENTS
AND METHODS
18 patients on regular haemodialysis therapy were studied before and immediately after dialysis. Erythrocyte ALAD was assayed by the method of Berlin and Schaller [2]. Whole blood lead and zinc and serum aluminium concentrations were determined by flameless atomic absorption spectrophotometry, using a Perkin Elmer 306 instrument with HGA72 graphite furnace and deuterium background correction; lead was measured by the method of Meredith et al. [4], zinc by that of Fjerdingstad et al. [ll] and serum aluminium by that of Fuchs et al. [12], These parameters were also measured in a group of 7 control subjects with no known excessive exposure to aluminium, zinc or lead. Statistical analysis was carried out using the parametric Student’s ‘t’ test and paired Student’s ‘t’ test and the non-parametric Wilcoxon matched-pairs signed-rank test. Regression analyses were carried out using the Kendal ranking test and the parametric least squares regression analysis of Pearson. RESULTS
The results of the determination of the various parameters in the 18 patients on haemodialysis are shown in Table I and those from the control subjects are shown in Table II. Blood zinc and blood lead figures shown in brackets are those corrected for packed cell volume (PCV). All the parameters measured show a statistically significant rise associated with haemodialysis (Table I). Rises in blood zinc and blood lead concentrations remain statistically significant when corrected for PCV. Comparison of the control group with the patient group predialysis reveals that the controls have a significantly higher PCV and erythrocyte ALAD activity but a significantly lower serum aluminium concentration (Table II). Whole blood zinc and whole blood lead concentrations were not significantly different. If these parameters are corrected. for PCV, it is clear that both zinc and lead concentrations are higher in the group of patients than in the control group (Table II). Analysis of the inter-relationships between the parameters measured failed to reveal any statistically significant association between erythrocyte ALAD activity and serum aluminium and blood lead concentrations. There was a significant relationship between 5%change in blood zinc and % change in erythrocyte ALAD associated with haemodialysis. The non-parametric KendalRanking test gave a correlation coefficient r of 0.74 (P < O.OOl), whilst the parametric linear regression analysis gave a correlation coefficient r = 0.84 (P < 0.001). This relationship is illustrated graphically in Fig. 1.
s-AMINOLAE-
-
0.01
P for Wilcoxon matched pairs test
23.6 * 4.1
21.5 r 4.4
0.01
post dialysis
dialysis
Pre
P for paired t-test
Haemodialysis patients n = 18
%
Packed cell volume
0.005
0.01
9.8 ?r 4.6
7.1 + 2.9
Erythrocyte ALAD activits (nmol ALA utilised/nG/ml RBC)
0.005
0.001
9.0 + 4.5
4.8 f 2.2
0.005 (0.01)
0.005 (0.01)
0.005 (0.01)
1.4 + 0.55 (6.2 c 2.6)
1.2 * 0.47 (5.7 f 2.4)
Blood lead Gmolll)
1762 41 (760 + 180)
139’S 44 (620 * 140)
Blood zinc bmol/l)
0.005 (0.01) - ~_____
Serum aluminium (Hmol/l)
All figures represent the mean 2 standard deviation. Figures in brackets represent those corrected for packed cell volume
SERUM ALUMINIUM, BLOOD ZINC AND BLOOD LEAD CONCENTRATIONS AND ERYTHROCYTE VULINIC ACID DEHYDRATASE (ALAD) MEASURED IN PATIENTS PRE AND POST DIALYSIS
TABLE I
422 TABLE
II
COMPARISON
OF THE CONTROL
All figures represent cell volume
AND THE PATIENT
the mean f standard
deviation.
GROUP
Figures
PREDIALYSIS
in brackets
are those corrected
for packed
__Packed cell volume (%)
Erythrocyte ALAD activity (run01 ALA utilised/min/ml RBC)
Patients predialysis (n = 18)
21.5
Controls (n= 7)
43 f 1.8
27 f 7.4
0.001
0.001
* 4.4
7.1 * 2.9
Serum aluminium @mol/l) ___-
Blood zinc (/Jmol/l)
Blood lead @mow)
4.8 + 2.2
139 (620
1.2 * 0.47 (5.7 f 2.4)
1.8 + 0.4
111 f (257 f
0.005
n.s. (P < 0.001)
f 44 f 140)
19 44)
1.2 + 0.5 (2.9 + 1.2)
P for t-test relative to pre dialysis patient group
%
;i”;
0.005)
-
increase m blood Zinc
Fig. 1. The correlation of the percentage increase in erythrocyte ALAD activity (y) and the percentage increase in blood zinc (x) concentration associated with haemodialysis; y = 0.62 (x) + 42 (r = 0.84, P < 0.001). DISCUSSION
As noted previously [8-lo] haemodialysis is associated with increases in serum aluminium and blood zinc concentrations; blood lead concentrations also rise following haemodialysis. Both aluminium and zinc are capable of activating ALAD [4-61, whilst it has long been recognised that lead can profoundly inhibit the activity of ALAD [13]. The small but significant rise in erythrocyte ALAD activity associated with haemodialysis described in the present study is paralleled by rises in serum aluminium and blood zinc
423
concentrations together with a small rise in blood lead concentrations. It is possible that the rise in erythrocyte ALAD activity following haemodialysis is the result of a complex interaction associated with the increased concentrations of these three metals, There was, however, no correlation between the fairly large rises in serum aluminium concentrations and changes in ALAD activity, although there was a significant linear correlation between the rise in blood zinc concentrations and the rise in erythrocyte ALAD activity (Fig. 1). It seems probable that the net activation of the enzyme by the relatively large increase in blood zinc concentration masks the inhibitory effects of the increased blood lead concentration, since the latter is relatively small. The results of the present study confirm our earlier findings that zinc has a small but si~ific~t effect on erythrocyte ALAD activity at physiological concentrations [6]. There is still no evidence to suggest that this negates the value of the enzyme assay as a bioanalytical measure of environmental lead exposure. Comparison of the control group of subjects with the group of patients prior to haemodialysis reveals that the activity of erythrocyte ALAD is considerably depressed in the patients (P < 0.001). It is not possible on the basis of the metal concentrations measured to attribute this depression in activity to any one of these metals. The concentration of lead as related to PCV is significantly greater in the patient group than in the control group. If the activity of erythrocyte ALAD reflects the activity of this enzyme in the major sites of haem synthesis, e.g. liver and bone marrow, the depression may account to some extent for the decline in haemoglobin concentration associated with haemodialysis. ACKNOWLEDGEMENTS
The authors
wish to thank Miss M.A: Hughes for skilled technical
assistance.
REFERENCES 1 P.A. Meredith, M.R. Moore and A. Goldberg, Erythrocyte ALAD activity and blood protoporphyrin concentrations as indices of lead exposure and altered haem biosynthesis, Clin. Sci., 56 (1979) 61-69. 2 A. Berlin and K.H. Schaller, European standardised method for the determination of ALAD activity in blood, Z. Klin. Cbem. Biochem., 12 (1974) 389-390. 3 European Economic Community, Council Directive of 29th March, 1977, on Biological Screening of the population for lead, Off. J. Eur. Commun., 20 (1977) 10-17. 4 P.A. Meredith, M.R. Moore and A. Goldberg, Effects of aluminium, lead and zinc on ALAD, Enzyme, 22 (1977) 22-27. 6 M. Abdulla, ALAD activity in red blood cells: Influence of metals on its activity with special reference to lead and zinc, Student Litteratur, Lund, 1978. 6 P.A. Meredith and M.R. Moore, The effects of zinc and lead on ALAD, Biochem. Sot. Trans., 6 (1978) 760-762. 7 H.L. Haust, D.S.M. Haines and C.V. Braun, Some factors affecting red cell ALAD activity in human subjects, in M. Doss (Ed.) Porphyrins in Human Diseases, Karger, Basel, 1976.
424 8 J. Blomfield, J. McPherson and C.R.P. George, Active uptake of copper and zinc during haemodialysis, Br. Med. J., 1 (1969) 141-145. 9 W.D. Kaehny, A.C. Alfrey, R.E. Holman and W.J. Shorr, Aluminium transfer during haemodialysis, Kidney Int., 12 (1977) 361-365. 10 H.L. Elliott, F. Dryburgh, G.S. Fell, S. Sabet and A.I. McDougall, Aluminium toxicity during regular haemodialysis, Br. Med. J., 1 (1978) 1101-1103. 11 E. Fjerdingstad, G. Danscher, G. and E.J. Fjerdingstad, Zinc content in hippocampus and whole brain of normal rats, Brain Res., 79 (1974) 338-342. 12 C. Fuchs, M. Brasche, K. Paschen, H. Nordbeck and E. Quellhorst, Aluminium determination in serum by flameless atomic absorption spectrophotometry, Clin. Chim. Acta, 52 (1974) 71-80. 13 K.D. Gibson, A. Neuberger and J.J. Scott, The purification and properties of ALAD, Biochem. J., 61 (1955) 618-629.
419
CHANGES IN SERUM ALUMINIUM, BLOOD ZINC, BLOOD LEAD AND ERYTHROCYTE 6-AMINOLAEVULINIC ACID DEHYDRATASE ACTIVITY DURING HAEMODIALYSIS
P.A. MEREDITH*,
H.L. ELLIOTT, B.C. CAMPBELL and M.R. MOORE**
Department of Materia Medica, University of Glasgow, Stobhill General Hospital, Glasgow, G21 3UW, and **Department of Medicine, University of Glasgow, Gardiner Institute, Western Infirmary, Glasgow, Gll (U.K.) (Received July 9th, 1979) (Accepted July 12th, 1979)
SUMMARY
In a study of 18 patients on haemodialysis, erythrocyte 6 -aminolaevulinic acid dehydratase (ALAD), serum aluminium, blood zinc and blood lead concentrations were increased significantly following haemodialysis. The increase in erythrocyte ALAD activity was found to be significantly linearly correlated with the increase in blood zinc concentrations. In comparison with control subjects, the patients had significantly lower activities of ALAD and significantly increased serum aluminium concentrations. When zinc and lead concentrations were corrected for packed cell volume they were found to be significantly higher in the patient group than in the control group.
INTRODUCTION
The activity of the second enzyme of the haem biosynthetic pathway, ALAD measured in erythrocytes is now widely accepted as an accurate bioanalytical measure of environmental lead exposure [l].This has been aided in Europe by the development of a standardised assay [2] for use as indicated in the Council Directives of the Commission of the European Communities [3]. Other metals, in particular aluminium and zinc, can alter the activity of ALAD [4, 51. Both metals have been shown to activate the enzyme, but only zinc has been shown to have a significant effect on its activity at physiological concentrations [6] and a greater effect following a poisoning episode [7]. There is evidence of active uptake of both zinc [8] and aluminium [9] during haemodialysis and the dialysis encephalopathy, which often includes an increasing anaemia, has been shown to be due to uptake of aluminium from the *Correspondence to Dr. P.A. Meredith. Abbreviation: ALAD, 6 -aminolaevulinic acid dehydratase.
420
water used in the preparation of the dialysis fluid [lo]. The present study was designed to measure any changes in erythrocyte ALA dehydratase activity, blood lead, blood zinc and serum aluminium concentrations associated with haemodialysis and to evaluate possible inter-relationships between the changes in these parameters. PATIENTS
AND METHODS
18 patients on regular haemodialysis therapy were studied before and immediately after dialysis. Erythrocyte ALAD was assayed by the method of Berlin and Schaller [2]. Whole blood lead and zinc and serum aluminium concentrations were determined by flameless atomic absorption spectrophotometry, using a Perkin Elmer 306 instrument with HGA72 graphite furnace and deuterium background correction; lead was measured by the method of Meredith et al. [4], zinc by that of Fjerdingstad et al. [ll] and serum aluminium by that of Fuchs et al. [12], These parameters were also measured in a group of 7 control subjects with no known excessive exposure to aluminium, zinc or lead. Statistical analysis was carried out using the parametric Student’s ‘t’ test and paired Student’s ‘t’ test and the non-parametric Wilcoxon matched-pairs signed-rank test. Regression analyses were carried out using the Kendal ranking test and the parametric least squares regression analysis of Pearson. RESULTS
The results of the determination of the various parameters in the 18 patients on haemodialysis are shown in Table I and those from the control subjects are shown in Table II. Blood zinc and blood lead figures shown in brackets are those corrected for packed cell volume (PCV). All the parameters measured show a statistically significant rise associated with haemodialysis (Table I). Rises in blood zinc and blood lead concentrations remain statistically significant when corrected for PCV. Comparison of the control group with the patient group predialysis reveals that the controls have a significantly higher PCV and erythrocyte ALAD activity but a significantly lower serum aluminium concentration (Table II). Whole blood zinc and whole blood lead concentrations were not significantly different. If these parameters are corrected. for PCV, it is clear that both zinc and lead concentrations are higher in the group of patients than in the control group (Table II). Analysis of the inter-relationships between the parameters measured failed to reveal any statistically significant association between erythrocyte ALAD activity and serum aluminium and blood lead concentrations. There was a significant relationship between 5%change in blood zinc and % change in erythrocyte ALAD associated with haemodialysis. The non-parametric KendalRanking test gave a correlation coefficient r of 0.74 (P < O.OOl), whilst the parametric linear regression analysis gave a correlation coefficient r = 0.84 (P < 0.001). This relationship is illustrated graphically in Fig. 1.
s-AMINOLAE-
-
0.01
P for Wilcoxon matched pairs test
23.6 * 4.1
21.5 r 4.4
0.01
post dialysis
dialysis
Pre
P for paired t-test
Haemodialysis patients n = 18
%
Packed cell volume
0.005
0.01
9.8 ?r 4.6
7.1 + 2.9
Erythrocyte ALAD activits (nmol ALA utilised/nG/ml RBC)
0.005
0.001
9.0 + 4.5
4.8 f 2.2
0.005 (0.01)
0.005 (0.01)
0.005 (0.01)
1.4 + 0.55 (6.2 c 2.6)
1.2 * 0.47 (5.7 f 2.4)
Blood lead Gmolll)
1762 41 (760 + 180)
139’S 44 (620 * 140)
Blood zinc bmol/l)
0.005 (0.01) - ~_____
Serum aluminium (Hmol/l)
All figures represent the mean 2 standard deviation. Figures in brackets represent those corrected for packed cell volume
SERUM ALUMINIUM, BLOOD ZINC AND BLOOD LEAD CONCENTRATIONS AND ERYTHROCYTE VULINIC ACID DEHYDRATASE (ALAD) MEASURED IN PATIENTS PRE AND POST DIALYSIS
TABLE I
422 TABLE
II
COMPARISON
OF THE CONTROL
All figures represent cell volume
AND THE PATIENT
the mean f standard
deviation.
GROUP
Figures
PREDIALYSIS
in brackets
are those corrected
for packed
__Packed cell volume (%)
Erythrocyte ALAD activity (run01 ALA utilised/min/ml RBC)
Patients predialysis (n = 18)
21.5
Controls (n= 7)
43 f 1.8
27 f 7.4
0.001
0.001
* 4.4
7.1 * 2.9
Serum aluminium @mol/l) ___-
Blood zinc (/Jmol/l)
Blood lead @mow)
4.8 + 2.2
139 (620
1.2 * 0.47 (5.7 f 2.4)
1.8 + 0.4
111 f (257 f
0.005
n.s. (P < 0.001)
f 44 f 140)
19 44)
1.2 + 0.5 (2.9 + 1.2)
P for t-test relative to pre dialysis patient group
%
;i”;
0.005)
-
increase m blood Zinc
Fig. 1. The correlation of the percentage increase in erythrocyte ALAD activity (y) and the percentage increase in blood zinc (x) concentration associated with haemodialysis; y = 0.62 (x) + 42 (r = 0.84, P < 0.001). DISCUSSION
As noted previously [8-lo] haemodialysis is associated with increases in serum aluminium and blood zinc concentrations; blood lead concentrations also rise following haemodialysis. Both aluminium and zinc are capable of activating ALAD [4-61, whilst it has long been recognised that lead can profoundly inhibit the activity of ALAD [13]. The small but significant rise in erythrocyte ALAD activity associated with haemodialysis described in the present study is paralleled by rises in serum aluminium and blood zinc
423
concentrations together with a small rise in blood lead concentrations. It is possible that the rise in erythrocyte ALAD activity following haemodialysis is the result of a complex interaction associated with the increased concentrations of these three metals, There was, however, no correlation between the fairly large rises in serum aluminium concentrations and changes in ALAD activity, although there was a significant linear correlation between the rise in blood zinc concentrations and the rise in erythrocyte ALAD activity (Fig. 1). It seems probable that the net activation of the enzyme by the relatively large increase in blood zinc concentration masks the inhibitory effects of the increased blood lead concentration, since the latter is relatively small. The results of the present study confirm our earlier findings that zinc has a small but si~ific~t effect on erythrocyte ALAD activity at physiological concentrations [6]. There is still no evidence to suggest that this negates the value of the enzyme assay as a bioanalytical measure of environmental lead exposure. Comparison of the control group of subjects with the group of patients prior to haemodialysis reveals that the activity of erythrocyte ALAD is considerably depressed in the patients (P < 0.001). It is not possible on the basis of the metal concentrations measured to attribute this depression in activity to any one of these metals. The concentration of lead as related to PCV is significantly greater in the patient group than in the control group. If the activity of erythrocyte ALAD reflects the activity of this enzyme in the major sites of haem synthesis, e.g. liver and bone marrow, the depression may account to some extent for the decline in haemoglobin concentration associated with haemodialysis. ACKNOWLEDGEMENTS
The authors
wish to thank Miss M.A: Hughes for skilled technical
assistance.
REFERENCES 1 P.A. Meredith, M.R. Moore and A. Goldberg, Erythrocyte ALAD activity and blood protoporphyrin concentrations as indices of lead exposure and altered haem biosynthesis, Clin. Sci., 56 (1979) 61-69. 2 A. Berlin and K.H. Schaller, European standardised method for the determination of ALAD activity in blood, Z. Klin. Cbem. Biochem., 12 (1974) 389-390. 3 European Economic Community, Council Directive of 29th March, 1977, on Biological Screening of the population for lead, Off. J. Eur. Commun., 20 (1977) 10-17. 4 P.A. Meredith, M.R. Moore and A. Goldberg, Effects of aluminium, lead and zinc on ALAD, Enzyme, 22 (1977) 22-27. 6 M. Abdulla, ALAD activity in red blood cells: Influence of metals on its activity with special reference to lead and zinc, Student Litteratur, Lund, 1978. 6 P.A. Meredith and M.R. Moore, The effects of zinc and lead on ALAD, Biochem. Sot. Trans., 6 (1978) 760-762. 7 H.L. Haust, D.S.M. Haines and C.V. Braun, Some factors affecting red cell ALAD activity in human subjects, in M. Doss (Ed.) Porphyrins in Human Diseases, Karger, Basel, 1976.
424 8 J. Blomfield, J. McPherson and C.R.P. George, Active uptake of copper and zinc during haemodialysis, Br. Med. J., 1 (1969) 141-145. 9 W.D. Kaehny, A.C. Alfrey, R.E. Holman and W.J. Shorr, Aluminium transfer during haemodialysis, Kidney Int., 12 (1977) 361-365. 10 H.L. Elliott, F. Dryburgh, G.S. Fell, S. Sabet and A.I. McDougall, Aluminium toxicity during regular haemodialysis, Br. Med. J., 1 (1978) 1101-1103. 11 E. Fjerdingstad, G. Danscher, G. and E.J. Fjerdingstad, Zinc content in hippocampus and whole brain of normal rats, Brain Res., 79 (1974) 338-342. 12 C. Fuchs, M. Brasche, K. Paschen, H. Nordbeck and E. Quellhorst, Aluminium determination in serum by flameless atomic absorption spectrophotometry, Clin. Chim. Acta, 52 (1974) 71-80. 13 K.D. Gibson, A. Neuberger and J.J. Scott, The purification and properties of ALAD, Biochem. J., 61 (1955) 618-629.