BIOLOGICAL TRACE ELEMENT RESEARCH 4, 105-115 (1982)
Copper, Manganese, Zinc, and Cadmium in Tissues from New Zealanders C L A R E E . C A S E Y , * BARBARA E . G U T H R I E , AND M A R I O N F . R O B I N S O N
Department of Nutrition, University of Otago, PO Box 56, Dunedin, New Zealand Received October 6, 1981; Accepted January 7, 1982
Abstract Concentrations of Cu, Mn, Zn, and Cd were measured in 13 different tissues collected at autopsy from 55 New Zealanders, aged 1 week to 74 years. All analyses were done by atomic absorption spectrophotometry. In general, concentrations of Mn and Zn were similar to those reported elsewhere, but Cu levels were slightly lower. Concentrations of Cd were low in all tissues except kidney. Median values were in accordance with those reported for other "unexposed" populations. A significant trend of increasing concentrations with age was found for Cu in cartilage, Zn in kidney cortex and medulla, and Cd in all tissues except bone, fat, and hair. Declines with age were observed for Cu in liver, aorta, and skeletal muscle, for Mn in heart, aorta, and cartilage and for Zn in lung and muscle. There were no obvious relationships between tissue trace element levels and cause of death assigned according to three groups: sudden accidental, cardiovascular, or respiratory. Index Entries: Trace elements, levels in human tissues; copper, in human tissues; manganese, in human tissues; zinc, in human tissues; cadmium, in human tissues; atomic absorption spectrophotometry, of trace elements in human tissues; human tissues, trace elements in; New Zealand, trace elements in human tissues in.
Introduction Investigations of the role of trace elements in health and disease are inhibited to a large extent by the lack of suitable indices of status in the livihg organism. How9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/6900--0105 $02.20
105
106
CASEY, GUTHRIE, AND ROBINSON
ever, analysis of tissues from healthy persons dying suddenly, (i.e., those without a disease or cause of death that may be expected significantly to alter trace element metabolism), may allow some assessment of the nutritional status of such elements on a population basis. Iron status, for example, can be readily assessed in the living population and the incidence of anemia thus determined agrees well with the incidence determined from analysis of storage Fe in the liver of accident victims (1). Such a correlation is not possible at present for most other elements; nonetheless, autopsy data is being used with increasing frequency to examine the metabolism of trace elements that have nutritional importance (2, 3) or that are of environmental concern (4, 5). New Zealand has an interesting history with respect to trace elements. Deficiencies of a number of essential minerals, including copper and selenium, have caused major problems in agriculture (6). The Se status of the human population is low compared with other countries (7). Less is known of the status of other trace elements of interest in human health and nutrition. The study reported here examined copper, manganese, zinc, and cadmium in various tissues obtained at autopsy from a group of New Zealanders. It was devised to provide information on the expected normal range of values for these elements in the New Zealand population. Such information may be used to examine differences within the population arising from nutritional, environmental, or disease factors, and to identify differences in nutritional status by comparison with values for populations in other countries.
Materials and Methods Tissue samples were obtained at autopsy from a total of 55 subjects who had died in Dunedin Hospital or were subject to a Coroner's Inquiry. At time of death, all except for neonates (obtained from Auckland) had been living in or near Dunedin. (Dunedin is a city of 113,000, situated in the south of the South Island of New Zealand. It has little heavy industry and the bulk of the food supply is produced locally.) No cancer-related deaths were included and none of the subjects was known to have had an occupational exposure to heavy metals. The age range varied from 1 week to 74 years and the age and sex distributions of subjects are given in Table 1. Table 1 also gives the age distribution of the adult subjects grouped according to the principal cause of death (COD). The groups included: A. Sudden accidental death: 16 subjects, mean age 33 years. B. Cardiovascular: 22 subjects, 57 years (includes heart failure, myocardial infarction, aneurysm and cerebral accident). C. Respiratory: 6 subjects, 58 years (includes pneumonia and chronic obstructive respiratory disease). Material was collected by the pathologist during the normal course of the postmortem, which was performed within 24 h of death. The pathologist wore disposable rubber gloves and used stainless steel scissors, forceps, and scalpel blades. Except for avoiding rinsing tissues in tap water, no other precautions could readily
107
TRACE ELEMENTS IN HUMAN TISSUES
TABLE 1 Distribution of Subjects According to Age, Sex, and Cause of Death Subjects, age range in years
Cause of deatha Males
Females
SA
Neonates (1-16 weeks) 5 3 1-5 3 15-29 7 2 9 30-39 2 1 40-49 3 3 2 50-59 9 4 2 60-69 7 4 1 70--79 2 1 I %A, sudden, accidental;CV, cardiovascular;RE, respiratory.
CV
RE
1
4 7 8 2
4 2
be taken. Samples of liver, lung, heart, spleen, and pancreas were taken from random parts of the organ, but were as large as possible, usually in the range of 30-50 g. Kidney was divided into cortex and medulla for separate analysis. Sources of other tissues included: aorta, 2-5 cm of ascending aorta; bone, rib; cartilage, knee cap; skeletal muscle, diaphragm; fat, abdominal wall. Hair was cut as close as possible to the scalp in the sub-occipital region. Tissues were placed in acid-washed plastic containers and taken immediately to the laboratory where they were stored frozen at -20~ until analysis. In preparation for analysis, organs, aorta, muscle, and fat were thawed and trimmed of extraneous tissue. Liver, heart, and kidney were freeze-dried; other tissues were dried under infrared lamps. Samples were then ground in a glass Waring Blender with stainless steel blades that had previously been found not to be a significant source of contamination of any of the trace elements. Bone and cartilage were prepared for ashing as described by McKenzie (8). All samples were ashed in silica crucibles in a muffle furnace at 450~ for 2-5 days. Where necessary, HNO3 was added as an ashing aid. Ash was dissolved in concentrated HC1 and diluted as appropriate with 0.1M HCI. Hair samples were washed with a nonionic detergent and EDTA, according to the procedure established by McKenzie (9), and then dry-ashed. Analyses for Cu, Mn, Zn, and Cd were performed by flame atomic absorption spectrophotometry, using appropriate aqueous standards diluted from 1000 Ixg/mL Certified Atomic Absorption Standard Solutions (Fisher Scientific Company, New Jersey, USA). Lung, spleen, pancreas, aorta, and muscle were analyzed on the AA5 (Varian Techtron Pty Ltd, Springvale, Victoria, Australia). Background correction was made for Cd by serial absorbance readings with a H2 lamp. All other tissues were analysed using an IL 251 (Instrumentation Laboratories, Wilmington, MA 01887, USA), with automatic continuous background correction for all elements. The analyses spanned a 4-year period and precision of technique was checked periodically by analyzing stock samples of liver, heart, and kidney. These were
108
CASEY, GUTHRIE, AND ROBINSON
prepared at the beginning of the study by pooling 2-3 kg of bovine tissue that was then dried and ground. At regular intervals throughout the study period, duplicates of the stocks were carried through the analytical procedures with samples. Recovery studies were also done by adding the element as a standard solution to the stock samples prior to ashing. Table 2 gives the mean values and coefficients of variation over the 4 years for the concentrations of Cu, Mn, Zn, and Cd in the stocks, and the recovery of added metal. Bovine liver (Standard Reference Material No. 1577, National Bureau of Standards, Washington, DC) was also used as an analytical control. Concentrations of Mn, Zn, and Cd were not different from the certified values. Results for Cu (mean - SD: 180 -+ 1 Ixg) were lower than the certified value (mean - 95% confidence limits: 193 --- 10 I~g/g) early in the study, but not when the bovine liver was reanalyzed 18 months later. No change was found in Cu concentrations in the stock tissues over this period. Manipulation of data and statistical analyses were carried out on a Burroughs B6,700 Computer using job-tailored programmes. A significance level of 1% or better was used in the interpretation of all statistical tests. TABLE 2 Concentrations of Cu, Mn, Zn, and Cd, and Recoveries of Added Metal, in Stock Samples of Liver, Heart, and Kidney, Analyzed over a 4 Year Period
Liver Concentration, txg/g
Recovery, %
Heart Concentration, ixg/g
Recovery, %
Kidney Concentration, Ixg/g
Recovery, %
Cu
Mn
Zn
Cd
n Mean CV n Mean CV
40 25.4 6.7 19 102 6
31 9.5 7.4 19 102 5
36 112 8.0 19 100 3
32 0.15 13.3 13 94 7
n Mean CV n Mean CV
47 13.1 6.9 27 98 5
48 1.2 8.3 22 97 2
42 67.7 4.1 30 101 3
48 0.026 15.4 15 99 2
n Mean CV n Mean CV
31 11.0 10.5 26 99 4
32 3.8 8.2 24 99 3
32 31 110 3.1 5.5 6.5 28 25 101 100 3 2
109
TRACE ELEMENTS IN H U M A N TISSUES
Results The concentration of Cu, Mn, Zn, and Cd in the 13 tissues examined are given in Table 3. Since most values were not normally distributed, the results are presented as the medians and 10th and 90th centiles for the whole group. The largest deviations from a normal distribution were found, not unexpectedly, for Cd. ConcentraTABLE 3 Concentrations of Cu, Mn, Zn, and Cd, and Water, in Human Tissues Water (g/g wet weight)
Cu
50
0.71 a (0.65-0.76) 0.81 (0.79-0.86) 0.77 (0.75-0.80) 0.77 (0.73-0.80) 0.79 (0.74-0.82) 0.77 (0.71-0.79) 0.71 (0.53-0.78) 0.62 (0.51-0.75) b
Cartilage
47
b
Skeletal muscle
48
Fat
41
0.75 (0.71-0.80) b
Hair
45
b
23.4 (12.6-109) 4.97 (3.8%8.77) 12.3 (9.38-15.0) 10.1 (8.27-12.4) 7.05 (5.47-8.45) 2.91 (2.19-4.28) 3.70 (2.01-5.40) 2.86 (1.87-4.36) 2.09 (1.03-3,57) 2.47 (0.88-4.04) 3.00 (1.59-5.13) 0.37 (0.16-1.05) 13.0 (8.24-24.8)
Tissue
n
Liver
53
Lung
52
Heart
46
Kidney cortex
46
Kidney medulla
45
Spleen
48
Pancreas
43
Aorta
43
Bone
aMedian (10-90th centiles). bWet and dry weight not different. Cn = 49. dn = 51. en = 45. fn = 47.
Mn Zn (txg/g dry weight) 3.89 (2.73-5.92) 0.58 (0.43-0.88) 0.68 (0.44--1.03) 3.54 (2.08-5.31) 1.49 (0.63-2.62) 0.53 (0.37-0.79) 2.43 (1.42-3.86) 0.32 (0.20-0.49) 0.36 (0.19-0.77) 0.16 (0.08-0.71) 0.45 (0.31-0.77) 0.05 (0.02-0.11) 0.80 (0.25-2.26)
Cd
199 4.65 (123-298)(0.06-17.0) 50.2 0.57 a (3.93-68.4)(0.06-4.56) 99.1 0.12 e (87.7-125) (0.04-0.34) 176 86.1 (99.1-267) (5.84--297) 81.0 25.8 (58.6-128) (1.15-100) 61.4 0.40 (43.9-78.7)(0.0%1.80) 89.0 1.85 (44.2-131) (0.32-6.99) 35.9 0.30 (23.7-51.8)(0.10-1.28) 151 0.20 (114-205) (0.10--0.50) 26.2 0.07 (15.0-47.4) (0.03-0.18) 170c 0.19f (143-239) (0.06-0.64) 2.26 0.03 (1.59-3.89) (0.00-0.16) 144 0.40 (74.0-172) (0.11-1.27)
110
CASEY, GUTHRIE, AND ROBINSON
tions of all elements are given on a dry weight basis. The water content is also given, as g/g fresh tissue, except for bone, cartilage, and hair in which wet weights were not different from dry weights. Highest levels of Cu, Mn, and Zn were in the liver, followed by heart and hair for Cu, cortex and pancreas for Mn, and cortex, bone and muscle for Zn. Kidney cortex contained the highest levels of Cd, the median being threefold greater than the next highest in kidney medulla and 20 times greater than in liver. The lowest concentrations of all elements were in fat; Cd levels in five samples were below the level of detection (
Copper, Manganese, Zinc, and Cadmium in Tissues from New Zealanders C L A R E E . C A S E Y , * BARBARA E . G U T H R I E , AND M A R I O N F . R O B I N S O N
Department of Nutrition, University of Otago, PO Box 56, Dunedin, New Zealand Received October 6, 1981; Accepted January 7, 1982
Abstract Concentrations of Cu, Mn, Zn, and Cd were measured in 13 different tissues collected at autopsy from 55 New Zealanders, aged 1 week to 74 years. All analyses were done by atomic absorption spectrophotometry. In general, concentrations of Mn and Zn were similar to those reported elsewhere, but Cu levels were slightly lower. Concentrations of Cd were low in all tissues except kidney. Median values were in accordance with those reported for other "unexposed" populations. A significant trend of increasing concentrations with age was found for Cu in cartilage, Zn in kidney cortex and medulla, and Cd in all tissues except bone, fat, and hair. Declines with age were observed for Cu in liver, aorta, and skeletal muscle, for Mn in heart, aorta, and cartilage and for Zn in lung and muscle. There were no obvious relationships between tissue trace element levels and cause of death assigned according to three groups: sudden accidental, cardiovascular, or respiratory. Index Entries: Trace elements, levels in human tissues; copper, in human tissues; manganese, in human tissues; zinc, in human tissues; cadmium, in human tissues; atomic absorption spectrophotometry, of trace elements in human tissues; human tissues, trace elements in; New Zealand, trace elements in human tissues in.
Introduction Investigations of the role of trace elements in health and disease are inhibited to a large extent by the lack of suitable indices of status in the livihg organism. How9 1982 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/82/6900--0105 $02.20
105
106
CASEY, GUTHRIE, AND ROBINSON
ever, analysis of tissues from healthy persons dying suddenly, (i.e., those without a disease or cause of death that may be expected significantly to alter trace element metabolism), may allow some assessment of the nutritional status of such elements on a population basis. Iron status, for example, can be readily assessed in the living population and the incidence of anemia thus determined agrees well with the incidence determined from analysis of storage Fe in the liver of accident victims (1). Such a correlation is not possible at present for most other elements; nonetheless, autopsy data is being used with increasing frequency to examine the metabolism of trace elements that have nutritional importance (2, 3) or that are of environmental concern (4, 5). New Zealand has an interesting history with respect to trace elements. Deficiencies of a number of essential minerals, including copper and selenium, have caused major problems in agriculture (6). The Se status of the human population is low compared with other countries (7). Less is known of the status of other trace elements of interest in human health and nutrition. The study reported here examined copper, manganese, zinc, and cadmium in various tissues obtained at autopsy from a group of New Zealanders. It was devised to provide information on the expected normal range of values for these elements in the New Zealand population. Such information may be used to examine differences within the population arising from nutritional, environmental, or disease factors, and to identify differences in nutritional status by comparison with values for populations in other countries.
Materials and Methods Tissue samples were obtained at autopsy from a total of 55 subjects who had died in Dunedin Hospital or were subject to a Coroner's Inquiry. At time of death, all except for neonates (obtained from Auckland) had been living in or near Dunedin. (Dunedin is a city of 113,000, situated in the south of the South Island of New Zealand. It has little heavy industry and the bulk of the food supply is produced locally.) No cancer-related deaths were included and none of the subjects was known to have had an occupational exposure to heavy metals. The age range varied from 1 week to 74 years and the age and sex distributions of subjects are given in Table 1. Table 1 also gives the age distribution of the adult subjects grouped according to the principal cause of death (COD). The groups included: A. Sudden accidental death: 16 subjects, mean age 33 years. B. Cardiovascular: 22 subjects, 57 years (includes heart failure, myocardial infarction, aneurysm and cerebral accident). C. Respiratory: 6 subjects, 58 years (includes pneumonia and chronic obstructive respiratory disease). Material was collected by the pathologist during the normal course of the postmortem, which was performed within 24 h of death. The pathologist wore disposable rubber gloves and used stainless steel scissors, forceps, and scalpel blades. Except for avoiding rinsing tissues in tap water, no other precautions could readily
107
TRACE ELEMENTS IN HUMAN TISSUES
TABLE 1 Distribution of Subjects According to Age, Sex, and Cause of Death Subjects, age range in years
Cause of deatha Males
Females
SA
Neonates (1-16 weeks) 5 3 1-5 3 15-29 7 2 9 30-39 2 1 40-49 3 3 2 50-59 9 4 2 60-69 7 4 1 70--79 2 1 I %A, sudden, accidental;CV, cardiovascular;RE, respiratory.
CV
RE
1
4 7 8 2
4 2
be taken. Samples of liver, lung, heart, spleen, and pancreas were taken from random parts of the organ, but were as large as possible, usually in the range of 30-50 g. Kidney was divided into cortex and medulla for separate analysis. Sources of other tissues included: aorta, 2-5 cm of ascending aorta; bone, rib; cartilage, knee cap; skeletal muscle, diaphragm; fat, abdominal wall. Hair was cut as close as possible to the scalp in the sub-occipital region. Tissues were placed in acid-washed plastic containers and taken immediately to the laboratory where they were stored frozen at -20~ until analysis. In preparation for analysis, organs, aorta, muscle, and fat were thawed and trimmed of extraneous tissue. Liver, heart, and kidney were freeze-dried; other tissues were dried under infrared lamps. Samples were then ground in a glass Waring Blender with stainless steel blades that had previously been found not to be a significant source of contamination of any of the trace elements. Bone and cartilage were prepared for ashing as described by McKenzie (8). All samples were ashed in silica crucibles in a muffle furnace at 450~ for 2-5 days. Where necessary, HNO3 was added as an ashing aid. Ash was dissolved in concentrated HC1 and diluted as appropriate with 0.1M HCI. Hair samples were washed with a nonionic detergent and EDTA, according to the procedure established by McKenzie (9), and then dry-ashed. Analyses for Cu, Mn, Zn, and Cd were performed by flame atomic absorption spectrophotometry, using appropriate aqueous standards diluted from 1000 Ixg/mL Certified Atomic Absorption Standard Solutions (Fisher Scientific Company, New Jersey, USA). Lung, spleen, pancreas, aorta, and muscle were analyzed on the AA5 (Varian Techtron Pty Ltd, Springvale, Victoria, Australia). Background correction was made for Cd by serial absorbance readings with a H2 lamp. All other tissues were analysed using an IL 251 (Instrumentation Laboratories, Wilmington, MA 01887, USA), with automatic continuous background correction for all elements. The analyses spanned a 4-year period and precision of technique was checked periodically by analyzing stock samples of liver, heart, and kidney. These were
108
CASEY, GUTHRIE, AND ROBINSON
prepared at the beginning of the study by pooling 2-3 kg of bovine tissue that was then dried and ground. At regular intervals throughout the study period, duplicates of the stocks were carried through the analytical procedures with samples. Recovery studies were also done by adding the element as a standard solution to the stock samples prior to ashing. Table 2 gives the mean values and coefficients of variation over the 4 years for the concentrations of Cu, Mn, Zn, and Cd in the stocks, and the recovery of added metal. Bovine liver (Standard Reference Material No. 1577, National Bureau of Standards, Washington, DC) was also used as an analytical control. Concentrations of Mn, Zn, and Cd were not different from the certified values. Results for Cu (mean - SD: 180 -+ 1 Ixg) were lower than the certified value (mean - 95% confidence limits: 193 --- 10 I~g/g) early in the study, but not when the bovine liver was reanalyzed 18 months later. No change was found in Cu concentrations in the stock tissues over this period. Manipulation of data and statistical analyses were carried out on a Burroughs B6,700 Computer using job-tailored programmes. A significance level of 1% or better was used in the interpretation of all statistical tests. TABLE 2 Concentrations of Cu, Mn, Zn, and Cd, and Recoveries of Added Metal, in Stock Samples of Liver, Heart, and Kidney, Analyzed over a 4 Year Period
Liver Concentration, txg/g
Recovery, %
Heart Concentration, ixg/g
Recovery, %
Kidney Concentration, Ixg/g
Recovery, %
Cu
Mn
Zn
Cd
n Mean CV n Mean CV
40 25.4 6.7 19 102 6
31 9.5 7.4 19 102 5
36 112 8.0 19 100 3
32 0.15 13.3 13 94 7
n Mean CV n Mean CV
47 13.1 6.9 27 98 5
48 1.2 8.3 22 97 2
42 67.7 4.1 30 101 3
48 0.026 15.4 15 99 2
n Mean CV n Mean CV
31 11.0 10.5 26 99 4
32 3.8 8.2 24 99 3
32 31 110 3.1 5.5 6.5 28 25 101 100 3 2
109
TRACE ELEMENTS IN H U M A N TISSUES
Results The concentration of Cu, Mn, Zn, and Cd in the 13 tissues examined are given in Table 3. Since most values were not normally distributed, the results are presented as the medians and 10th and 90th centiles for the whole group. The largest deviations from a normal distribution were found, not unexpectedly, for Cd. ConcentraTABLE 3 Concentrations of Cu, Mn, Zn, and Cd, and Water, in Human Tissues Water (g/g wet weight)
Cu
50
0.71 a (0.65-0.76) 0.81 (0.79-0.86) 0.77 (0.75-0.80) 0.77 (0.73-0.80) 0.79 (0.74-0.82) 0.77 (0.71-0.79) 0.71 (0.53-0.78) 0.62 (0.51-0.75) b
Cartilage
47
b
Skeletal muscle
48
Fat
41
0.75 (0.71-0.80) b
Hair
45
b
23.4 (12.6-109) 4.97 (3.8%8.77) 12.3 (9.38-15.0) 10.1 (8.27-12.4) 7.05 (5.47-8.45) 2.91 (2.19-4.28) 3.70 (2.01-5.40) 2.86 (1.87-4.36) 2.09 (1.03-3,57) 2.47 (0.88-4.04) 3.00 (1.59-5.13) 0.37 (0.16-1.05) 13.0 (8.24-24.8)
Tissue
n
Liver
53
Lung
52
Heart
46
Kidney cortex
46
Kidney medulla
45
Spleen
48
Pancreas
43
Aorta
43
Bone
aMedian (10-90th centiles). bWet and dry weight not different. Cn = 49. dn = 51. en = 45. fn = 47.
Mn Zn (txg/g dry weight) 3.89 (2.73-5.92) 0.58 (0.43-0.88) 0.68 (0.44--1.03) 3.54 (2.08-5.31) 1.49 (0.63-2.62) 0.53 (0.37-0.79) 2.43 (1.42-3.86) 0.32 (0.20-0.49) 0.36 (0.19-0.77) 0.16 (0.08-0.71) 0.45 (0.31-0.77) 0.05 (0.02-0.11) 0.80 (0.25-2.26)
Cd
199 4.65 (123-298)(0.06-17.0) 50.2 0.57 a (3.93-68.4)(0.06-4.56) 99.1 0.12 e (87.7-125) (0.04-0.34) 176 86.1 (99.1-267) (5.84--297) 81.0 25.8 (58.6-128) (1.15-100) 61.4 0.40 (43.9-78.7)(0.0%1.80) 89.0 1.85 (44.2-131) (0.32-6.99) 35.9 0.30 (23.7-51.8)(0.10-1.28) 151 0.20 (114-205) (0.10--0.50) 26.2 0.07 (15.0-47.4) (0.03-0.18) 170c 0.19f (143-239) (0.06-0.64) 2.26 0.03 (1.59-3.89) (0.00-0.16) 144 0.40 (74.0-172) (0.11-1.27)
110
CASEY, GUTHRIE, AND ROBINSON
tions of all elements are given on a dry weight basis. The water content is also given, as g/g fresh tissue, except for bone, cartilage, and hair in which wet weights were not different from dry weights. Highest levels of Cu, Mn, and Zn were in the liver, followed by heart and hair for Cu, cortex and pancreas for Mn, and cortex, bone and muscle for Zn. Kidney cortex contained the highest levels of Cd, the median being threefold greater than the next highest in kidney medulla and 20 times greater than in liver. The lowest concentrations of all elements were in fat; Cd levels in five samples were below the level of detection (
