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Reduced plasma and white blood cell selenium levels in haemodialysis patients
323
Clinica Chimica Acta, 164 (1987) 323-328 Elsevier
CCA 03780
Reduced plasma and white blood cell selenium levels in haemodialysis patients John W. Foote, Lesley J. Hinks and Barbara
Lloyd
University Department of Chemical Pathology and Human Metabolism, Level D, South Laboratory and Pathology Block, Southampton General Hospital, Southampton (UK) (Received
6 September
1986; revision
Key words: Selenium;
12 January
Plasma;
1987; accepted
Leucocyte;
after revision
Hemodialysis
14 January
1987)
patients
Selenium concentrations have been measured in plasma and in blood leucocytes from 29 haemodialysis patients and from 25 healthy men. The selenium contents of the plasma and white blood cells of the dialysis patients were significantly reduced ( p < 0.001). Selenium deficiency in humans results in a congestive cardiomyopathy and is associated with increased risks of accelerated atherosclerosis and cancer. Each of these is found with abnormal frequency in haemodialysis patients.
Introduction
The survival of most patients with chronic renal disease is now ensured as a result of improved dialysis methods. However, the present treatment of these patients is not yet optimal since many continue to experience widespread disturbances of body function such as anaemia, endocrine dysfunctions, osteodystrophy, cardiomyopathy, and accelerated atherosclerosis, despite the most scrupulous dialysis technique. These refractory uraemic features have generally been assumed to result from the retention of specific, but unidentified, toxins. Recently, however, attention has been directed to the possibility that they result from disturbances of trace elements; reports in the literature implicate abnormalities of aluminium [l-3], zinc [4-61, arsenic [7], and cobalt [8] metabolism in the pathogenesis of the uraemic syndrome.
Correspondence Medical Centre,
to: Dr. J.W. Foote, Department Nottingham, NG7 2UH, UK.
0009-8981/87/%03.50
of Clinical
0 1987 Elsevier Science Publishers
Chemistry,
B.V. (Biomedical
University
Division)
Hospital,
Queens
324
Selenium is acknowledged to be an essential micro-nutrient for man. Naturally occurring selenium deficiency has been found in China in association with a fatal congestive cardiomyopathy [9], and skeletal and cardiac myopathies have also been attributed to acute selenium deficiency during total parenteral nutrition [lO,ll]. There is, additionally, increasing epidemiological evidence linking reduced selenium availability with increased risks of cardiovascular disease 1121 and cancer [13]. As these manifestations of selenium depletion correspond with features seen in uraemia we have assessed the selenium reserves of a group of haemodialysis patients. The results of our study are presented here. Patients and methods Dialysis patients Twenty-nine consecutive male patients, aged 22-61 (mean 44.9) yr, who attended a nephrology clinic were investigated. Each had stable end-stage renal failure treated by haemodialysis for 8-10 h twice weekly, and all but three had been established on dialysis for more than one year. Daily treatment with aluminium hydroxide, ferrous sulphate and a multivitamin preparation (BC 500, Ayers& UK) was routinely given to all patients. Three also received l&H vitamin D, in doses ranging from 1 pg/wk to 1 pg,/day, and one other was given propranolol for systemic hypertension. Control subjects Twenty-five healthy controls.
men aged 21-62
(mean 41.5) yr volunteered
to act as
Samples Samples (20 ml) of venous blood were collected using disposable plastic syringes and stainless steel needles. Each sample was divided as follows: 15 ml was transferred to a siliconised glass tube for leucocyte separation, and two further portions of 2.5 ml were added to polycarbonate tubes containing EDTA as anti~ag~~t. Plasma was separated from one of these by centrifugation at 1000 x g for 10 mm at room temperature, whilst the contents of the other were reserved for the dete~nation of the white cell count. Samples of plasma awaiting selenium analysis were stored in trace element-free polycarbonate tubes at - 20 o C. Plasma selenium Selenium concentrations in plasma were determined by hydride generation and atomic-absorption spectrophotometry as previously described 1141. The betweenbatch imprecision of this assay was 4.9% at a concentration of 1.52 pmol/l (120 M/l). L.eucacyte separation and analysis Blood leucocytes were separated using a dextran sedimentation technique described elsewhere [15]. The cells were subs~uendy digested with nitric acid and their selenium content measured by hydride generation and ato~c-abso~t~on
325
spectrophotometry using methods given previously for the measurement of selenium in red blood cells [14]. The between-batch coefficient of variation of this method was 5.9% at a level of 2.05 pmol/106 cells (162 pg/106 cells). Statistical analysis The differences between data sets were assessed using the Mann-Whitney
U test.
Data are expressed in this paper as mean f 1 SD. Ethical considerations
All of the subjects participating in this study gave informed consent to venepuncture which was performed with the approval of the relevant Ethical Committees. Results Plasma selenium
Plasma selenium concentrations are shown in Fig. 1. The values measured in the samples from the dialysis patients were lower than those of the healthy controls, 1.12 rt 0.16 pmol/l (88.7 f 12.9 pg/l) and 1.46 f 0.21 pmol/l (115.6 f 16.5 pg/l), respectively ( p < 0.001). L.eucocyte selenium The selenium contents of the leucocytes obtained from the dialysis patients were
also reduced when compared
150-
0-
to those found in cells from the control subjects,
00 0
1 Dialysis patients
I NOITfXll subjects
Fig. 1. Plasma selenium concentrations in haemodialysis patients and healthy control subjects. Horizontal bars indicate median values of data sets. Conversion of traditional to SI units - plasma selenium: 1 pg/l = 12.7 nmol,‘l.
326
Dialysic patients
Normal subjects
Fig. 2. Selenium contents of blood leucocytes in haemodialysis Horizontal bars indicate median values of data sets. Conversion selenium: 1 pg/106 cells = 12.7 fmol/106 cells.
patients and healthy control subjects. of traditional to SI units - leucocyte
1.49 5 0.46 pmol/106 cells (117.4 f 36.4 pg/106 cells) and 2.24 * 0.62 pmol/106 cells (176.7 f 49.2 pg/106 cells), respectively (p < 0.001). These data are shown in Fig. 2. Discussion
Severe selenium deficiency in man results in potentially fatal skeletal and cardiac myopathies [g-11]. More moderate deficiency states appear to be associated with increased risks of cardiovascular death and myocardial infarction [12], and with an increased incidence of cancer of the gastrointestinal tract and the prostate [13]. Features similar to these are also found with abnormal frequency in uraemic patients maintained with haemodialysis. Congestive cardiomyopathy, for example, is detectable by echocardiography or ventriculography without evidence of ischaemic heart disease in a large proportion of haemodialysis patients [16,17], and accelerated atherosclerosis is a major cause of morbidity and mortality in these cases [18]. Patients treated by haemodialysis are also reported to suffer an increased incidence of cancer [19]. It is difficult to ignore the possibility that these complications result from a previously unrecognised deficiency of selenium. The assessment of selenium status is complicated since no single measurement provides a reliable indication of body selenium reserves in all circumstances [20]. Plasma selenium concentrations are commonly used however, and they respond rapidly to deficiency states induced by a low dietary intake of the element [21,22].
The reduced plasma concentrations of selenium found in the uraemic patients investigated in this study are clearly relevant. Erythrocyte selenium concentrations and glutathione peroxidase activities are widely used to complement plasma measurements during the clinical assessment of selenium availability. However, we have chosen not to use these measurements because of the well known abnormalities of red blood cell production and survival that are found with advanced renal failure. Instead, we have determined the selenium content of blood leucocytes which have the advantage of being a metabolically active tissue that can be repeatedly sampled without undue trauma. The use of Ieucocyte analyses to assess body selenium status has not been evaluated as far as we are aware, but similar measurements of white blood cell composition have proved helpful in the diagnosis of deficiencies of other trace elements, such as zinc, both in normal individuals and in uraemic patients [23,24]. It is therefore interesting that the selenium contents of the leucocytes obtained from the haemodialysis patients investigated in this study should be so low. Abnormalities of trace element metabolism in advanced renal failure have only attracted significant attention during the last decade. Uraemia was previously thought to result largely from the retention of organic toxins, and the persistence of uraemic symptoms after the institution of haemodialysis was held to be due to the poor clearance of some of these chemical species through the dialysis membranes that were currently available. Recent reports challenge this view. Thus, uraemic encephalopathy and dialysis oste~ystrophy are thought to be associated with the accumulation of aluminium [l-3], arsenic has been implicated in the pathogenesis of renal anaemia [7], and cobalt excess has been suggested as a contributory factor in uraemic heart failure [8]. Uraemic abnormalities of trace element metabolism do not necessarily take the form of toxicity states; zinc deficiency is thought to occur in end-stage renal failure and to cause testicular failure [4], impaired cell-mediated immunity [5], and anorexia and distaste for dietary protein [6]. It is conceded that the reduced plasma and leucocyte selenium levels reported here do not provide unequivocal evidence of selenium deficiency. However their occurrence in patients who suffer unduely from atherosclerosis, cardiomyopa~y, and malignant disease is int~guing, and they indicate a need for further research. It is only possible to speculate on the pathogenesis of this putative selenium deficiency state. However, their anorexia and distaste for dietary protein frequently cause uraemic patients to eat inadequate amounts of meat and fish, and these are the most effective dietary sources of selenium [25]. Acknowledgements
We are grateful to Professor B.E. Clayton for her helpful discussions and support, and to Professor A. Polak, Professor H.A. Lee and ‘Dr. D.J. Warren for permission to study patients in their care at the Wessex Regional Renal Unit at St. Mary’s Hospital, Portsmouth. J.W.F. also gratefully acknowledges financial support from BDH Chemicals Ltd, the Wessex Regional Health Authority, the Wessex Medical School Trust and the Wellcome Trust.
328
References 1 Parkinson IS, Ward MK, Kerr DNS. Dialysis encephalopathy, bone disease and anaemia: the ahuninium intoxication syndrome during regular dialysis. J Clin Path01 1981;34;1285-1294. 2 Davidson AM, Walker GS, Oli H, Lewins AM. Water supply aluminium concentration, dialysis dementia, and effect of reverse-osmosis water treatment. Lancet 1982;%785-787. 3 Nathan E, Pederson S. Dialysis encephalopathy in a non-dialysed uraemic boy treated with almninium hydroxide orally. Acta Paediatr Stand 1980;69:793-796. 4 Antoniou LD, Shalboub RJ, Sudakar T, Smith JC. Reversal of uraemic impotence by zinc. Lancet 1977;ii:895-898. 5 Antoniou LD, Shalhoub RJ, Schechter GP. The effect of zinc on cellular immunity in chronic uremia. Am J Clin Nutr 1981;34:1912-1917. 6 A&in-Thor E, Goddard BW, O’Nion J, Stephen RL, Kolff WJ. Hypogeusia and zinc depletion in chronic dialysis patients. Am J Clin Nutr 1978;31:1948-1951. 7 Pershagen G, Hast R, Lins LE, Pehrsson K. Increased arsenic concentration in the bone marrow in chronic renal failure - a contributor to anaemia? Nephron 1982;30:250-252. 8 Pehrsson K, Lms LE. Cobalt in uraemic c~diomyopathy. Lancet 1978;ii:51-52. 9 ~onymous. Selenium in the heart of China {atone). Lancet 1979;~:889-8#. 10 Johnson RA, Baker SS, Fallon JT, et al. An occidental case of ~r~omyopathy and selenium deficiency. N Engl J Med 1981;3~:1210-1212. 11 Fleming CR, Lie JT, McCall JT, O’Brien JF, Baillie EE, Thistle JL. Selenium deficiency and fatal ~rdiomyopa~y in a patient on home parenteral nutrition. Gastroenterology 1982;83:689-693. 12 Salonen JT, Alfthan G, Huttunen JK, Piiarainen J, Puska P. Association between cardiovascular death and myocardial infarction and serum selenium in a matched-pair longitudinal study. Lancet 1982;&175-179. 13 Willet WC, Polk BF, Morris JS, et al Prediagnostic serum selenium and risk of cancer. Lancet 1983;ii:130-134. 14 Lloyd B, Holt P, Delves HT. Determination of selenium in biological samples by hydride generation and atomic-absorption spectroscopy. Analyst 1982;107:927-933. 15 Hinks LJ, Colmsee M, Delves HT. Determination of zinc and copper in isolated leucocytes. Analyst 1982;107:815-823. 16 Drneke T, Le Pailleur C, Meilhac B, et al. Congestive cardiomyopathy in uraemic patients on long term haemodialysis. B Med J 1977;i:350-353. 17 Lai KN, Barnden L, Mathew TH. Effect of renal transplantation on left ventricular function in hemodialysis patients. Clin Nephrol 1982;18:74-78. 18 Lindner A, Charra B, Sherrard DJ, Scribner BH. Accelerated atherosclerosis in prolonged maintenance hemodialysis. N Engl J Med 1974;290:697-701. 19 Curtis JR. Cancer and patients with end-stage renal failure. B Med J 1982;284:69-70. 20 Neve J, Vertongen F, Molle L. Selenium deficiency. Clin Endocrinol Metab 1985;14:629-656. 21 Van Rij AM, Thomson CD, McKenzie JM, Robinson MF. Selenium deficiency in total and parenteral nutrition. Am J Clin Nutr 1979;32:2076-2085. 22 Janghorbani M, Kasper L, Young V. Dynamics of selenite metabolism in young men: studies with the stable isotope tracer method. Am J Clin Nutr 1984;#:208-218. 23 Jones RB, Keeling PWN, Hilton PJ, Thompson RPH. The relationship between leucocyte and muscle zinc in health and disease. Clin Sci 1981;60:237-239. 24 Mahajan SK, Prasad AS, Rabbani P, Briggs WA, Mc~n~d FD. Zinc metabotism in uraemia. J Lab Clin Med 1979;94:693-698. 25 Zabel NL, Harland J, Gormican AT, Ganther HE. Selenium content of commercial formula diets. Am J Clin Nutr 1978;31:850-858.
Clinica Chimica Acta, 164 (1987) 323-328 Elsevier
CCA 03780
Reduced plasma and white blood cell selenium levels in haemodialysis patients John W. Foote, Lesley J. Hinks and Barbara
Lloyd
University Department of Chemical Pathology and Human Metabolism, Level D, South Laboratory and Pathology Block, Southampton General Hospital, Southampton (UK) (Received
6 September
1986; revision
Key words: Selenium;
12 January
Plasma;
1987; accepted
Leucocyte;
after revision
Hemodialysis
14 January
1987)
patients
Selenium concentrations have been measured in plasma and in blood leucocytes from 29 haemodialysis patients and from 25 healthy men. The selenium contents of the plasma and white blood cells of the dialysis patients were significantly reduced ( p < 0.001). Selenium deficiency in humans results in a congestive cardiomyopathy and is associated with increased risks of accelerated atherosclerosis and cancer. Each of these is found with abnormal frequency in haemodialysis patients.
Introduction
The survival of most patients with chronic renal disease is now ensured as a result of improved dialysis methods. However, the present treatment of these patients is not yet optimal since many continue to experience widespread disturbances of body function such as anaemia, endocrine dysfunctions, osteodystrophy, cardiomyopathy, and accelerated atherosclerosis, despite the most scrupulous dialysis technique. These refractory uraemic features have generally been assumed to result from the retention of specific, but unidentified, toxins. Recently, however, attention has been directed to the possibility that they result from disturbances of trace elements; reports in the literature implicate abnormalities of aluminium [l-3], zinc [4-61, arsenic [7], and cobalt [8] metabolism in the pathogenesis of the uraemic syndrome.
Correspondence Medical Centre,
to: Dr. J.W. Foote, Department Nottingham, NG7 2UH, UK.
0009-8981/87/%03.50
of Clinical
0 1987 Elsevier Science Publishers
Chemistry,
B.V. (Biomedical
University
Division)
Hospital,
Queens
324
Selenium is acknowledged to be an essential micro-nutrient for man. Naturally occurring selenium deficiency has been found in China in association with a fatal congestive cardiomyopathy [9], and skeletal and cardiac myopathies have also been attributed to acute selenium deficiency during total parenteral nutrition [lO,ll]. There is, additionally, increasing epidemiological evidence linking reduced selenium availability with increased risks of cardiovascular disease 1121 and cancer [13]. As these manifestations of selenium depletion correspond with features seen in uraemia we have assessed the selenium reserves of a group of haemodialysis patients. The results of our study are presented here. Patients and methods Dialysis patients Twenty-nine consecutive male patients, aged 22-61 (mean 44.9) yr, who attended a nephrology clinic were investigated. Each had stable end-stage renal failure treated by haemodialysis for 8-10 h twice weekly, and all but three had been established on dialysis for more than one year. Daily treatment with aluminium hydroxide, ferrous sulphate and a multivitamin preparation (BC 500, Ayers& UK) was routinely given to all patients. Three also received l&H vitamin D, in doses ranging from 1 pg/wk to 1 pg,/day, and one other was given propranolol for systemic hypertension. Control subjects Twenty-five healthy controls.
men aged 21-62
(mean 41.5) yr volunteered
to act as
Samples Samples (20 ml) of venous blood were collected using disposable plastic syringes and stainless steel needles. Each sample was divided as follows: 15 ml was transferred to a siliconised glass tube for leucocyte separation, and two further portions of 2.5 ml were added to polycarbonate tubes containing EDTA as anti~ag~~t. Plasma was separated from one of these by centrifugation at 1000 x g for 10 mm at room temperature, whilst the contents of the other were reserved for the dete~nation of the white cell count. Samples of plasma awaiting selenium analysis were stored in trace element-free polycarbonate tubes at - 20 o C. Plasma selenium Selenium concentrations in plasma were determined by hydride generation and atomic-absorption spectrophotometry as previously described 1141. The betweenbatch imprecision of this assay was 4.9% at a concentration of 1.52 pmol/l (120 M/l). L.eucacyte separation and analysis Blood leucocytes were separated using a dextran sedimentation technique described elsewhere [15]. The cells were subs~uendy digested with nitric acid and their selenium content measured by hydride generation and ato~c-abso~t~on
325
spectrophotometry using methods given previously for the measurement of selenium in red blood cells [14]. The between-batch coefficient of variation of this method was 5.9% at a level of 2.05 pmol/106 cells (162 pg/106 cells). Statistical analysis The differences between data sets were assessed using the Mann-Whitney
U test.
Data are expressed in this paper as mean f 1 SD. Ethical considerations
All of the subjects participating in this study gave informed consent to venepuncture which was performed with the approval of the relevant Ethical Committees. Results Plasma selenium
Plasma selenium concentrations are shown in Fig. 1. The values measured in the samples from the dialysis patients were lower than those of the healthy controls, 1.12 rt 0.16 pmol/l (88.7 f 12.9 pg/l) and 1.46 f 0.21 pmol/l (115.6 f 16.5 pg/l), respectively ( p < 0.001). L.eucocyte selenium The selenium contents of the leucocytes obtained from the dialysis patients were
also reduced when compared
150-
0-
to those found in cells from the control subjects,
00 0
1 Dialysis patients
I NOITfXll subjects
Fig. 1. Plasma selenium concentrations in haemodialysis patients and healthy control subjects. Horizontal bars indicate median values of data sets. Conversion of traditional to SI units - plasma selenium: 1 pg/l = 12.7 nmol,‘l.
326
Dialysic patients
Normal subjects
Fig. 2. Selenium contents of blood leucocytes in haemodialysis Horizontal bars indicate median values of data sets. Conversion selenium: 1 pg/106 cells = 12.7 fmol/106 cells.
patients and healthy control subjects. of traditional to SI units - leucocyte
1.49 5 0.46 pmol/106 cells (117.4 f 36.4 pg/106 cells) and 2.24 * 0.62 pmol/106 cells (176.7 f 49.2 pg/106 cells), respectively (p < 0.001). These data are shown in Fig. 2. Discussion
Severe selenium deficiency in man results in potentially fatal skeletal and cardiac myopathies [g-11]. More moderate deficiency states appear to be associated with increased risks of cardiovascular death and myocardial infarction [12], and with an increased incidence of cancer of the gastrointestinal tract and the prostate [13]. Features similar to these are also found with abnormal frequency in uraemic patients maintained with haemodialysis. Congestive cardiomyopathy, for example, is detectable by echocardiography or ventriculography without evidence of ischaemic heart disease in a large proportion of haemodialysis patients [16,17], and accelerated atherosclerosis is a major cause of morbidity and mortality in these cases [18]. Patients treated by haemodialysis are also reported to suffer an increased incidence of cancer [19]. It is difficult to ignore the possibility that these complications result from a previously unrecognised deficiency of selenium. The assessment of selenium status is complicated since no single measurement provides a reliable indication of body selenium reserves in all circumstances [20]. Plasma selenium concentrations are commonly used however, and they respond rapidly to deficiency states induced by a low dietary intake of the element [21,22].
The reduced plasma concentrations of selenium found in the uraemic patients investigated in this study are clearly relevant. Erythrocyte selenium concentrations and glutathione peroxidase activities are widely used to complement plasma measurements during the clinical assessment of selenium availability. However, we have chosen not to use these measurements because of the well known abnormalities of red blood cell production and survival that are found with advanced renal failure. Instead, we have determined the selenium content of blood leucocytes which have the advantage of being a metabolically active tissue that can be repeatedly sampled without undue trauma. The use of Ieucocyte analyses to assess body selenium status has not been evaluated as far as we are aware, but similar measurements of white blood cell composition have proved helpful in the diagnosis of deficiencies of other trace elements, such as zinc, both in normal individuals and in uraemic patients [23,24]. It is therefore interesting that the selenium contents of the leucocytes obtained from the haemodialysis patients investigated in this study should be so low. Abnormalities of trace element metabolism in advanced renal failure have only attracted significant attention during the last decade. Uraemia was previously thought to result largely from the retention of organic toxins, and the persistence of uraemic symptoms after the institution of haemodialysis was held to be due to the poor clearance of some of these chemical species through the dialysis membranes that were currently available. Recent reports challenge this view. Thus, uraemic encephalopathy and dialysis oste~ystrophy are thought to be associated with the accumulation of aluminium [l-3], arsenic has been implicated in the pathogenesis of renal anaemia [7], and cobalt excess has been suggested as a contributory factor in uraemic heart failure [8]. Uraemic abnormalities of trace element metabolism do not necessarily take the form of toxicity states; zinc deficiency is thought to occur in end-stage renal failure and to cause testicular failure [4], impaired cell-mediated immunity [5], and anorexia and distaste for dietary protein [6]. It is conceded that the reduced plasma and leucocyte selenium levels reported here do not provide unequivocal evidence of selenium deficiency. However their occurrence in patients who suffer unduely from atherosclerosis, cardiomyopa~y, and malignant disease is int~guing, and they indicate a need for further research. It is only possible to speculate on the pathogenesis of this putative selenium deficiency state. However, their anorexia and distaste for dietary protein frequently cause uraemic patients to eat inadequate amounts of meat and fish, and these are the most effective dietary sources of selenium [25]. Acknowledgements
We are grateful to Professor B.E. Clayton for her helpful discussions and support, and to Professor A. Polak, Professor H.A. Lee and ‘Dr. D.J. Warren for permission to study patients in their care at the Wessex Regional Renal Unit at St. Mary’s Hospital, Portsmouth. J.W.F. also gratefully acknowledges financial support from BDH Chemicals Ltd, the Wessex Regional Health Authority, the Wessex Medical School Trust and the Wellcome Trust.
328
References 1 Parkinson IS, Ward MK, Kerr DNS. Dialysis encephalopathy, bone disease and anaemia: the ahuninium intoxication syndrome during regular dialysis. J Clin Path01 1981;34;1285-1294. 2 Davidson AM, Walker GS, Oli H, Lewins AM. Water supply aluminium concentration, dialysis dementia, and effect of reverse-osmosis water treatment. Lancet 1982;%785-787. 3 Nathan E, Pederson S. Dialysis encephalopathy in a non-dialysed uraemic boy treated with almninium hydroxide orally. Acta Paediatr Stand 1980;69:793-796. 4 Antoniou LD, Shalboub RJ, Sudakar T, Smith JC. Reversal of uraemic impotence by zinc. Lancet 1977;ii:895-898. 5 Antoniou LD, Shalhoub RJ, Schechter GP. The effect of zinc on cellular immunity in chronic uremia. Am J Clin Nutr 1981;34:1912-1917. 6 A&in-Thor E, Goddard BW, O’Nion J, Stephen RL, Kolff WJ. Hypogeusia and zinc depletion in chronic dialysis patients. Am J Clin Nutr 1978;31:1948-1951. 7 Pershagen G, Hast R, Lins LE, Pehrsson K. Increased arsenic concentration in the bone marrow in chronic renal failure - a contributor to anaemia? Nephron 1982;30:250-252. 8 Pehrsson K, Lms LE. Cobalt in uraemic c~diomyopathy. Lancet 1978;ii:51-52. 9 ~onymous. Selenium in the heart of China {atone). Lancet 1979;~:889-8#. 10 Johnson RA, Baker SS, Fallon JT, et al. An occidental case of ~r~omyopathy and selenium deficiency. N Engl J Med 1981;3~:1210-1212. 11 Fleming CR, Lie JT, McCall JT, O’Brien JF, Baillie EE, Thistle JL. Selenium deficiency and fatal ~rdiomyopa~y in a patient on home parenteral nutrition. Gastroenterology 1982;83:689-693. 12 Salonen JT, Alfthan G, Huttunen JK, Piiarainen J, Puska P. Association between cardiovascular death and myocardial infarction and serum selenium in a matched-pair longitudinal study. Lancet 1982;&175-179. 13 Willet WC, Polk BF, Morris JS, et al Prediagnostic serum selenium and risk of cancer. Lancet 1983;ii:130-134. 14 Lloyd B, Holt P, Delves HT. Determination of selenium in biological samples by hydride generation and atomic-absorption spectroscopy. Analyst 1982;107:927-933. 15 Hinks LJ, Colmsee M, Delves HT. Determination of zinc and copper in isolated leucocytes. Analyst 1982;107:815-823. 16 Drneke T, Le Pailleur C, Meilhac B, et al. Congestive cardiomyopathy in uraemic patients on long term haemodialysis. B Med J 1977;i:350-353. 17 Lai KN, Barnden L, Mathew TH. Effect of renal transplantation on left ventricular function in hemodialysis patients. Clin Nephrol 1982;18:74-78. 18 Lindner A, Charra B, Sherrard DJ, Scribner BH. Accelerated atherosclerosis in prolonged maintenance hemodialysis. N Engl J Med 1974;290:697-701. 19 Curtis JR. Cancer and patients with end-stage renal failure. B Med J 1982;284:69-70. 20 Neve J, Vertongen F, Molle L. Selenium deficiency. Clin Endocrinol Metab 1985;14:629-656. 21 Van Rij AM, Thomson CD, McKenzie JM, Robinson MF. Selenium deficiency in total and parenteral nutrition. Am J Clin Nutr 1979;32:2076-2085. 22 Janghorbani M, Kasper L, Young V. Dynamics of selenite metabolism in young men: studies with the stable isotope tracer method. Am J Clin Nutr 1984;#:208-218. 23 Jones RB, Keeling PWN, Hilton PJ, Thompson RPH. The relationship between leucocyte and muscle zinc in health and disease. Clin Sci 1981;60:237-239. 24 Mahajan SK, Prasad AS, Rabbani P, Briggs WA, Mc~n~d FD. Zinc metabotism in uraemia. J Lab Clin Med 1979;94:693-698. 25 Zabel NL, Harland J, Gormican AT, Ganther HE. Selenium content of commercial formula diets. Am J Clin Nutr 1978;31:850-858.