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Quantification of trace elements in normal human brain by inductively coupled plasma atomic emission spectrometry
JOURNALOFTNE
NEUROLOGICAL SCIENCES Journal of the Neurological Sciences 146 (1997) 153-166
Quantificationof trace elements in normal human brain by inductively coupled plasma atomic emission spectrometry M.T. Rajan a, K.S. JagannathaRao a’*,B.M. Mamathaa,R.V. Rao b,P. Shanmugavelub, Rani. B. Menonb,M.V.Pavithranb aDepartment oj’Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore-570 013, India b Analytical Control Section, Rare Materials Project, Bhabha Atomic Research Centre, Mysore-571
130, India
Received 22 April 1996; revised 6 August 1996; accepted 21 August 1996
Abstract Eight normal human brain autopsy samples were analyzed for Na, K, P, Ca, Mg, Si, Cr, Cu, Ni, Zn, Fe, Al, Cd, Pb and As in 12 regions of brain (frontal cerebrum, temporal cerebrum, parietal cerebrum, somatosensory cortex, occipital cerebrum, cerebellum, mid-brain, pens, hypothalamus, thalamus, hippocampus and medulla oblongata) using inductively coupled plasma atomic emission spectrometry (lCPAES). The distribution of these 15 elements varied significantly from region to region of the brain. Potassium was most abundant in nearly all regions of the brain, followed by sodium and phosphorus (mg/g). The concentration of Al was found to be comparatively high and varied in different areas of the brain (58–196 Wg/g). Moderate levels of Pb, Cd and As were observed in different regions. Ratios of Al to Fe were found to be high in temporal cerebrum (8.07) and hippocampus (9.05) and these two regions are significantly involved in Alzheimer’s disease. The concentration of Na in mole percentage showed an inverse correlation with that of K, Ca, Mg, Fe and Cr. Direct correlation was observed in the concentration of all analyzed elements, which indicated for the first time the direct dependency of concentration of trace elements in one brain region to other regions. The mole ratios between different elements in different brain regions and total amounts of the elements in an average weight of 1.4 kg human brain were also computed. The present study provides new and in-depth data which may be used as base line data for normal human brains. 0 1997 Elsevier Science B.V. All rights reserved. Keywords: Human brain; Trace element; Inductively coupled plasma atomic emission spectrometry
1. Introduction Trace elements play a dual role in the biological system through their interaction with biomolecules. They regulate a number of celhtlar metabolic reactions, with a few of
them acting as etiological agents in many environmentally induced neurological diseases (Strong and Garruto, 1994; Garruto et al., 1993). Elements like sodium, magnesium, potassium, calcium, and phosphorus serve as structural components of tissues, as constituents of the body fluids and therefore, are essential for the function of cells. (Mertz, 1981). Trace elements in optimum levels are required for a number of metabolic and physiological processes in human body. Even though these are required for many biological
“ Corresponding author. Present address: Department of Pathology, University of Virginia, Box 168, Charlottesville, Virginia 22908, USA. Fax: + 1 (804) 924 2574. Tel: + 1 (804) 924 5682. E-mail: [email protected] 0022-510X\ 97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIJ S0022-510X(96)O03 OO-O
functions and their deficiencies would cause various metabolic disorders, the amount of their requirement in most of the cases is very less (Mertz, 1995). Essential trace elements at high levels are toxic and adversely affect the biological activities. Recently, it has been reported that Zn which is an essential trace element is capable of inducing A ~ protein clump formation; similar to the amyloid plaque found in the brains of Alzheimer’s patients (Kaiser, 1994). Non-essential trace elements like Al, Pb and Cd are also present in the biological tissues. They are highly toxic and are reported to be associated with a number of neurological disorders found in man (Crapper Mc Lachlan and DeBoni, 1980; Barrington et al., 1993). The role of these metals is suspected in many diseases whose etiology is still unknown. For example, Al is suspected to be involved in the pathogenesis of Alzheimer’s disease (Crapper Mc Lachlan et al., 1990). The increasing amounts of trace elements, particularly heavy metals, in animal tissues have been a cause of great concern, since their deposition in
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different vital organs is capable of inducing cellular injuries. Heavy metal ions are extremely toxic due to their diverse mode of action on various levels of biological organization. They are known as carcinogens and mutagenes by their interaction with DNA and also induce cellular damage through lipid peroxidation (Goering et al., 1987). Because of the wide-spread contamination of life sustaining factors like food, water and air with metals, the total body burden of trace metals has been increasing alarmingly (Jagannatha Rao and Valeswara Rae, 1992). The absorbed metals are metabolically non-biodegradable and their accumulation in the body leads to chronic effects. The only way of their elimination from the body is by excretion. The major organs in human body where metals are reported to be accumulated are kidney, liver, bone marrow, blood spleen, brain and muscle, etc. Recent studies have shown that there is a potential relation between the levels of heavy metals and incidence of neurological disorders (Martyn et al., 1989). Very limited studies are available on the levels of metals in human brain in normal and pathological conditions (ICRP report, 1959; Crapper et al., 1973; Markesbery et al., 1984; Uitti et al., 1987 and Uitti et al., 1989; Luckiw et al., 1991; Wender et al., 1992; Yoshida et al., 1993; Zecca and Swartz, 1993; Zecca et al., 1994). Most of this information is limited to a few selected regions of the brain and neuromelanin pigments (Zecca et al., 1994). There is also no study which examines a cross-relation of metal to metal-dependent retention in brain tissues. To generate normalized values in brain, a base-line data bank on trace elements in different regions of the brain from different countries need to be computed. Since there is no report in this direction from elsewhere and particularly from India, the present investigation was undertaken. Furthermore, in developing countries like India the magnitude of environmental contamination is very high, implying that the chance is higher for human brain to be burden by environmental substances. The progress in understanding the role of metals in disease of the nervous system has been hampered to a I&ge extent by lack of data on normal metal concentrations in the human brain. Since several metals interact metabolically, concurrent metal levels are essential for clinical correlation (Uitti et al., 1987). Therefore, an attempt has been made to generate data on the levels of trace metals in 12 important regions of the human brain using Inductively Coupled Plasma Atomic Emission Spectrometer (ICPAES) and attempts were also made to correlate values between elements in different regions of the brain. 2. Materials and methods 2.1. Brain tissue Autopsies of human brain taken from eight postmortem bodies were obtained from the Department of Forensic
.%iences 146 (1997) 153–166
Sciences, Government Medical College, Mysore, India. The whole brain was removed during post-mortem from eight male subjects in the age group of 50–60 years. The subjects had no history of psychiatric, neurological or any other neuro-degenerative disorder as per their medical records. They were neurologically quite normal. Autopsies were carried out within 24 h after death. The whole brain was removed, rinsed in ultra-pure water with resistivity of 18 ML?-cm (collected by using ELGA UHQ II system), wrapped with pre-washed polyethylene sheets and stored in a pre-washed plastic container and frozen at – 20°C until dissection. Special care was taken to avoid contamination with metals during handling and dissection, as described by Savory and Wills (1986) and Moody and Lind Strom (1977). The brain was dissected on a polyethylene plate with stainless steel tools (knives, scissors, forceps etc.) coated with titanium nitride (Zecca et al., 1994). Prior to dissection of the brain, the meninges along with blood vessels were carefully removed using a forceps, followed by rinsing of the dissected brain samples in ultra-pure water in order to minimize contamination due to cerebrospinal fluid and blood. Talc-free rubber surgical gloves, pre-treated with 2% quartz distilled nitric acid and washed thoroughly in ultra-pure water were used during handling and dissection of the brain. Twelve important regions of the brain based on anatomical lines viz., (1) frontal cerebrum (2) temporal cerebrum (3) parietal cerebrum (4) somatosensory cortex (5) occipital cerebrum (6) cerebellum (7) mid-brain (8) pens (9) hypothalamus (10) thalamus (1 1) hippocampus (12) medulla oblongata were dissected using a brain atlas (Gross Clark, 1956). The brain regions were dissected under the supervision of an anatomy professor from J.S.S. Medical College, Mysore, India. Brain samples taken from the cerebral regions contained both white and grey matters. The internal structures were also present in the brain samples taken from the mid brain, pens, thalamus, medulla oblongata etc. 2.2. Sample preparation All glass wares and silica crucibles were treated with di- sodium EDTA solution (0.1%), rinsed in distilled water and were kept immersed in a beaker containing 10910nitric acid (BDH analytical grade) for 24 h, followed by rinsing thoroughly with ultra-pure water. Pre-weighed brain samples were taken into silica crucibles and charred completely. The charred samples were ashed in a muffle furnace at 600° C for 8 h. The crucibles that had a spout were covered with lids in order to avoid the contamination from the wall of the muffle furnace. The ashed brain samples were treated with con. HNO~ (BDH analytical grade, quartz distilled) and mixture was heated for 30 min in a water bath. The mixture was diluted with ultra-pure water (collected by using ELGA UHQ 11system), filtered, and made up to 20 ml so as to get a final acid concentra-
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tion of 5%. Since the sample weights were different and concentrations in which elements are present are also different, appropriate dilutions were made before analysis. Sample preparation and analysis were carried out in a dust-free air-conditioned laboratory at Rare Materials Project, BARC, Mysore.
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155
15 elements and their concentrations were found to be very low. The blank value for Al was estimated to be 0.5–0.9 ppm, which was found to be very low when compared with concentrations of Al in brain samples. The blank values were deducted from the sample values. 2.5. Recovery study and reference material analysis
2.3. Instrumentation Inductively coupled plasma atomic emission spectrometer (ICPAES) model JY 38 was used for the sequential analysis of Na, K, Ca, P, Mg, Fe, Zn, Cu, Ni, Si, Al, Pb, Cd, As, and Cr. Argon plasma was used for excitation of the elemental atoms. The lines were selected for each element in such a way that interference from other elements was minimum. The wave length used and detection limit of the elements are summarized below. Element
Wavelength (rim)
Detection limit * (ppm)
Al As Ca Cd Cr Cu Fe K Mg Na Ni P Pb Si Zn
396.152 193.759 393.366 226.502 267.716 224.7 259.94 766.49 279.806 588.995 221.67 213.618 220.353 251.611 213.856
0.002 0.1 0.002 0.001 0.004 0.002 0.005 0.06 0.001 0.03 0.002 0.05 0.01 0.08 0.002
* Detection limit for each element was calculated by running a multi- element standard solution containing 500 ppb of each of the above cited elements. The instrumental conditions were as follows Incident power: 1.0 KW (57.6 MHz) Plasma Ar gas: 20 l/rein Auxiliary Ar gas: 0.6 l/rein Nebulizer Ar gas: 0.5 l/rein Spectral observation: Optics: 1 m Czerny-Turner Grating: Holographic 3600 l/rein Resolution: 0.009 nm Entrance slit width: 30 pm Exit slit width: 30 p,m Integration time: 400 ms 2.4. Blank The blanks (quartz-distilled A.R. grade nitric acid and ultra-pure water) were processed in the same way as the brain samples were processed. They were analysed for the
Known concentrations of multi-standard elements were added to the analyzed brain samples and the percentage of recovery for each element was computed. Low-cadmium reference material (unpolished rice flour) NIES-10 obtained from Japan was also analyzed to confirm the validity of the results. A multi element synthetic mixture (prepared by using Spex Indust. Inc. and Johnson Matthey England) containing all the 15 analyzed elements with the concentration equivalent to analyzed concentrations of elements in brain samples was also prepared and analyzed to confirm the validity of the results. The concentrations of trace elements were calculated as per gram of the wet tissue of the brain regions. The percentage elemental concentrations in mole were also calculated. 2.6. Statistics Mean and SD values were calculated from the total number of brains analyzed. Concentrations of element to element and brain region to region correlation were analyzed by linear regression analysis. One way analysis of variance (ANOVA) followed by Duncan’s t-test was also employed to check the variations in the trace element concentrations from one region of the brain to other.
3. Results The distribution of trace elements was not homogeneous in different regions of the brain and their concentrations varied significantly in different regions as shown in Tables 1 and 2. Potassium was more abundant in most of the regions, followed by Na and P, while Cd was found in the lowest concentration. The analyzed elements were classified into four sets based on the ranges of concentrations. First set of elements consisted of Na, K and P and their concentrations ranged from 1.17 to 4.4 mg/g. Second set of elements were Ca, Mg, and Al and their levels ranged from 58 to 196 ~g/g. Fe, Zn, Si, and Cu formed the third set and their concentrations varied from 3.5 to 59.5 ~g/g in all regions of the brain. Fourth set of elements consisted of As,. Ni, Pb, Cd, and Cr and their concentrations were comparatively very less and were found to be within a concentration limit of 0.015–2.64 ~g/g. The Al was found comparatively in high concentration and its levels varied greatly in different regions of the brain (58–196
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Elemental cone ( ~mol/g) = Elemental conc( Kg/g) /Atomic wt.
77 90 88 69 82 57 79 64 113 71 64 77
87 96 83 73 96 60 152 91 151 I02 117 170
6.5 5.7 7.6 5.I 6.5 6,7 5.1 5.3 4.2 4.6 4.8 5.6
Mg
Frontal cerebrum Temporal cerebrum Parietal cerebrum Somatosensory cortex Cerebrum occipital Cerebellum Mid-brain Pens Hypothalamus Tbalamus Hippocampus Medulla oblongata
Ca
K
Na
Brain regions P
in different regions of human brain
Table 3 Trace metal concentrations ( pmole/g)
0.9 0.9 I,0 0.7 0.7 0.8 0.5 0.3 0.5 0.7 0.5 0.3
Fe 0. I 0.1 0.4 0. I 0.2 0.1 0. I 0.1 0. I 0. I 0.1 0.3
Zn o. I 0.1 0. I 0.1 0.1 0. I 0.1 0. I o. I 0.1 o. I 0.1
Cu 4.2 7.3 4.1 2.6 2,2 2,3 3.4 2.I 3.4 4.8 4.2 3.9
Al 0.9 I.0 1.1 0.6 0.6 0.5 1.1 0.4 I.0 1.0 1.5 1.3
Si 0.011 0.020 0.009 0.009 0,008 0.007 0.016 0.009 0.017 0.013 0.015 0.016
As 0.005 I 0.0102 0.0034 0.005 I 0.0051 0.0034 0.0017 — 0.0034 0.005 I 0.0102
Ni
Cd 0.00018 0.00027 0.00018 0.00018 0.00318 0.00018 0.00027 0.00018 0.00027 0.00044 0.00018 0.00036
Pb 0.00193 0.00193 0,00193 0.00193 0.0029 0.00145 0.00338 0.0029 0.01255 0.00338 0.00434 0.0Q386
0.0029 0.0035 0.0031 0.0025 0.0027 0.0021 0.0023 0.0025 0.0021 0.0027 0.0019 0.0023
Cr
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34.0 37.2 33.2 34.8 36.8 35.5 46.7 38.9 42.5 38.4 45.7 48.9
Frontal cerebrum Temporal cerebrum Parietal cerebrum Somatosensory cortex Occipital cerebrum Cerebellum Mid-brain Pens Hypothalamus Tbalamus Hippocampus Medulla oblongata
30.0 34.8 35.4 32.9 3 I.5 33.7 24.3 27.2 31.8 26.5 24.9 22.1
K 29.3 20.4 24.0 25.8 26.1 22.4 24.8 28.9 21.9 29.I 23.9 24.2
P I.7 1.8 1.7 2.1 1.6 2.I I.2 1.4 1.3 I.5 1.2 1.4
Ca 2.54 2.21 3.04 2.43 2.52 3.99 1.58 2.28 1.19 1.75 1.86 1.60
Mg 0.34 0.35 0.40 0.32 0.27 0.45 0.16 o.13 0. I3 0.27 0.18 0.08
Fe 0.04 0.05 0. I5 0.07 0.08 0.07 0.04 0.05 0.03 0.04 0.04 0.09
Zn 0.02 0.03 0.04 0.03 0.03 0.07 0.03 0.02 0.02 0.02 0.02 0.02
Cu 1.64 2.83 1.64 1.22 0.84 1.34 1.03 0.91 0.97 1.80 1.62 1.13
Al 0.35 0.37 0.43 0.29 0.22 0.28 0.35 0.19 0.29 0.37 0.58 0.38
Si
Elemental cone (mole%) = Elemental conc( &mole/g)X 100/Total elemental cone ( pmole/g) of analysed elements in each region.
Na
Brain regions
Table 4 Mole percentage of analysed elements in different regions of human brain
0.004 0.008 0.004 0.004 0.003 0.004 0.005 0.004 0.005 0.005 0.006 0.005
As
0.032 0.004 0.001 0.002 0.002 0.002 0.001 — 0.001 0.002 0.003
Ni
0.00 I 0.031 0.001 0.001 0.001 0.001 0.001 0.001 0.004 0.001 0.002 0.001 I
Pb
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0002 0.0001 0.0001
Cd
➤ 0.001 I 0.0013 0.0012 0.0012 0.oo1o 0.0013 0.0007 0.001 I 0.0006 0.oo1o 0.0007 0.0007
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M.T. Rajan et al. /Journal
Sciences 146 (1997) 153–166 6.0
●
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Fig. 1. Inverse correlation of Na with K, Ca and Mg in different regions of humarr brain.
were highest in hypothalamus and the levels of Mg, Fe and Zn wer~ found ~~ be highest in ~arietal cerebrum. The concentrations of Cd, Cu and Si were highest in thalamus, cerebellum and hippocampus, respectively. Compared to other regions of the brain, in cerebellum Na, K, P, Cd, Pb and As were found in lowest concentrations.
Pg/g). Similady, concentrations of Fe also showed great variations in its levels in different regions of brain (16–59 Pg/g). Tbe concentrations of Na, P-and Ca were highest in medulla oblongata. In temporal cerebrum, the concentrations of Al, As, Ni, and Cr were found to be highest compared to other regions of brain. The levels of K and Pb
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2, Inverse correlation of Na with Fe and Cr in different regions of human brain.
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The most of the analyzed elements serve as structural components of the major biomolecules and also found in the bound form. The data are presented (Table 3) in mole concentrations in order to calculate mole/mole ratios and also to explain metal to metal correlation. It is evident from the Table 3 that the concentrations of Na in mole ( pmol/g) were found to be highest in all regions compared to those of other analyzed elements. This observation is quite contrary to that presented in Table 1, where the concentrations of K (mg/g) were found to be highest. This dissimilarity is attributed to the differences in the atomic weights of the elements. The percentage of mole elemental concentration of each analyzed elements in different regions of the brain is shown in Table 4. On the total mole elemental concentrations of all analyzed elements, the percentage of Na was highest and the value ranged from 33.2 to 48.9% in different regions of the brain. The percentage concentrations of Ca, Mg, Fe and Cu were highest in cerebellum compared to other regions. It is interesting to note that in the total mole elemental concentrations of all analyzed elements, the percentage concentrations of Na showed an inverse correlation with that of K, Ca, Mg, Fe and Cr (Figs. 1 and 2). This indicates that increases in concentrations of Na in different regions of the brain decrease the level of K, Ca, Mg, Fe and Cr and vice versa. In biological system there is an interdependency in the concentrations of certain elements to maintain homeostasis of trace elements pool. Based on this, element/element mole ratio in different regions for few elements having biological importance was calculated. Element to element mole ratio for Na/K, Na/P and K/P ranged from 0.9 to 2.2 in different regions of the brain (Table 5). Na/K and Na/P mole ratios were highest in medulla oblongata and ratios were 2.2 and 2.0, respectively. Al/Fe mole ratio showed great variations in different regions of the brain and ranged from a lowest value of 2.9 in occipital cerebrum to a highest value of 14.45 in the medulla oblongata. It is interesting to mention that Al/Fe mole ratio was comparatively high in temporal cerebrum (8.07) and hippocampus (9.05) and these two regions are highly susceptible to neuropathology in Alzheimer’s disease. In the pari-
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Table 7 Estimated total amounts of trace elements in an average weight of 1.4 kg of human brain K Na P Ca Mg Al Fe Si Zn Cu As Pb Ni Cr Cd
4.22c 0.77 g 3.36 + 1.0 g 2.8 + 0.56 g 227 +28 mg 192+ 32 mg 140+ 52 mg 52* 19 mg 36* 13 mg
15A 8 mg 6.5 + 1.5 mg l.3 + 0.4 mg 1~ 0.8 mg 0.4+ 0.2 mg 0.19 * 0.03 mg 0.04 + 0.01 mg
etal cerebrum Al and Ca were present in almost equal proportions and mole ratio was found to be 1:0.98. When A1/Mg ratio ranged from 0.33 to 1.28, A1/Si mole ratio varied from 2.8 in hippocampus to 7.64 in temporal cerebrum. Cu/Zn mole ratio was found to be highest in cerebellum (1:0.95) and lowest in medulla oblongata
(1:0.23). Interestingly, a direct correlation in the concentration of trace elements between some regions of brain was observed as shown in Table 6. The correlation coefficient r was found to be 0.99 in most of the cases. This indicates that a direct relationship is existing between the concentrations of trace elements in one region and those in the other regions. Estimated total amounts of trace elements in an average weight of 1.4 kg of human brain are shown in Table 7. This shows that amounts of K, Na and P present in total human brain are comparatively higher than those of other elements. The validation of the analysis was done by conducting recovery study and reference materials analysis. The recovery study showed 92–1027o recovery of added elements to the sample as shown in Table 8. Similarly, the analysis of rice flour reference material containing low level of Cd showed 0.023 ppm Cd against the certified
Table 6 Direct correlation in the concentration of trace metals between one brain region to other
Table 8 Recovery study of added elements
Brain regions
Correlation co-efficient r
Element
Frontal vs. temporal cerebrum Temporal vs. parietal cerebrum Parietal cerebrum vs. Somatosensory cortex Somatosensory cortex vs. occipital cerebrum Mid-brain vs. pens Hypothalamus vs. thalamus Tbalamus vs. hippocampus
0.97 a 0.99 a 0.99 a 0.99 a 0.98 a 0.97 a 0.98 ‘
Pa Mg ‘ Ca Zn b Al a Fe b
‘ P <0.05.
Sample cone. ppm
Added cone. ppm
Recovered ppm
Recovery (%)
52.4
20 I I 2 I I
70.7 3.27 2.92 4.38 I .99 10.69
91.5 100 100 100 Iol 102
2.27 1.92 2.38 0.98 9.67
a Stocksolution was diluted 100 times and analysed. b Stock solutions was diluted 10 times and analysed.
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Table 9 Analysis of synthetic mixture containing Na (0.35%) and K (0.52%) as matrix with following trace elements SLNO
Element
Actual (ppm)
Analysed (ppm)
1
Al As Ca Cd Cr Cu Fe Mg Ni P Pb Zn
2 2
2.3 2.2 19.99
2 3 4 5 6 7 8 9
10 11 12
20 0.05 0.3 5 20 0.4 26 2 2
0.07 0.34 0.94 4.9 21.7 0.39 26.3 2.2 2.1
value of 0.023 ppm, indicating the reliability of our reported vah.tes. Further, the analysis of a synthetic mixture containing all the analyzed elements, having a known concentration ( ~g/ml) equal to that we already analyzed in different brain regions, produced the values very near to the known concentrations of the elements (Table 9). This indicates the good line selection and less interference from other elements which are present in the matrix.
4. Discussion The levels of trace elements in different vital organs in normal and pathological conditions are very essential to establish a cause and effect relationship between nutritional deficiencies and many metabolic disorders in which trace elements are involved. The need to generate a clinical data on normal trace elemental level in brain tissues is highly emphasized, while considering the role of non-essential trace elements like Al (Garruto et al., 1993; Strong and Garruto, 1994) and Pb (WHO, 1986) in many neurological disorders. The recent advances in analytical instrumentation made it possible to analyze the biological samples for their elemental concentrations even at ppb level. In the present study we have used ICPAES for the detection of Na, K, Ca, Mg, Fe, Si, Pb, Cd, Cr, P, Al, Ni, Zn, As and Cu in different regions of brain. ICPAES is very much preferred in elemental analysis because of its high elemental sensitivity and low detection limit (ppb level). We could detect micro elements like Pb, Cd, Cr, As at ppb levels using ICPAES. The quality assurance test was done through recovery studies and also by analyzing reference material (unpolished rice flour) obtained from NIEH, Japan. We compared our results with literature values available for some regions of the brain and found to be in good agreement in many cases (ICRP report, 1959; Long et al., 1961; Dexter et al., 1991; Markesbery et al., 1984; Riederer et al., 1989). The comparison of our results with the published literature values is summarized in Table 10. The
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reported values of trace elemental concentrations in the normal human brain for Western population need not to be same for Indian population, as the living conditions, life style and environmental conditions are quite dissimilar for different countries and populations. There are some interesting observations with reference to Fe levels in brain. Zecca et al. (1994) detected highest concentrations of Fe in neuromelanin, substarttia nigra, and putamen of brain. The concentrations of Fe reported by Long et al. (1961) in grey matter and white matter were 89 pg/g md 89 Kg/g (wet tissue) respectively. In the present study, the concentrations of Fe were found to be comparatively low in different brain regions (16–59 Kg/g). The Fe values ( pg/g wet weight) in hippocampus (26.6), thalamus (41.8) and hypothalamus (26.6) found in the present study are in agreement with the reported values of Sofic et al. (1988) and Riederer et al. (1989). However, our values are low when compared to those reported by Dexter et al. (1991). The low amounts of Fe observed in different brain regions may be due to the loss and displacement of Fe by Al. It has been shown that Al binds with Fe carrier proteins like transfemin and ferritin and competes with Fe for binding (Flemming and Joshi, 1991). The displacement of Fe by toxic metal may affect the neuronal activity. Because, Fe is intimately involved in dopamine receptor synthesis and dopaminergic neuro-trartsmission (Youdim and Ben Shachar, 1987). In our study, the concentration of Al was found to be very high and varied greatly in different areas of the brain (58-196 pg/g). Xu et al. (1992) reported 6.2 to 9.8 Kg/g (dry mass basis) of Al in human brain. The mean value of Al in normal human brain reported by Crapper et al. (1973) was 1.1 pg/g with a range of 0.23 to 2.7 #g/g dry weight. Crapper et al. (1976) reported elevated Al concentrations in some regions of the brains of subjects with Alzheimer’s disease. On the other hand, McDermott et al. (1979) could not detect any statistically significant difference in brain Al concentrations between the 10 patients with Alzheimer’s disease (mean 2.7 pg/g dry weight of tissue, mean age 81 years) and the 9 non- neurological controls (mean 2.5 pg/g mean age 73 years). Markesbery et al. (1984) reported Al concentrations ( Kg/g wet weight) in different regions of brain (globus pallidus, 0.893, putamen, 0.663, temporal lobe, 0.654). McDermott et al. (1979) found the highest Al concentration in hippocampus. Contrary to this, in the present study we observed highest Al concentration in temporal cerebrum (mean value 196 Pg/g). The high level of Al which we detected in the brain may be due to the high intake of Al through food water and drugs. In India, the rural population largely uses low grade A1-Pb alloy vessels for cooking and storing foods. Jagannatha Rao and Valeswara Rao (1995) have shown high concentrations of Al leaching from low grade A1-Pb alloy vessels and also found very high leaching into acidic foodstuffs. Aluminum salts are also used extensively in water purification and is a common water contaminant
Cu
Zn
4.17 + 0.36 3 1.25+ 3.6 5.3 + 0.4 I6.86+ 0.7 6.6+ 0.8 14.43* 0.7 8.8 k 0.8 13.2+ 1.0 18.53~ 0.76 8.5 k 0.8 Markesbery et al. (1984) Fe Cr — 0.265 136 0.26 43.8 0.081 — 169 Riederer et al. ( 1989) Fe Cu Zn — 105.5 8.0 68.6 5. I 6.3 — — 21.1 — 31.65 4.4 84.4 I I.6 3.8 84.4 9.5 Sofic et al. (1988) Fe 48 k 8,2 96+ 19 81 + 18 24* 3.3 28+ 5.4 Ca 173 131.8 — — 97. I I21,3
Dry weight values cited in thetiterature were converted into wet weight (pg/g) ‘Average vahreof8 cerebral regions.
Substantialnigra Putamen Globus pallidus Hippocampus Cortex
Globus pallidus Substantialnigra Hypothalamus Thalamus Caudate nucleus Putamen
Cerebella hemisphere Putamen Hippocampus Globus pallidus
Cerebral cortex Cacsdatenucleus Putamen Substantialnigra Cerebellum
Dexter et al. ( 1991) Fe
— 126.6 94.95
Mg 100.2 105.5
16.1 12
Zn —
58.92 141.41 164.98 164.98 76.60
Zn —
—
7 * 0.5 7.5 + 0.5 —
26.6 k 12.2 41.8 k 18.5
—
Fe
26.6 + 7.6
(Ehmann and Markesbery, 1994)
Present study Cu — — 4.2 + 1.9 3.5 + I.4 — — Present study Fe — — — 26.6* 7.6 48.92 k 7.5 a
12.7+6. 1‘
Fe 45.4 * 19.3
Zn
Cu 4.54 * 0.5 a — — 7.4 ~ 2.5 Present study Cr 0.1 I A0.06 — 0. 10+ 0.08
Present study
by using FD/wetratioofO.211
Table 10 Comparison of values obtained from the present study with published literature values
—
179*90 158+72
Ca
Fe
Zn 8il — 6.5+0.5
48.92 k 7.5 ‘
% :Y
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in India. It has been shown that small amounts of Al can accumulate during a life span with an increment of about 10 mg per year which corresponds approximately with the observed AI(IH) content in human brain after 70 years of life (McDermott et al., 1979). Zecca et al. (1994) found high level of Al in Neuromelanin isolated from brain (220 ng/mg dry weight). Luckiw et al. (1991) have shown increased amounts of Al in chromatin and they detected high concentration of Al (885.4 pg/g DNA) in DNA isolated from the neuronal nuclei. Since we could detect comparatively high concentrations of Al in the brains of eight subjects, there is an urgent need for a large scale screening of brain tissues for Al from different localities of India to get an exact nature of Al accumulation in human brain. Future studies should focus on measurement of Al in samples from different geographical area. Studies of Arunachalam et al. (1979) and Gangadharan et al. (1973) showed the variations in the trace elements (As, Zn, Cr, Mn, Fe, Br) levels in human hair with reference to sex and dietary habits of Indian populations. Jagamatha Rao and Valeswara Rao (1992), while reviewing the literature indicated higher levels of Cd, Mn, Pb, As and Co in food, water and air in India. According to GEMS (1988), the total intakes of Pb and Cd in Asian countries were 9 and 3 ~g\kg of body weight. Further, it was reported that Pb (130 Vg of blood) and Cd (20 mg/Kg of kidney cortex) levels were high in human tissues in India. Jagamatha Rao (1994) reported 30 mg per day of Al consumed by Indian populations, while WHO (1986) set limit was 7 mg/kg of body weight per week. These factors might have contributed for high levels of Al, Cd and Pb in human brain reported in the present study. According to Ehmann and Markesbery, 1994 the brain matrix contains major concentrations of alkali metals and phosphorus containing compounds that have now been shown to interfere with many instrumental analytical procedures. In order to avoid interference from the alkali metals, in the present study we made appropriate dilutions of the samples before analysis and suitable lines were also selected. A standard matrix having aluminium, alkali metals and phosphorus were analyzed to determine the interference from these elements on the aluminium signal. Interference from these elements was not significant. Further, in our studies, total regions of brain in gms were taken to get homogeneous vah.te of aluminium while in other studies only small portions (mg) of the regions were taken for the analysis. This will not reflect the homogeneity of the sample with reference to trace elements distribution in that region. The present study reports for the first time a direct correlation between the concentration of trace elements in one region of the brain to other. This region to region correlation is interesting and indicates a direct relationship between the concentration of trace elements in one region of the brain to other. The functional activity of the specific brain region with regard to the elemental concentrations is
165
not completely understood. Our findings provide a new and an in-depth data, which can be used as base line figures for normal brains.
Acknowledgements The authors thank Dr. V. Prakash, Director, CITRI and Mr. B. Bhattacharjee, Project Director and Mr. N.D. Sharma, Project Manager, RMP, BARC, Mysore for their encouragement. We thank Dr. Bhargava, Professor of Anatomy, J.S.S. Medical College and Head, Dept. of forensic Science, Government Medical College, Mysore for their help. Dr. M.T. Rajan acknowledges CSIR, New Delhi, Govt. of India for a research associateship.
References Arunachalam, J., Gangadhamrt, S. and Yengnasubramanium, S. (1979) Elemental data from human hair sampled from Indian population. Symposium on Nuclear Activation Technique in Life Sciences, Vienna, 499–5 13. Barrington, C.D., Sheehan, D.M. and Bolger, P.M. (1993) Hazard assessment of lead. Food Addit. Contamin. 10: 325–335. Crapper, D.R., Krishnan, S.S. and Dalton, A.J. (1973) Brain aluminium distribution in Alzheimer’s disease and experimental neuro fibnllary degeneration. Science 180, 51 1–513. Crapper Mc Lachlan, D.R., Lukiw, W.J. and Kruck, T.P.A. (1990) Aluminium, altered transcription and pathogenesis of Alzheimer’s disease. Environ. Geochem. Health 12: 103–I 14. Crapper Mc Lachlan, D.R. and DeBoni, U. (1980) Ahrminium in human brain disease — An overview. Neurotoxicology 1: 3–16. Crapper, D.R., Krishnan, S.S. and Quittkat, S. (1976) Ahrminium, Neurofibrillary degeneration and Alzheimer’s disease. Brain 99: 67–80. Dexter, D.T., Carayon, A., Agid, F.J., Agid, Y., Wells, F.R., Daniel, S.E. Lees, A.J. Jenner, P. and Mardsen C.D. (1991) Alterations in the levels of iron, ferritin and other trace metals in Parkinson’s disease and other neurodegenerative disease affecting the basal ganglia. Brain 114: 1953-1975. Ehmann, W.D. and Markesbery, W.R. (1994) A multi-technique approach to the study of aluminium in an Alzheimer’s disease brain. Life Chem. Rep. 2: 11-28. Flemming, J.T. and Joshi, J. (1991) Ferritin: The role of aluminium in ferntin fnnction. Neurobiol. Aging, 12: 413-418. Gangadhamn, S., Lakshmi, V.V. and Sankar Das, M. (1973) Growth of hair and the trace element profile, a study by sectional analysis. J. Radioanal. Chem., 15: 287-304. Garnrto, R.M., Flaten, T.P. and Wakayama, I. (1993) Natural and experimental models of environmentally induced neurogeneration: Implications for Alzheimer’s disease. In: Alzheimer’s Disease: Advances in Clinical and Basic Research. B. Corain, H. Wisneiwski and P. Zatta (Eds.), PP. 257-266 John Wiley and Sons Ltd. Global Environmental monitoring Survey (GEMS) Report (1988) Global Pollution and Health 1–23, WHO, Geneva. Goering, P.L., Mistry, P. and Fowler, B.A. (1987) Mechanism of metalinduced cell injury. In: T.J. Haley and W.O. Bemdt (Eds.), Hand Book of Toxicology, Hemisphere Publishing Corp. Washington. Gross Clark, W.E.L (1956) Central nervous system. In: W.J. Hamilton (Ed.), Text BOOk of Human Anatomy, MacMillan and Co Ltd. St.Martin’s Press, New York. International Commission on Radiological Protection (ICRP) (1959) Report of Committee of il on Permissible Dose for International Radiation, pp 146–151, Pergamon Press.
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Jagarrnatha Rae, K.S and Valeswara Rae, G. (1995) Aluminium leaching from utensils — A kinetic study. Int. J. Food Sci. Nrrtr. 46: 31-38. Jagamratha Rae, K.S. (1994) Ahrminium content in tea leaves and in different prepared tea infusions. Nahrrmg 38: 533-537. Jagannatha Rae, K.S. and Valeswara Rae, G. (1992) A review of metal distribution in Indian environment. Indian J. Environ. Toxicol. 2: 1-13. Kaiser, J. (1994) Alzheimer’s: Could there be a zinc link? Science, 265: 1365. Long, C., King, E.J. and Sperry, W.M. (1961) Chemical composition of brain. Biochemist’s Handbook pp 639-655 E and N F.N. SPON Ltd. Ltrkiw, W.J., Krishnan, B., Wong, L., Kruck, T.P.A., Bergerson, C. and Crapper McLachlarr, D.R. (1991) Nuclear compartmentalization of Ahrminium in Alzheimer’s disease. Neurobiol. Aging. 13: 115–121. Markesbery, W.R., Ehmann, W.D., Alauddin, M. and Hossain, T.I.A. (1984) Brain trace elementconcentrations in aging. Neurobiol. Aging 5: 19-28. Martyn, C.N., Osmond, C., Edwardson, J.A., Barker, D.J.P., Hams, E.C. and Lacey, R.F. (1989) Geographical relation between Alzhemer’s disease and aluminium in drinking water. Lancet, 1: 59-62. McDermott, J.R., Smith, A.L, Iqbal, K. and Wisniewski, H.M.(1979) Brain aluminium in aging and Alzheimer’s disease. Neurology 29: 809-814. Merfz, W. (1981) The essential trace elements. Science, 213: 1332–1338. Mertz, W.(1995) Risk assessment of essential mace elements: New approaches to setting recommended dietary allowances and safety limits. Nrrtrit. Rev. 53: 179–185. Moody, J.R. and Lind Strom, R.M. (1977) Selection and cleaning of plastic containers for storage of trace element samples. Anal. Chem. 49: 2264–2267. Riederer, P., Sofic, E., Rausch, W.D., Schmidt, B., Reynolds, G.P., Jellinger, K. and Youdim, M.B.H. (1989) Transition metals, ferritin, ghrtatfrione and ascorbic acid in Parkinsonian brain. J. Neurochem. 52: 515-520. Savory, J. and Wills, M.R. (1986) Analytical methods for aluminium measurement. Kidney Int. 29, Suppl. 18: S24 – S27. Sotic, E., Riederer, P., Heinsen, H., Beckmann, H., Reynolds, C.P.,
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Hebenstreit, G. and Youdim, M.B.H. (1988) Increased iron(III) and total iron content in Post-mortem substantia-nigra of parkinsonirm Brain. J. Neural. Transm. 74: 199–205. Strong, M.J. and Garruto, R.M. (1994) Experimental paradigms of motor neuron degeneration. In: M.L. Woodruff. and A.J. Nonrremarr(Eds.), Toxin-induced models of neurological disorders. pp. 39–88, Plenum Press New York. Uitti, R.J., Rajput, A.H., Rozdilsky, B. and Yuen, W.K.(1987) Regional distribution of metals in human brain. Clin. Invest. Med. 10:10-13. Uitti, R.J., Rajput, A.H., Rozdilsky, B., Bickis, M., Wollin, T, and Yuen, W.K. ( 1989) Regional metal concentration in Parkinson’s disease, ofher chronic neurological disease and control brains. Can. J. Neurol. Sci. 16: 310-314. Wender, M., Szczeech, J., Hoffmarm, S. and Hilezer, W.(1992) Electron paramagnetic resonance analysis of heavy metals in aging human hair. Neuropathol. Pol. 30(1): 65-72. World Health Organization (WHO) (1986) Toxicological evaluation of certain food additives and contaminants. WHO food additive series 21, prepared by the 30fh meeting of the Joint FAO/WHO Expert Committee on Food Additives. pp. 223–255. Cambridge University Press, Cambridge. Xu, N., Majidi, V., Ehmann, W.D. and Markesbery, W.R. (1992) Determination of alrrminium in human brain by electrothermal atomic-absorption spectrometry. Anal. At. Spectrom. 7: 749–751. Yoshida, D., Ikeda, Y. and Nakazawa, S. (1993) Quantitative analysis of copper, zinc and copper\zinc ratio in selected human brain turners. J. Neurol. Oncol. 16:109-115. Youdim, M.B.H. and Ben Shachar, D. ( 1987) Minimal brain damage induced by early iron-deficiency: modified dopaminergic neurotransmission. lsr. J. Med. Sci. 23: 19–25. Zecca, L. and Swartz, H.M. ( 1993) Total and paramagnetic metals in human substantial nigra and its neuromelanin. J. Neural. Transm. 5: 203-213. Zecca, L., Pietra, R., Goj, C., Mecacci, C., Radice, D. and Sabbioni, E.(1994) Iron and other metals in neuro melanin, substarrtia nigra and putamen of human brain. J. Neurochem. 62: 1097- 110I.
NEUROLOGICAL SCIENCES Journal of the Neurological Sciences 146 (1997) 153-166
Quantificationof trace elements in normal human brain by inductively coupled plasma atomic emission spectrometry M.T. Rajan a, K.S. JagannathaRao a’*,B.M. Mamathaa,R.V. Rao b,P. Shanmugavelub, Rani. B. Menonb,M.V.Pavithranb aDepartment oj’Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore-570 013, India b Analytical Control Section, Rare Materials Project, Bhabha Atomic Research Centre, Mysore-571
130, India
Received 22 April 1996; revised 6 August 1996; accepted 21 August 1996
Abstract Eight normal human brain autopsy samples were analyzed for Na, K, P, Ca, Mg, Si, Cr, Cu, Ni, Zn, Fe, Al, Cd, Pb and As in 12 regions of brain (frontal cerebrum, temporal cerebrum, parietal cerebrum, somatosensory cortex, occipital cerebrum, cerebellum, mid-brain, pens, hypothalamus, thalamus, hippocampus and medulla oblongata) using inductively coupled plasma atomic emission spectrometry (lCPAES). The distribution of these 15 elements varied significantly from region to region of the brain. Potassium was most abundant in nearly all regions of the brain, followed by sodium and phosphorus (mg/g). The concentration of Al was found to be comparatively high and varied in different areas of the brain (58–196 Wg/g). Moderate levels of Pb, Cd and As were observed in different regions. Ratios of Al to Fe were found to be high in temporal cerebrum (8.07) and hippocampus (9.05) and these two regions are significantly involved in Alzheimer’s disease. The concentration of Na in mole percentage showed an inverse correlation with that of K, Ca, Mg, Fe and Cr. Direct correlation was observed in the concentration of all analyzed elements, which indicated for the first time the direct dependency of concentration of trace elements in one brain region to other regions. The mole ratios between different elements in different brain regions and total amounts of the elements in an average weight of 1.4 kg human brain were also computed. The present study provides new and in-depth data which may be used as base line data for normal human brains. 0 1997 Elsevier Science B.V. All rights reserved. Keywords: Human brain; Trace element; Inductively coupled plasma atomic emission spectrometry
1. Introduction Trace elements play a dual role in the biological system through their interaction with biomolecules. They regulate a number of celhtlar metabolic reactions, with a few of
them acting as etiological agents in many environmentally induced neurological diseases (Strong and Garruto, 1994; Garruto et al., 1993). Elements like sodium, magnesium, potassium, calcium, and phosphorus serve as structural components of tissues, as constituents of the body fluids and therefore, are essential for the function of cells. (Mertz, 1981). Trace elements in optimum levels are required for a number of metabolic and physiological processes in human body. Even though these are required for many biological
“ Corresponding author. Present address: Department of Pathology, University of Virginia, Box 168, Charlottesville, Virginia 22908, USA. Fax: + 1 (804) 924 2574. Tel: + 1 (804) 924 5682. E-mail: [email protected] 0022-510X\ 97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIJ S0022-510X(96)O03 OO-O
functions and their deficiencies would cause various metabolic disorders, the amount of their requirement in most of the cases is very less (Mertz, 1995). Essential trace elements at high levels are toxic and adversely affect the biological activities. Recently, it has been reported that Zn which is an essential trace element is capable of inducing A ~ protein clump formation; similar to the amyloid plaque found in the brains of Alzheimer’s patients (Kaiser, 1994). Non-essential trace elements like Al, Pb and Cd are also present in the biological tissues. They are highly toxic and are reported to be associated with a number of neurological disorders found in man (Crapper Mc Lachlan and DeBoni, 1980; Barrington et al., 1993). The role of these metals is suspected in many diseases whose etiology is still unknown. For example, Al is suspected to be involved in the pathogenesis of Alzheimer’s disease (Crapper Mc Lachlan et al., 1990). The increasing amounts of trace elements, particularly heavy metals, in animal tissues have been a cause of great concern, since their deposition in
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different vital organs is capable of inducing cellular injuries. Heavy metal ions are extremely toxic due to their diverse mode of action on various levels of biological organization. They are known as carcinogens and mutagenes by their interaction with DNA and also induce cellular damage through lipid peroxidation (Goering et al., 1987). Because of the wide-spread contamination of life sustaining factors like food, water and air with metals, the total body burden of trace metals has been increasing alarmingly (Jagannatha Rao and Valeswara Rae, 1992). The absorbed metals are metabolically non-biodegradable and their accumulation in the body leads to chronic effects. The only way of their elimination from the body is by excretion. The major organs in human body where metals are reported to be accumulated are kidney, liver, bone marrow, blood spleen, brain and muscle, etc. Recent studies have shown that there is a potential relation between the levels of heavy metals and incidence of neurological disorders (Martyn et al., 1989). Very limited studies are available on the levels of metals in human brain in normal and pathological conditions (ICRP report, 1959; Crapper et al., 1973; Markesbery et al., 1984; Uitti et al., 1987 and Uitti et al., 1989; Luckiw et al., 1991; Wender et al., 1992; Yoshida et al., 1993; Zecca and Swartz, 1993; Zecca et al., 1994). Most of this information is limited to a few selected regions of the brain and neuromelanin pigments (Zecca et al., 1994). There is also no study which examines a cross-relation of metal to metal-dependent retention in brain tissues. To generate normalized values in brain, a base-line data bank on trace elements in different regions of the brain from different countries need to be computed. Since there is no report in this direction from elsewhere and particularly from India, the present investigation was undertaken. Furthermore, in developing countries like India the magnitude of environmental contamination is very high, implying that the chance is higher for human brain to be burden by environmental substances. The progress in understanding the role of metals in disease of the nervous system has been hampered to a I&ge extent by lack of data on normal metal concentrations in the human brain. Since several metals interact metabolically, concurrent metal levels are essential for clinical correlation (Uitti et al., 1987). Therefore, an attempt has been made to generate data on the levels of trace metals in 12 important regions of the human brain using Inductively Coupled Plasma Atomic Emission Spectrometer (ICPAES) and attempts were also made to correlate values between elements in different regions of the brain. 2. Materials and methods 2.1. Brain tissue Autopsies of human brain taken from eight postmortem bodies were obtained from the Department of Forensic
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Sciences, Government Medical College, Mysore, India. The whole brain was removed during post-mortem from eight male subjects in the age group of 50–60 years. The subjects had no history of psychiatric, neurological or any other neuro-degenerative disorder as per their medical records. They were neurologically quite normal. Autopsies were carried out within 24 h after death. The whole brain was removed, rinsed in ultra-pure water with resistivity of 18 ML?-cm (collected by using ELGA UHQ II system), wrapped with pre-washed polyethylene sheets and stored in a pre-washed plastic container and frozen at – 20°C until dissection. Special care was taken to avoid contamination with metals during handling and dissection, as described by Savory and Wills (1986) and Moody and Lind Strom (1977). The brain was dissected on a polyethylene plate with stainless steel tools (knives, scissors, forceps etc.) coated with titanium nitride (Zecca et al., 1994). Prior to dissection of the brain, the meninges along with blood vessels were carefully removed using a forceps, followed by rinsing of the dissected brain samples in ultra-pure water in order to minimize contamination due to cerebrospinal fluid and blood. Talc-free rubber surgical gloves, pre-treated with 2% quartz distilled nitric acid and washed thoroughly in ultra-pure water were used during handling and dissection of the brain. Twelve important regions of the brain based on anatomical lines viz., (1) frontal cerebrum (2) temporal cerebrum (3) parietal cerebrum (4) somatosensory cortex (5) occipital cerebrum (6) cerebellum (7) mid-brain (8) pens (9) hypothalamus (10) thalamus (1 1) hippocampus (12) medulla oblongata were dissected using a brain atlas (Gross Clark, 1956). The brain regions were dissected under the supervision of an anatomy professor from J.S.S. Medical College, Mysore, India. Brain samples taken from the cerebral regions contained both white and grey matters. The internal structures were also present in the brain samples taken from the mid brain, pens, thalamus, medulla oblongata etc. 2.2. Sample preparation All glass wares and silica crucibles were treated with di- sodium EDTA solution (0.1%), rinsed in distilled water and were kept immersed in a beaker containing 10910nitric acid (BDH analytical grade) for 24 h, followed by rinsing thoroughly with ultra-pure water. Pre-weighed brain samples were taken into silica crucibles and charred completely. The charred samples were ashed in a muffle furnace at 600° C for 8 h. The crucibles that had a spout were covered with lids in order to avoid the contamination from the wall of the muffle furnace. The ashed brain samples were treated with con. HNO~ (BDH analytical grade, quartz distilled) and mixture was heated for 30 min in a water bath. The mixture was diluted with ultra-pure water (collected by using ELGA UHQ 11system), filtered, and made up to 20 ml so as to get a final acid concentra-
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tion of 5%. Since the sample weights were different and concentrations in which elements are present are also different, appropriate dilutions were made before analysis. Sample preparation and analysis were carried out in a dust-free air-conditioned laboratory at Rare Materials Project, BARC, Mysore.
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155
15 elements and their concentrations were found to be very low. The blank value for Al was estimated to be 0.5–0.9 ppm, which was found to be very low when compared with concentrations of Al in brain samples. The blank values were deducted from the sample values. 2.5. Recovery study and reference material analysis
2.3. Instrumentation Inductively coupled plasma atomic emission spectrometer (ICPAES) model JY 38 was used for the sequential analysis of Na, K, Ca, P, Mg, Fe, Zn, Cu, Ni, Si, Al, Pb, Cd, As, and Cr. Argon plasma was used for excitation of the elemental atoms. The lines were selected for each element in such a way that interference from other elements was minimum. The wave length used and detection limit of the elements are summarized below. Element
Wavelength (rim)
Detection limit * (ppm)
Al As Ca Cd Cr Cu Fe K Mg Na Ni P Pb Si Zn
396.152 193.759 393.366 226.502 267.716 224.7 259.94 766.49 279.806 588.995 221.67 213.618 220.353 251.611 213.856
0.002 0.1 0.002 0.001 0.004 0.002 0.005 0.06 0.001 0.03 0.002 0.05 0.01 0.08 0.002
* Detection limit for each element was calculated by running a multi- element standard solution containing 500 ppb of each of the above cited elements. The instrumental conditions were as follows Incident power: 1.0 KW (57.6 MHz) Plasma Ar gas: 20 l/rein Auxiliary Ar gas: 0.6 l/rein Nebulizer Ar gas: 0.5 l/rein Spectral observation: Optics: 1 m Czerny-Turner Grating: Holographic 3600 l/rein Resolution: 0.009 nm Entrance slit width: 30 pm Exit slit width: 30 p,m Integration time: 400 ms 2.4. Blank The blanks (quartz-distilled A.R. grade nitric acid and ultra-pure water) were processed in the same way as the brain samples were processed. They were analysed for the
Known concentrations of multi-standard elements were added to the analyzed brain samples and the percentage of recovery for each element was computed. Low-cadmium reference material (unpolished rice flour) NIES-10 obtained from Japan was also analyzed to confirm the validity of the results. A multi element synthetic mixture (prepared by using Spex Indust. Inc. and Johnson Matthey England) containing all the 15 analyzed elements with the concentration equivalent to analyzed concentrations of elements in brain samples was also prepared and analyzed to confirm the validity of the results. The concentrations of trace elements were calculated as per gram of the wet tissue of the brain regions. The percentage elemental concentrations in mole were also calculated. 2.6. Statistics Mean and SD values were calculated from the total number of brains analyzed. Concentrations of element to element and brain region to region correlation were analyzed by linear regression analysis. One way analysis of variance (ANOVA) followed by Duncan’s t-test was also employed to check the variations in the trace element concentrations from one region of the brain to other.
3. Results The distribution of trace elements was not homogeneous in different regions of the brain and their concentrations varied significantly in different regions as shown in Tables 1 and 2. Potassium was more abundant in most of the regions, followed by Na and P, while Cd was found in the lowest concentration. The analyzed elements were classified into four sets based on the ranges of concentrations. First set of elements consisted of Na, K and P and their concentrations ranged from 1.17 to 4.4 mg/g. Second set of elements were Ca, Mg, and Al and their levels ranged from 58 to 196 ~g/g. Fe, Zn, Si, and Cu formed the third set and their concentrations varied from 3.5 to 59.5 ~g/g in all regions of the brain. Fourth set of elements consisted of As,. Ni, Pb, Cd, and Cr and their concentrations were comparatively very less and were found to be within a concentration limit of 0.015–2.64 ~g/g. The Al was found comparatively in high concentration and its levels varied greatly in different regions of the brain (58–196
‘
156
M.T. Rajan et aL/.Journal
.
of the Neurological
---------e=
Sciences 146 (1997) 153–166
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Elemental cone ( ~mol/g) = Elemental conc( Kg/g) /Atomic wt.
77 90 88 69 82 57 79 64 113 71 64 77
87 96 83 73 96 60 152 91 151 I02 117 170
6.5 5.7 7.6 5.I 6.5 6,7 5.1 5.3 4.2 4.6 4.8 5.6
Mg
Frontal cerebrum Temporal cerebrum Parietal cerebrum Somatosensory cortex Cerebrum occipital Cerebellum Mid-brain Pens Hypothalamus Tbalamus Hippocampus Medulla oblongata
Ca
K
Na
Brain regions P
in different regions of human brain
Table 3 Trace metal concentrations ( pmole/g)
0.9 0.9 I,0 0.7 0.7 0.8 0.5 0.3 0.5 0.7 0.5 0.3
Fe 0. I 0.1 0.4 0. I 0.2 0.1 0. I 0.1 0. I 0. I 0.1 0.3
Zn o. I 0.1 0. I 0.1 0.1 0. I 0.1 0. I o. I 0.1 o. I 0.1
Cu 4.2 7.3 4.1 2.6 2,2 2,3 3.4 2.I 3.4 4.8 4.2 3.9
Al 0.9 I.0 1.1 0.6 0.6 0.5 1.1 0.4 I.0 1.0 1.5 1.3
Si 0.011 0.020 0.009 0.009 0,008 0.007 0.016 0.009 0.017 0.013 0.015 0.016
As 0.005 I 0.0102 0.0034 0.005 I 0.0051 0.0034 0.0017 — 0.0034 0.005 I 0.0102
Ni
Cd 0.00018 0.00027 0.00018 0.00018 0.00318 0.00018 0.00027 0.00018 0.00027 0.00044 0.00018 0.00036
Pb 0.00193 0.00193 0,00193 0.00193 0.0029 0.00145 0.00338 0.0029 0.01255 0.00338 0.00434 0.0Q386
0.0029 0.0035 0.0031 0.0025 0.0027 0.0021 0.0023 0.0025 0.0021 0.0027 0.0019 0.0023
Cr
s ~
e. s m $ = 2 & m ~, B # ~. z~ % k & m
Q < Q = 3n
34.0 37.2 33.2 34.8 36.8 35.5 46.7 38.9 42.5 38.4 45.7 48.9
Frontal cerebrum Temporal cerebrum Parietal cerebrum Somatosensory cortex Occipital cerebrum Cerebellum Mid-brain Pens Hypothalamus Tbalamus Hippocampus Medulla oblongata
30.0 34.8 35.4 32.9 3 I.5 33.7 24.3 27.2 31.8 26.5 24.9 22.1
K 29.3 20.4 24.0 25.8 26.1 22.4 24.8 28.9 21.9 29.I 23.9 24.2
P I.7 1.8 1.7 2.1 1.6 2.I I.2 1.4 1.3 I.5 1.2 1.4
Ca 2.54 2.21 3.04 2.43 2.52 3.99 1.58 2.28 1.19 1.75 1.86 1.60
Mg 0.34 0.35 0.40 0.32 0.27 0.45 0.16 o.13 0. I3 0.27 0.18 0.08
Fe 0.04 0.05 0. I5 0.07 0.08 0.07 0.04 0.05 0.03 0.04 0.04 0.09
Zn 0.02 0.03 0.04 0.03 0.03 0.07 0.03 0.02 0.02 0.02 0.02 0.02
Cu 1.64 2.83 1.64 1.22 0.84 1.34 1.03 0.91 0.97 1.80 1.62 1.13
Al 0.35 0.37 0.43 0.29 0.22 0.28 0.35 0.19 0.29 0.37 0.58 0.38
Si
Elemental cone (mole%) = Elemental conc( &mole/g)X 100/Total elemental cone ( pmole/g) of analysed elements in each region.
Na
Brain regions
Table 4 Mole percentage of analysed elements in different regions of human brain
0.004 0.008 0.004 0.004 0.003 0.004 0.005 0.004 0.005 0.005 0.006 0.005
As
0.032 0.004 0.001 0.002 0.002 0.002 0.001 — 0.001 0.002 0.003
Ni
0.00 I 0.031 0.001 0.001 0.001 0.001 0.001 0.001 0.004 0.001 0.002 0.001 I
Pb
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0002 0.0001 0.0001
Cd
➤ 0.001 I 0.0013 0.0012 0.0012 0.oo1o 0.0013 0.0007 0.001 I 0.0006 0.oo1o 0.0007 0.0007
Cr
.
< g ~ = 2 6w ~. E ~ ~. g * k s m — .’ U u v
n Y > ~ zQ
16)0
M.T. Rajan et al. /Journal
Sciences 146 (1997) 153–166 6.0
●
35.0
of the Neurological
●
r = -0.82 30.0- -
●
●
+
25.0- A ●
% %
20.0- -
s ~ ~
r = -0.71
A
A
15.0- -
A
●
A
.
r = -0.77 ■
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10.0- -
A
■
‘m H
F
■
5.0 i-
0.01
t 0.0
30.0
32.0
34.0
38.0
36.0
40.0 Mole %
42.0
44.0
46.0
43.0
50.0
of Ma
Fig. 1. Inverse correlation of Na with K, Ca and Mg in different regions of humarr brain.
were highest in hypothalamus and the levels of Mg, Fe and Zn wer~ found ~~ be highest in ~arietal cerebrum. The concentrations of Cd, Cu and Si were highest in thalamus, cerebellum and hippocampus, respectively. Compared to other regions of the brain, in cerebellum Na, K, P, Cd, Pb and As were found in lowest concentrations.
Pg/g). Similady, concentrations of Fe also showed great variations in its levels in different regions of brain (16–59 Pg/g). Tbe concentrations of Na, P-and Ca were highest in medulla oblongata. In temporal cerebrum, the concentrations of Al, As, Ni, and Cr were found to be highest compared to other regions of brain. The levels of K and Pb
0.60
0.0014 ■
r = -0.87 ■
0.0012
0.50
■
●
, ■
0.40
r = -0.84
~ 5 $ 0.30 ; z
0.0010
●
0.0008 G % s Z 0.0006 ~
● ● ●
0.20 0.0004 .
●
0.10
0.0002
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30.0
32.0
34.0
36.0
38.0
40.0
42.0
44.0
46.0
48.0
50.0
Mole % of Na Fig.
2, Inverse correlation of Na with Fe and Cr in different regions of human brain.
M.T. Rajan et aL/Journal
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162
M.T. Rajan et ul. /Journul
of the Neurological
The most of the analyzed elements serve as structural components of the major biomolecules and also found in the bound form. The data are presented (Table 3) in mole concentrations in order to calculate mole/mole ratios and also to explain metal to metal correlation. It is evident from the Table 3 that the concentrations of Na in mole ( pmol/g) were found to be highest in all regions compared to those of other analyzed elements. This observation is quite contrary to that presented in Table 1, where the concentrations of K (mg/g) were found to be highest. This dissimilarity is attributed to the differences in the atomic weights of the elements. The percentage of mole elemental concentration of each analyzed elements in different regions of the brain is shown in Table 4. On the total mole elemental concentrations of all analyzed elements, the percentage of Na was highest and the value ranged from 33.2 to 48.9% in different regions of the brain. The percentage concentrations of Ca, Mg, Fe and Cu were highest in cerebellum compared to other regions. It is interesting to note that in the total mole elemental concentrations of all analyzed elements, the percentage concentrations of Na showed an inverse correlation with that of K, Ca, Mg, Fe and Cr (Figs. 1 and 2). This indicates that increases in concentrations of Na in different regions of the brain decrease the level of K, Ca, Mg, Fe and Cr and vice versa. In biological system there is an interdependency in the concentrations of certain elements to maintain homeostasis of trace elements pool. Based on this, element/element mole ratio in different regions for few elements having biological importance was calculated. Element to element mole ratio for Na/K, Na/P and K/P ranged from 0.9 to 2.2 in different regions of the brain (Table 5). Na/K and Na/P mole ratios were highest in medulla oblongata and ratios were 2.2 and 2.0, respectively. Al/Fe mole ratio showed great variations in different regions of the brain and ranged from a lowest value of 2.9 in occipital cerebrum to a highest value of 14.45 in the medulla oblongata. It is interesting to mention that Al/Fe mole ratio was comparatively high in temporal cerebrum (8.07) and hippocampus (9.05) and these two regions are highly susceptible to neuropathology in Alzheimer’s disease. In the pari-
Sciences 146 (1997) 153–166
Table 7 Estimated total amounts of trace elements in an average weight of 1.4 kg of human brain K Na P Ca Mg Al Fe Si Zn Cu As Pb Ni Cr Cd
4.22c 0.77 g 3.36 + 1.0 g 2.8 + 0.56 g 227 +28 mg 192+ 32 mg 140+ 52 mg 52* 19 mg 36* 13 mg
15A 8 mg 6.5 + 1.5 mg l.3 + 0.4 mg 1~ 0.8 mg 0.4+ 0.2 mg 0.19 * 0.03 mg 0.04 + 0.01 mg
etal cerebrum Al and Ca were present in almost equal proportions and mole ratio was found to be 1:0.98. When A1/Mg ratio ranged from 0.33 to 1.28, A1/Si mole ratio varied from 2.8 in hippocampus to 7.64 in temporal cerebrum. Cu/Zn mole ratio was found to be highest in cerebellum (1:0.95) and lowest in medulla oblongata
(1:0.23). Interestingly, a direct correlation in the concentration of trace elements between some regions of brain was observed as shown in Table 6. The correlation coefficient r was found to be 0.99 in most of the cases. This indicates that a direct relationship is existing between the concentrations of trace elements in one region and those in the other regions. Estimated total amounts of trace elements in an average weight of 1.4 kg of human brain are shown in Table 7. This shows that amounts of K, Na and P present in total human brain are comparatively higher than those of other elements. The validation of the analysis was done by conducting recovery study and reference materials analysis. The recovery study showed 92–1027o recovery of added elements to the sample as shown in Table 8. Similarly, the analysis of rice flour reference material containing low level of Cd showed 0.023 ppm Cd against the certified
Table 6 Direct correlation in the concentration of trace metals between one brain region to other
Table 8 Recovery study of added elements
Brain regions
Correlation co-efficient r
Element
Frontal vs. temporal cerebrum Temporal vs. parietal cerebrum Parietal cerebrum vs. Somatosensory cortex Somatosensory cortex vs. occipital cerebrum Mid-brain vs. pens Hypothalamus vs. thalamus Tbalamus vs. hippocampus
0.97 a 0.99 a 0.99 a 0.99 a 0.98 a 0.97 a 0.98 ‘
Pa Mg ‘ Ca Zn b Al a Fe b
‘ P <0.05.
Sample cone. ppm
Added cone. ppm
Recovered ppm
Recovery (%)
52.4
20 I I 2 I I
70.7 3.27 2.92 4.38 I .99 10.69
91.5 100 100 100 Iol 102
2.27 1.92 2.38 0.98 9.67
a Stocksolution was diluted 100 times and analysed. b Stock solutions was diluted 10 times and analysed.
M.T. Rajan er al. /Journal
of the Neurological
Table 9 Analysis of synthetic mixture containing Na (0.35%) and K (0.52%) as matrix with following trace elements SLNO
Element
Actual (ppm)
Analysed (ppm)
1
Al As Ca Cd Cr Cu Fe Mg Ni P Pb Zn
2 2
2.3 2.2 19.99
2 3 4 5 6 7 8 9
10 11 12
20 0.05 0.3 5 20 0.4 26 2 2
0.07 0.34 0.94 4.9 21.7 0.39 26.3 2.2 2.1
value of 0.023 ppm, indicating the reliability of our reported vah.tes. Further, the analysis of a synthetic mixture containing all the analyzed elements, having a known concentration ( ~g/ml) equal to that we already analyzed in different brain regions, produced the values very near to the known concentrations of the elements (Table 9). This indicates the good line selection and less interference from other elements which are present in the matrix.
4. Discussion The levels of trace elements in different vital organs in normal and pathological conditions are very essential to establish a cause and effect relationship between nutritional deficiencies and many metabolic disorders in which trace elements are involved. The need to generate a clinical data on normal trace elemental level in brain tissues is highly emphasized, while considering the role of non-essential trace elements like Al (Garruto et al., 1993; Strong and Garruto, 1994) and Pb (WHO, 1986) in many neurological disorders. The recent advances in analytical instrumentation made it possible to analyze the biological samples for their elemental concentrations even at ppb level. In the present study we have used ICPAES for the detection of Na, K, Ca, Mg, Fe, Si, Pb, Cd, Cr, P, Al, Ni, Zn, As and Cu in different regions of brain. ICPAES is very much preferred in elemental analysis because of its high elemental sensitivity and low detection limit (ppb level). We could detect micro elements like Pb, Cd, Cr, As at ppb levels using ICPAES. The quality assurance test was done through recovery studies and also by analyzing reference material (unpolished rice flour) obtained from NIEH, Japan. We compared our results with literature values available for some regions of the brain and found to be in good agreement in many cases (ICRP report, 1959; Long et al., 1961; Dexter et al., 1991; Markesbery et al., 1984; Riederer et al., 1989). The comparison of our results with the published literature values is summarized in Table 10. The
Sciences 146 (1997) 153-166
163
reported values of trace elemental concentrations in the normal human brain for Western population need not to be same for Indian population, as the living conditions, life style and environmental conditions are quite dissimilar for different countries and populations. There are some interesting observations with reference to Fe levels in brain. Zecca et al. (1994) detected highest concentrations of Fe in neuromelanin, substarttia nigra, and putamen of brain. The concentrations of Fe reported by Long et al. (1961) in grey matter and white matter were 89 pg/g md 89 Kg/g (wet tissue) respectively. In the present study, the concentrations of Fe were found to be comparatively low in different brain regions (16–59 Kg/g). The Fe values ( pg/g wet weight) in hippocampus (26.6), thalamus (41.8) and hypothalamus (26.6) found in the present study are in agreement with the reported values of Sofic et al. (1988) and Riederer et al. (1989). However, our values are low when compared to those reported by Dexter et al. (1991). The low amounts of Fe observed in different brain regions may be due to the loss and displacement of Fe by Al. It has been shown that Al binds with Fe carrier proteins like transfemin and ferritin and competes with Fe for binding (Flemming and Joshi, 1991). The displacement of Fe by toxic metal may affect the neuronal activity. Because, Fe is intimately involved in dopamine receptor synthesis and dopaminergic neuro-trartsmission (Youdim and Ben Shachar, 1987). In our study, the concentration of Al was found to be very high and varied greatly in different areas of the brain (58-196 pg/g). Xu et al. (1992) reported 6.2 to 9.8 Kg/g (dry mass basis) of Al in human brain. The mean value of Al in normal human brain reported by Crapper et al. (1973) was 1.1 pg/g with a range of 0.23 to 2.7 #g/g dry weight. Crapper et al. (1976) reported elevated Al concentrations in some regions of the brains of subjects with Alzheimer’s disease. On the other hand, McDermott et al. (1979) could not detect any statistically significant difference in brain Al concentrations between the 10 patients with Alzheimer’s disease (mean 2.7 pg/g dry weight of tissue, mean age 81 years) and the 9 non- neurological controls (mean 2.5 pg/g mean age 73 years). Markesbery et al. (1984) reported Al concentrations ( Kg/g wet weight) in different regions of brain (globus pallidus, 0.893, putamen, 0.663, temporal lobe, 0.654). McDermott et al. (1979) found the highest Al concentration in hippocampus. Contrary to this, in the present study we observed highest Al concentration in temporal cerebrum (mean value 196 Pg/g). The high level of Al which we detected in the brain may be due to the high intake of Al through food water and drugs. In India, the rural population largely uses low grade A1-Pb alloy vessels for cooking and storing foods. Jagannatha Rao and Valeswara Rao (1995) have shown high concentrations of Al leaching from low grade A1-Pb alloy vessels and also found very high leaching into acidic foodstuffs. Aluminum salts are also used extensively in water purification and is a common water contaminant
Cu
Zn
4.17 + 0.36 3 1.25+ 3.6 5.3 + 0.4 I6.86+ 0.7 6.6+ 0.8 14.43* 0.7 8.8 k 0.8 13.2+ 1.0 18.53~ 0.76 8.5 k 0.8 Markesbery et al. (1984) Fe Cr — 0.265 136 0.26 43.8 0.081 — 169 Riederer et al. ( 1989) Fe Cu Zn — 105.5 8.0 68.6 5. I 6.3 — — 21.1 — 31.65 4.4 84.4 I I.6 3.8 84.4 9.5 Sofic et al. (1988) Fe 48 k 8,2 96+ 19 81 + 18 24* 3.3 28+ 5.4 Ca 173 131.8 — — 97. I I21,3
Dry weight values cited in thetiterature were converted into wet weight (pg/g) ‘Average vahreof8 cerebral regions.
Substantialnigra Putamen Globus pallidus Hippocampus Cortex
Globus pallidus Substantialnigra Hypothalamus Thalamus Caudate nucleus Putamen
Cerebella hemisphere Putamen Hippocampus Globus pallidus
Cerebral cortex Cacsdatenucleus Putamen Substantialnigra Cerebellum
Dexter et al. ( 1991) Fe
— 126.6 94.95
Mg 100.2 105.5
16.1 12
Zn —
58.92 141.41 164.98 164.98 76.60
Zn —
—
7 * 0.5 7.5 + 0.5 —
26.6 k 12.2 41.8 k 18.5
—
Fe
26.6 + 7.6
(Ehmann and Markesbery, 1994)
Present study Cu — — 4.2 + 1.9 3.5 + I.4 — — Present study Fe — — — 26.6* 7.6 48.92 k 7.5 a
12.7+6. 1‘
Fe 45.4 * 19.3
Zn
Cu 4.54 * 0.5 a — — 7.4 ~ 2.5 Present study Cr 0.1 I A0.06 — 0. 10+ 0.08
Present study
by using FD/wetratioofO.211
Table 10 Comparison of values obtained from the present study with published literature values
—
179*90 158+72
Ca
Fe
Zn 8il — 6.5+0.5
48.92 k 7.5 ‘
% :Y
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in India. It has been shown that small amounts of Al can accumulate during a life span with an increment of about 10 mg per year which corresponds approximately with the observed AI(IH) content in human brain after 70 years of life (McDermott et al., 1979). Zecca et al. (1994) found high level of Al in Neuromelanin isolated from brain (220 ng/mg dry weight). Luckiw et al. (1991) have shown increased amounts of Al in chromatin and they detected high concentration of Al (885.4 pg/g DNA) in DNA isolated from the neuronal nuclei. Since we could detect comparatively high concentrations of Al in the brains of eight subjects, there is an urgent need for a large scale screening of brain tissues for Al from different localities of India to get an exact nature of Al accumulation in human brain. Future studies should focus on measurement of Al in samples from different geographical area. Studies of Arunachalam et al. (1979) and Gangadharan et al. (1973) showed the variations in the trace elements (As, Zn, Cr, Mn, Fe, Br) levels in human hair with reference to sex and dietary habits of Indian populations. Jagamatha Rao and Valeswara Rao (1992), while reviewing the literature indicated higher levels of Cd, Mn, Pb, As and Co in food, water and air in India. According to GEMS (1988), the total intakes of Pb and Cd in Asian countries were 9 and 3 ~g\kg of body weight. Further, it was reported that Pb (130 Vg of blood) and Cd (20 mg/Kg of kidney cortex) levels were high in human tissues in India. Jagamatha Rao (1994) reported 30 mg per day of Al consumed by Indian populations, while WHO (1986) set limit was 7 mg/kg of body weight per week. These factors might have contributed for high levels of Al, Cd and Pb in human brain reported in the present study. According to Ehmann and Markesbery, 1994 the brain matrix contains major concentrations of alkali metals and phosphorus containing compounds that have now been shown to interfere with many instrumental analytical procedures. In order to avoid interference from the alkali metals, in the present study we made appropriate dilutions of the samples before analysis and suitable lines were also selected. A standard matrix having aluminium, alkali metals and phosphorus were analyzed to determine the interference from these elements on the aluminium signal. Interference from these elements was not significant. Further, in our studies, total regions of brain in gms were taken to get homogeneous vah.te of aluminium while in other studies only small portions (mg) of the regions were taken for the analysis. This will not reflect the homogeneity of the sample with reference to trace elements distribution in that region. The present study reports for the first time a direct correlation between the concentration of trace elements in one region of the brain to other. This region to region correlation is interesting and indicates a direct relationship between the concentration of trace elements in one region of the brain to other. The functional activity of the specific brain region with regard to the elemental concentrations is
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not completely understood. Our findings provide a new and an in-depth data, which can be used as base line figures for normal brains.
Acknowledgements The authors thank Dr. V. Prakash, Director, CITRI and Mr. B. Bhattacharjee, Project Director and Mr. N.D. Sharma, Project Manager, RMP, BARC, Mysore for their encouragement. We thank Dr. Bhargava, Professor of Anatomy, J.S.S. Medical College and Head, Dept. of forensic Science, Government Medical College, Mysore for their help. Dr. M.T. Rajan acknowledges CSIR, New Delhi, Govt. of India for a research associateship.
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