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Micron and submicron nuclear probes in biomedicine
Nuclear Instruments North-Holland
MICRON
and Methods
in Physics
AND SUBMICRON
Research
NUCLEAR
B49 (1990) 451-464
PROBES
451
IN BIOMEDICINE
Nuclear or proton microprobes are now well-established analytical instruments, aith~)u~h the accessibility is rather low. The majority of applications are found in the biomedical field but the number of areas where the nuclear probes are utilised are steadily increasing: good examples are archaeology. geology and microelectronics. A series of typical and exciting applications in biomedicine will be presented and their biological and medical impact discussed.
1. I~~oduction
The exploitation of PIXE and other analytical techbased on the interaction between nuclear projectiles and the atoms and nuclei of the irradiated specimen has now reached a stage of development that allows a fruitful application to biomedical research. The awareness and acceptation of the nuclear microprobes in the biological and medical societies are, however, rather low. The reason for this is probably the low accessibility of instruments. The most prominent feature of nuclear microprobes. in co~lparison with other well-established techniques such as X-ray microanalysis exercised in the scanning electron microscope, is the capability of trace element determination. The designation trace elements developed because early workers were unable to measure their precise concentrations. This designation has remained in popular usage, although analytical methods today allow almost all trace elements in biological objects to be determined with some accuracy and precision. Lacking a logical classification of trace elements, the chemical characterisation of trace elements as those present in concentrations less then 100 ppm is adopted for this presentation. Trace elements, or micronutrients. are intimately involved in physiological function and dysfunction at all levels of organisation. Depending on its action the trace elements has been named either esserz~ia~ or toxic. The feature of being essential, of course, reflects the state of knowledge at a certain point of time. On the other hand. it does not seem logical to establish a group of toxic elements because toxicity is inherent in at1 elements and depends mainly on the concentration. The essentiality has been defined some years ago [l]. A trace element is considered to be essential if it meets the following criteria: niques
0168-583X/90/$03.50 (North-Holland)
8~ Elsevier Science Publishers
B.V
1) it is present in all healthy tissue of all living things, 2) its concentration from one animal to the next is fairly constant. 3) its withdrawal from the body induces reproducibly the same physiological and structural abnormalities, regardless of the species studied, 4) its addition either reverses or prevents these ahnormalities, 5) the abnorRlal~ties induced by deficiencies are always accompanied by specific biochemical changes and 6) these biological changes can be prevented or cured when deficiency is prevented or cured. Trace elements are often bound as important cofaetars in enzymes, the most prominent example being zinc. The importance of zinc as an essential trace elements is evident from the fact that more than 200 enzymes are zinc-dependent 171. Trace elements are necessary for maintenance and regulation of compnrtmentation of cell functions, gene regulation and regulation of membrane functions, although some of the elements, by definition. cannot be regarded as trace elements. The development of life has brought about elaborate, diverse and subtle control mechanisms to provide for trace elements homoeostasjs. The trace elements thus exert profound influence on human health and disease states. The homocostasis and interaction of trace elements with each other and with nonessential trace elements such as heavy metals in biological systems is very complex. In spite of the research efforts spent, detailed knowledge of trace element functions is rather scarce. A lot of information has been gained through the use of radioactive n&ides in cell cultures and in experimental animal models. With the advent of Positron Emission Tomography it has been possible to perform dynamic studies with short-lived radionuclides. The gross concentration of major and trace elements in human tissues VI11. MICROPROBE
APPLICATIONS
452
V. Lmdh / Nuclear probes in biomedicrne
is well characterised by various analytical methods, e.g. [3]. The literature, however, lacks a good compilation of major and trace element concentrations in animal tissues. Through the use of X-ray microanalysis in the scanning electron microscope and the nuclear microprobe, data are being gathered of trace element distributions in different tissues and cells. Trace element importance for the normal biological function and disease processes has hitherto only scarcely been studied. Approaches to study cell-to-cell interaction and communication are similarly scarce. mainly because techniques and instrumentation have been lacking. The adequate application of nuclear microprobes to cellular studies of trace-element biology should, therefore. constitute a valuable source of information. Some organs or organ structures. for example parts of the brain. are extraordinarily difficult to remove cleanly and reproducibly from the surrounding tissue. In addition. it is often necessary to perfuse the tissue to circumvent the influence from remaining blood corpuscles if the objective is to determine total concentrations of trace elements. The nucIear microprobe, in contrast, offers a unique possibility of trace element analysis providing organ and cell distributions. Thus it will be within reach to relate trace element concentration and distribution to function and dysfunction of almost any organismal structure. The medical cell biologist often wants to extrapolate data and conclusions obtained at one level of biological organisation to higher and lower levels. It is necessary, therefore. to carefully sample and prepare the specimens before analysis. This is the most critical part of trace element analysis and has to be performed with utmost expertise. Eventually it puts heavy demands on the analyst and the equipment. A valuable series of papers on the problem has been presented by Iyengar and Sansoni [4-61. The sampling will be dependent on the kind of problem under study. Preparation techniques should optimally preserve the elemental and morphological integrity of the sample. When the appropriate microprobe has been selected with adequate sensitivity and the appropriate spatial resolution the possible beam-induced damages should be monitored and the loss of volatile elements taken into account. The greatest impediment to the practical application of our knowledge of trace elements in human health is the inadequacy of status diagnosis. The beneficial effects of trace element supplementation. like those of other nutrients, depend on the existence of a deficiency: supplements do not further improve an adequate, element-dependent function. Since there are some very recent reviews [7-111 of nuclear microprobe applications to biomedicine the objective of this work is not to present an extensive review of these applications. A small sample of representative and exciting nuclear microprobe approaches to bio-
medical problems will be presented and discussed. The exploitation of elemental data is also dealt with in some depth.
2. Examples of nuclear microprobe applications With a somewhat arbitrarily chosen restriction of a beam intensity of 100 pA in a spot less than or equal to 10 x 10 l_trn’ there are roughly twenty nuclear microprobes over the world involved with biomedicine (table 1). For this presentation, the main applications are separated in: cell biology, neurology. immunology, toxicology. trace-element biology and internal medicine.
The average human and animal cells display a diameter of 5-10 pm in the tissue. Some other cells. for example white blood corpuscles, have diameters in the region lo-20 pm. This living cell comprises a very complicated structure with a lot of organelles of varying size. Most of the existing nuclear microprobes will, therefore, be excluded from detailed elemental mapping
Table 1 Nuclear microprobes used in biomedicine ( I 10 pm’) Location
Size of probe
SUNY. Albany. USA
1.3 x
1.3
pm’
Applications
biology. trace elements Vrije U. Amsterdam. NL 5x5 tXl1’ biology 40% U Birmingham, GB 5x5 [Lrn’ biological samples U Bochum. FRG medicine 50%, 10 pm biology 20% Bordeaux-Gradignan. F 1.8~ 1.8 pm2 biology ( z 1988) Eindhoven U of Tech, NL 10 x 10 pm’ biology 80% U Oregon. Eugene, Or. US 8 ym biology 70% Fame, South Africa 4x4 pm’ biology MPI. Heidelberg, FRG 3x2 pm’ biology 30% Karlsruhe, FRG 2x3 vrn’ medicine 80%. closed 1987 Queen’s U. Kingston. biology 30% Canada 10 Pm biomedicine 50% Lund U of Tech. S 5x5 1”“2 Manitoba, Canada medical and SxSpm’ biophysics U Melbourne. Australia biomedicine 50% 1 iLm U Oxford. GB ilxl pm’ biology 50%. medicine 30% 2 Kernforsch, Rossendorf, biology 10% GDK 6pm Schonland Res C, South Africa
453
U. Lindh / Nuclear probes in biomedicine
of cells. Even the best resolution achieved, = 1 ym. is barely adequate to extract detailed spatial information of the smallest organelles. Nuclear microprobe studies of individual cells include the works of Cookson and Pilling [12], Legge and Mazzolini [13], Blower 1141, Consior [15] and Heck and Rokita [16]. Part of the work on individual cells in our laboratory will be described in subsequent sections. Here we present a special case of elemental mapping (fig. 1) of individual erythrocytes performed with a beam of size 1.7 x 1.7 pm2 with only 35 pA proton current. An area of 25 X 25 pm’ was scanned in a raster pattern. The low current implied total irradiation times of nearly ten hours. In some of the elemental maps it is possible to discern the biconcave shape of the erythrocytes. The central part in, for example, the chlorine map displays lower intensity, thus reflecting the fact that the mass probed is less than in the periphery. The results from the experiment is in good agreement with those of O’Brien and Legge [17]. There is one exception, though, in that the zinc concentrations differ between the studies. The Melbourne group indicated appro~mately 1 pg/g Zn, whereas our study revealed 25-35 pg/g. The difference might be explained by differences in preparation techniques. It should also be added that nuclear microprobe analysis of red blood cells mainly has been used for calibration purposes. The elemental maps in fig. 1 also demonstrate that contamination can be controlled. The structure that is revealed in the aluminium and silicon maps is a contamination. Using bulk analytical methods
would not have been possible to attribute the aluminium and silicon signals to a contamination. Another exciting individual cell study was performed by the Oxford group [9} on the yeast Cundida afbicans. In addition to elemental maps with l-micron resolution, they presented quantitative elemental concentrations of ten elements ranging from sodium to zinc. The study of this pathogenic yeast has been brought into focus for various reasons, one of which is the possibility of a very frequent human disease candidiasis. it
2.2. Neurologv The number of applications to neurology and neurobiology is steadily increasing. Extensive research on trace elements alterations in ischaemic rat brain was carried out by the Lund group [18-201. Total calcium and potassium and some trace elements were measured in the stratum oriens, pyramidale, radiatum and moleculare of the CA1 and CA2 sectors of the hippocampus. using a 20 X 20 pm2 static microbeam. High resolution elemental mapping of honey bee brain was performed by Watt and Grime [9]. Senile plaques from the brain of patients with Alzheimer’s disease were examined by the Oxford group [21]. Aluminium and silicon were consistently found and appeared to be co-localised in the centre of the plaque cores. Fairly high concentrations of particulate titanium were also revealed. The authors present the hypothesis that the titanium in the senile piaque cores originate
Sulphw
Phosphorus ” ~:,..’
.,
ron
%gn&um Fig. 1. Elemental maps of eqthrocytes
Aluminium
Silicon
from a healthy individual. ‘The intensity scales are not equal for all elements. example, is at the 1 pg/g level or less, VIII. MICROPROBE
Manganese,
APPLICATIONS
for
from air-borne pollution entering the organism via olfactory bulb. Recent work by the Lund group include studies calcium accumulation in substantia nigra lesions patients suffering from epilepsy and trace elements astrocytomas [22,23]. The latter paper also includes relevant multivariate statistical approach.
the of in in a
17.2.1. Lead distribution in the cerebelhn The possibility of persisting impairments caused by an increased lead burden on the developing central nervous system has attracted an increasing clinical as well as experimental interest. However, relatively little is known about the distribution of lead in the central nervous system following early low-dose Lead exposure. Many developmental studies dealing with chronic lead exposure have focused on the cerebellum where a haemorrhagic encephalopathy has been reproduced, also in rats not showing concomittant growth retardation. The Studsvik Nuclear Microprobe, now transferred to Uppsala and presently being replaced by a new probe, was used to analyse the distribution of lead in the cerebellum of suckling rats exposed to two different doses of iead. the lower of which does not result in growth retardation 1241. Suckling Sprague-Dawley rats were given intraperitoneal injections with lead nitrate from age 1 to 14 days. The calculated daily dose was 1.8 ug/g body weight (b.w.) or 15.6 kg/g b.w. At 20 days the rats were sacrificed. After vascular perfusion with ammonium acetate, the cerebellar vermis was frozen in propane/ propylene (Gasol”) chilled with liquid nitrogen. Cryostate sections, 15 urn thick, were cut and air-dried on formvar coats. Adjoining sections were stretched on glass and stained with toluidine blue. The resulting elemental map of lead and the micrograph of the stained section are displayed in fig. 2 and the sum spectrum From white matter in one animal from the high-dose group is shown in fig. 3. The elemental map and the nlicro~raph do not show a one-to-one correspondence. This is probably caused by the stretching on glass of the stained section and such a procedure cannot be used for the unstained section. The major finding of this study was the greater accutnulation of lead in the white than in the cortical grey matter. The present data do not give information on whether lead was predominant in axons or in myelin sheeths. The relativeI:: sharp border between cortex and white matter does necessarily mean that accumulation was mainly in the myelin rather than in the axons since both these structural components are diluted when spreading into the cartex. Within the cortex. small differences were observed in lead concentration of the granular and the molecular layer. This may be related to an accumulation in nuclei and mitochondria since cell nuclei are low in number in the molecular layer but
Fig. 2. Lead elemental map of rat cerebellum (upper part) and corresponding micrograph of toluidine-blue stained section (lower part)
Fig. 3. An X-ray sum spectrum from nuclear microprobe analysis of white matter in one of the experimental animals.
numerous in the granular layer, whereas the opposite is the case for mitochondria. The low amounts in the Purkinje cell layer would favour an accumulation in mitochondria since Purkinje cells of the rate cerebellum are relatively widely spaced and the Bergmann glial cells exhibit low activity of oxidative enzymes indicating low number of mitochondria. With a submicron beam some of these questions could be answered. .?.3. Immunology In an extension of the studies performed by the Uppsala group on blood cells isolated from patients suffering from the metal syndrome [25,26], a group of patients with an Epstein-Barr virus associated disease was studied in the same manner [27]. The Epstein-Barr virus (EBV) has been thoroughly studied. It is a lymphotropic virus causing infectious mononucelosis. Chronic illness has been associated with serological evidence of persistent active EBV-infection. The terms Chronic Mononucleosis and Chronic Fatigue Syndrome (CFS) have been used. From venous blood, red and white (neutrophit granulocytes) cells were isolated by centrifugation [ZS] and placed on formvar foils as monolayers. After quench freezing in isopentane chilled with liquid nitrogen and freeze-drying, the cells were analysed in the nuclear microprobe to unveil their individual elemental concentrations. The measurements revealed the presence of mercury in one or more of 100 cells for 81% of the patients, whereas the age- and six-matched controls displayed no measurable mercury. The frequency of mercu~-containing cells is depicted in fig. 4. Our findings of mercury in blood cells from patients with CFS as compared to nothing detectable in controls point to a hitherto not observed relationship in man between abnormal immune responses and the presence in tissues of an immunomodulating heavy metal. Knowing the
:6-E
26-50
Fig. 4. The distribution of mercu~-containing white blood ceils in the CFS-study as detected by the nuclear microprobe. A total of 100 cells was studied.
properties of mercury even in minute quantities, the results are somewhat suggestive as to the possibility of mercury being the cause of part of or all the symptoms. Mercury, any how. has to be considered as an important cofactor for the development of the syndrome. This study clearly shows one of the potentials of nuclear microprobe analysis. Measuring elemental contents in individual cells is, of course, tedious and timeconsuming. Skew distributions of heavy metals could. however, remain undetected if the concentrations had been measured on bulk material because of the very small differences in total concentrations. toxic
-7.4. Toxicolog? The Uppsala group have devoted a lot of efforts to the study of interactions between essential trace elements and toxic heavy metals. As experimental model the Sprague-Dawly rat strain was used. Early experiments with selenium and mercury revealed co-localisation of these elements in periportal hepatocytes and epithelial cells in the proximal tubules in kidneys from animals exposed to both elements. i%o ~stopatholo~tally discernable damages could be detected. The conclusion was that selenium exerted a protective effect against mercury toxicity f29.111. The same conclusion was applicable also when zinc and lead were administered simultaneously 1111. In a follow-up study [30] it was shown with elemental maps (fig. 5) and histopathological studies that selenium has a protective effect against cadmium toxicity. In the cadmium-treated group of rats there were scattered signs. in the liver, of parenchymal cell necrosis and a low degree of Kupfer ceil vacuolation. In addition, degeneration of some hepatocytes was observed. The pathological alterations found in kidney sections comprised scarce signs of degeneration in proximal tubule cells exhibited as exfoliation. in the cadmiumselenium-treated group the signs of parenchymal cell necrosis and vacuolation of Kupfer ceils were not observed. Some Kupfer cells with refractile bodies within their cytoplasm, however, were found. In the kidney section the proximal renal tubular epithelial cells showed no signs of exfoliation but many of them contained pro~nent intranuclear refractiie inclusions. In the elemental maps from the cad~um-treated group it was found that cadmium appeared to accumulate in renal proximal tubule epithelial cells and also in hepatic parenchymal cells and Kupfer cells. When cadmium and selenium were administered in combination. the elements were found co-localised in renal proximal proximal tubule lining cells and in hepatic parenchymal and Kupfer cells. There appeared to be a fairly constant ratio of cadmium-to-selenium in these cells. The mechanism by which selenium protects against VIII. MICROPROBE APPLICATIONS
U. Lindh / Nuclear probes in hiomediclne
456
Cadmium map
Selenium
map
Cadmium
Selenium
map
map
Fig. 5. Elemental maps of a liver (upper part) section and a kidney (lower part) section from a rat in the cadmium-selenium Co-localisation
of the elements
cadmium toxicity probably involves a direct interaction of the two elements. Gasiewicz and Smith [31] showed that when SeOz- and Cd2+ were incubated in plasma containing erythrocytes, a protein of 130000 molecular weight, with cadmium and selenium bound in a 1 : 1 ratio, was formed. The same protein peak was found in gel filtration of plasma that had been incubated with H,Se and Cd’+ without erythrocytes, although erythrocytes were necessary to form the cadmiumselenium complex when selenium was present as SeO? The authors hypothesised that erythrocytes must absorb and release H,Se, which then and metabolise SeO:interacts with Cd2+. After SeOiand CdCl, were injected simultaneously into rats, two peaks representing 330000 and 130000 daltons with a Cd : Se stoichiometry of 1 : 1 were found in the gel filtrates of plasma, showing that a cadmium-selenium complex can also be formed in vivo. 2.5. Trace elements The field of trace-element biology has yet only been opened for the nuclear microprobes but it is certainly
group.
is evident.
well-founded to anticipate that a lot of exciting applications will be seen very soon. The characteristic concentrations and functional forms of trace elements must be maintained within narrow limits if the functional and structural integrity of the tissues is to be safeguarded and growth, health and fertility are to remain unimpaired. The degree of homoeostatic control varies from one element to another, but continued ingestion of diets, or continued exposure to total environments that are severely deficient, imbalanced, or excessively high in a particular trace element, induces changes in the functioning forms, activities, or concentrations of that element in the body tissues and fluids so that they fall below or rise above the permissible limits. In these circumstances biochemical defects develop, physiological functions are affected, and structural disordes may arise in ways which differ with different elements, with the degree and duration of the dietary deficiency or toxicity, and with the age, sex, and species of the animal involved. Most, but not all trace elements act primarily as catalysts in enzyme systems in the cells, where they serve a wide range of functions. Evidence is accumulat-
U. Lmdh / Nuclear probes tn bio~edjcj~e ing that the protein-metal interactions not only enhance the catalytic activity of enzymes but also may increase the stability of the protein moiety to metabolic turnover. The high concentrations of certain elements in nucleic acids [32] may be essential to maintain the structure. As an example of possible fields of nuclear microprobe application some thoughts about selenium metabolism are presented 1331. Selenium is known to be essential for mammals since 1957. It is an integral component of the enzyme glutathione peroxidase (GSH-Px). This enzyme has important tasks in the cellular protecting system against lipid peroxide-initiated damage. Animals suffering from selenium-deficiency diseases display decreased GSH-Px activities in organs. This suggests that peroxidative damage to cells and tissues is responsible for at least some of the symptoms. Although similarly well-defined primary selenium deficiency diseases have hitherto not been observed in humans, many researchers consider it to be a contributing factor in a variety of human pathological conditions (table 2). Albeit the available evidence often is only suggestive it must be taken seriously because marginal selenium deficiency is common in patients as well as clinically healthy subjects and a depletion of selenium body stores may occur during certain therapeutic interventions. 2.6. Infernal medicine Applications in this wide area include inflammatory connective-tissue diseases [34-361, neoplasias [37], hereditary diseases such as Down’s syndrome [38] and diabetes [39,40]. Applications to be discussed here are another hereditary disease, cystic fibrosis, and an inflammatory connective-tissue disease called ankylosing spondylitis or Bechterew’s disease. These two examples chosen will also serve as basis for a discussion of elemental data presentation and effective data handling. 2.6.1. Cystic fibrosis Although the cystic fibrosis (CF) gene is known to be localised on chromosome number 7, its gene product is still not known and the basic defect of the disease therefore remains an enigma. Eight children suffering from cystic fibrosis of varying severity and eight ageand sex-matched healthy children were included in the study. Furthermore, the parents of four of the CF children and eight age- and sex-matched healthy adults were included. Monolayers of individual red cells were prepared as described above. The results of the nuclear microprobe analysis are displayed in fig. 6. A closer look at the sodium and magnesium concentrations of erythrocytes is found in fig. 7. It was found 1411 that the CF children had significantly lower median erythrocyte con~ntrations of Na
457
Table 2 Human diseases for which selenium deficiency be a contributing
factor.
is considered
to
From ref. [33]
Malnutrition kwashiorkor malabsorption syndromes total parental nutrition Diseases requiring specia1 diets phenylketonuria maple-syrup disease Keshan disease Cardiovascular diseases myocardial infarction cardiomyopathy hypertension arteriosclerosis Muscular disorders Pancreatic diseases pancreatitis cystic fibrosis Cancer Alcoholism, cirrhosis Other diseases mucoviscidosis Glanzmann-thrombasteny coeliac disease haemolytic anemia neuronal ceroid lipofuscinosis infertility cataract diabetic retinopathy rheumatoid arthritis macular degeneration multiple sclerosis
and Mg than both the healthy children and the parents of the CF children. The parents of the CF children had significantly lower median concentrations of these elements than the healthy adults. In this study emphasis
Fig. 6. Elemental profiles (Na, Mg, Ct. Ca, Cu and Zn) in erythrocytes
from CF children, control children, children and healthy adults. VIII. MICROPROBE
parents
to CF
APPLICATIONS
I/. Lindh / Nuclear probes in biomedicine
458
a 1
Fig. 7. (a) Na-concentration
of erythrocytes in the CF-study. (b) Mg-concentration
was laid on Na, Mg, Cl, Ca, Cu and Zn. Albeit a lot of difference could be extracted by ordinary univariate nonparametric statistical methods, there was an interest to try to elucidate covariations between the elements. To do so one has to resort to multivariate statistical methods. Cluster analysis [42] was performed on the elemental data from control and CF children, respectively. The results from the control children are displayed in fig. 8, where three clusters have been found; Na + Mg + Cl being together in cluster 1, Ca + Cu in cluster 2 and Zn forms a cluster of its own. The corresponding clustering for the CF children (fig. 9) shows that Na + Mg + Cl constitue one cluster (#l), Ca + Zn another (# 2) and Cu a third (# 3). This is an indication
b
of erythrocytes in the CF-study
of differences in the handling of major and trace elements between the two groups. These covariations would have been very hard to extract using univariate methods. Another way of presenting data from cluster analysis is to construct dendrograms. The results of the hierarchical clustering by the nearest neighbour method are presented as dendrograms in fig. 10. The dendrograms may be more illustrative since they comprise all numbers of clusters in the process. The clustering methods thus constitute very important and powerful means in the design and interpretation of trace-element biological studies. Yet another powerful multivariate method that can be used for maximal group separation is the discrimi-
1
, t
.. i
5.
L
. I_, l
Fig. 8. Plot of clusters of elements measure in red cells of control children in the CF-study.
I__ I.
L
2.
0.
Fig. 9. Plot of clusters of elements measured children in the CF-study.
I
._--.
. .
J s.
in red cells of CF
459
U. Lindh / Nuclear probes in biomedicine Dendrogram
for control
although based on very small groups, indicate that erythrocyte concentrations of Na and Mg may be used to distinguish the CF heterozygotes from the healthy controls. As the concentrations of Na and Mg in the heterozygotes were between those of the CF patients and the healthy groups and did not seem to be influenced by nutritional factors, it is possible that the decreased erythrocyte concentrations of these elements reflect a primary defect of the CF disease.
chikben
Na Mg Cl Ca Cu Zn
w-r-I I Dendrognun Na
hfg
-T-
for CFchikiren Cl
Ca
Cu Zn
‘i Fig. 10. Dendrograms for control and CF children groups in the CF-study.
nant analysis [43]. The four groups involved in the CF study were merged to one. The merged group was analysed by discriminant analysis with respect to the Na- and Mg-concentrations in erythrocytes. The result of the analysis is displayed in fig. 11, where group 1 is the CF children, group 2 the control children, group 3 the parents of the CF children and group 4 the healthy adults. One can discern a separation of group 1. Group 2 and 4 constitute one cluster and group 3 is somewhere in between, i.e. the healthy control individuals separate from the CF children and their parents. These results,
2.62. Ankvlosing spon&litis The alterations of trace element metabolism in association with chronic inflammatory diseases has been a field under study for many decades. During recent years, clinical trace element research has come into focus partly due to the development of antirheumatic therapies which involve the manipulation of certain elements, but also due to the discovery of the central role certain metals have in free radical generation and lysosomal degranulation. So far. most works in chronic inflammatory diseases have concentrated on the study of single elements. However, this approach means certain limitations for the understanding of the complex redistribution of metals in chronic inflammation. In the study of ankylosing spondylitis four groups of individuals participated because of the genetic impact of the disease; it appears only in individuals who are HLA-B27 positive. Therefore, the study comprised patients (HLA-B27+), sexand age-matched controls, HLA-B27relatives and HLA-B27+ relatives.
6.
j_
.L ~~~~~
1
m Dircriminrnt
Fig. 11. Discriminant cerning
function
-1
C1
Mn
1
FI
--I
.2~
Zn
El=lllWlt
I
analysis diagram for the CF-study Na- and Mg-concentration.
L__
con-
Fig.
12. Multiple Box-and-Whisker plot of the granulocyte elemental profile of controls in the Bechterew study. VIII. MICROPROBE
APPLICATIONS
!
460
U. Lindh / Nuclear probes in biomedicine
I
I
Ha
c1
mn
Fr
Zn
Element
Fig. 13. Multiple Box-and-Whisker plot of the granulocyte elemental profile of patients in the Bechterew study.
Because of the elemental data nature Box-andWhisker plots were used for the presentation of the elemental profiles of neutrophil granulocytes. The analytical methods used in nuclear microprobes have detection limits of kg/g or slightly better. Therefore, some measurements of trace elements repeatedly result in not detectable concentrations. Ordinary bar charts consistently do not reflect this situation. To cope with this kind of limitation inherent in the analytical methods, Box-and-Whisker plots were chosen. Figs. 12-15 show the elemental profiles of neutrophil granulocytes from the four groups. Compared with the healthy controls the investigated 1st degree B27+ relatives showed significant accumulation of cellulazl,Mg, Ca, Mn and Fe. Compared with the patients, relatives and controls showed significantly lower values of Mg, Ca, Mn and Fe [44]. Zinc was significantly higher in relatives and controls. Granulocyte Sr was below the detection limit in relatives and controls but measurable in all patients. To study possible covariations between elements in neutrophil granulocytes, principal component analysis was resorted to [42]. Principal component analysis is a useful technique for reducing the number of variables in a data set by finding linear combinations of those variables that explain most of the variability. The principal component analysis can be visualised through biplots for the first two principal components accounting for the largest variance or spread in the data. Figs. 16-19 display such biplots for the control, patient, HLA-B27+ relative and HLA-B27relative groups, respectively. The lines intersecting at the origin (0. 0)
Fig. 14. Multiple elemental profile
Box-and-Whisker plot of the granulocyte of HLA-B27 positive relatives in the Bechterew study.
represent the original variables. The length of each vector is proportional to its contribution to the principal components. The angle between any two is in-
!-L----._.
A
no
CI
4 :
.I
mn
Fe
.-..-LA Zn
Elrmmnt
Fig. 15. Multiple elemental profile
3~x-and-W~sker of HLA-B27
plot of the granuiocyte negative relatives in the
Bechterew study.
U. Lindh / Nuclear probes In bromedicwze
N
_
Component.I
componqlt
Fig. 16. Biplot of the first two principal components trols in the Bechterew study.
versely can
proportional
be concluded
lation
between
relatives.
Zinc
to the correlation that
there
of con-
between
is a similar
them.
lack
It
of corre-
Mn and Zn in the control and HLA-B27and manganese seem to be similarly
1
Fig. 18. Biplot of the first two principal components of HLAB27 positive relatives to the patients in the Bechterew study.
correlated in the patient group and the group with HLA-B27+ relatives. In the same way Ca and Fe are similarly correlated in the patient group and in the
Mt-l
.I.,
FR
-
I
-3.7
-1.7
FI
a.,
._D
componrnt
1.3
1
Fig. 17. Biplot of the first two principal components tients in the Bechterew study.
of pa-
Fig. 19. Biplot of the first two p:incipal components of HLAB27 negative relatives to the patients in the Bechterew study. VIII. MICROPROBE
APPLICATIONS
462
U. Lindh / Nuclear probes VI hromedicine
to elucidate the controversial borders between health and disease. manifest or hidden disease in HLA-B27 positive individuals. Such a sensitive tool can also be of value for epidemiological studies concerning the propensity to inflammtory reactions in HLA-B27 positive individuals.
3. Exploitation
I
3
-2
Fig. 20. Discriminant analysis diagram for the Bechterew study concerning Mg. Ca, Mn, Fe and Zn.
group with HLA-B27+ relatives. Calcium and zinc correlate quite well in the control and HLA-B27relative groups. Our study was designed to recruit healthy HLA-B27 positive 1st degree relatives of HLA-B27 positive patients with ankylosing spondylitis. Thus, only relative that considered themselves healthy and had no obvious history of rheumatic disease were included. No radiographic examination was done at the time of inclusion for ethical reasons. These relatives, defined healthy by these subjective criteria, nevertheless, had altered cellular major and trace elements stores, similar to those observed with ankylosing spondylitis [45] in a qualitative way but quantitatively substantially lower. One hypothesis at this point was that the changes were of primary importance for disease susceptibility in HLAB27 positive individuals. The element pattern found in the granulocytes of the patients with ankylosing spondylitis and their relatives can be expected to stimulate the inflammatory activity of the cells. A discriminant analysis of the total study group of ankylosing spondylitis reveals that the patient group (# 1) is clearly separated from the other groups (fig. 20) and that the control group (#2) together with the group of HLA-B27relatives (#4) constitute an inseparable cluster. The HLA-B27+ relatives (#3) is closer to the latter cluster but seem to be separable and with a trend towards the patient group. Sophisticated analyses of major and trace elements in cells may be of signficant value in research in order
of elemental
data
The bar graph is probably the most common chart type for presenting elemental data. It has. however, serious drawbacks. Firstly. if the base level is zero it does not account for the detection limits of the analytical methods; to account for the detection limit it may be necessary to use different base lines for each element. Secondly, the bar chart displays neither range nor variation. The Box-and-Whisker plot meets all the demands on elemental data presentation and, furthermore. it is usually created from nonparametric values for central tendency and variation. Examples of Box-and-Whisker plots are found in figs. 12-15. The central box covers the middle 50% of the data values. between the lower and upper quartiles. The whiskers extend out to the extremes, while the central line is at the median. When unusual values occur far away from the bulk of the data, they are plotted as separate points. The whiskers extend only to those points that are within 1.5 times the interquartile range. One of the main advantages of multielemental analytic methods is that they are unbiased. This means that one can get information on elements that was not primarily sought. Multielemental methods can generate data of lo-15 elements when biological objects are analysed. The effective handling of such large volumes of elemental data calls fore multivariate statistical methods such as principal component analysis, discriminant analysis. cluster analysis and pattern recognition.
4. Caution Analysing very minute volumes in biological tissues causes problems in assessing the accuracy of determination. When probing volumes as small as 10m9 mm3. which corresponds to 1 pg or less. one has to bear in mind that trace element analysis can prove meaning-less. With an analytical sensitivity of 1 pg/g. the absolute mass detection limit approaches lo-” g. Pushing the limits further may imply practical obstacles. For example, 10~tx g of selenium contain some 76000 selenium atoms. Extending the spatial resolution of the nuclear microprobe to 0.1 pm, a goal which is within realistic reach, means that the probed volume can be as small as 10-l’ mm3 corresponding to a mass of 1 fg. This would
imply, if the detection limit can be pushed down to 100 rig/g.. the analysis of 8 selenium atoms. This situation is. of course, absurd. The cross sections will probably not permit such analyses within practical limits. If the lack of material to be analysed with very small probes is one obstacle, another is the possible beam-induced damages when current densities are improved. A current of 100 pA or more in a spot of 0.01-0.25 rJ.m’ implies current densities of at least 400-10000 PA/pm*. It can be questioned if a specimen can withstand such an intense radiation even if rapid beam scanning is employed. Some reassuring results were reviewed by Watt and Grime (91 but they extend only to current densities of 100 pA/l.tm*. Much more work is consequently necessary on the subject of radiation damage before firm conclusions can be reached. The problem of radiation damage has been addressed by Grodzins [46] and the temperature rise in irradiated specimens was recently investigated by Cholewa and Legge [47]. Unfortunately. there is no evident conclusion regarding nuclear probes with spatial resolution of 0.1 pm or better in nuclear microscopy and trace element mapping.
5. Conclusions The conclusions in an earlier review of nuclear microprobe applications in medicine [ll] are in practice still valid: the nuclear microprobe has not yet been fully appreciated by the biological and medical communities. It is now. however, the time to try to show the versatility of the instrument and choose scientifically sound applications. It is a very challenging and promising task for all nuclear microprobers. My sincere thanks go to all colleagues working with nuclear microprobes thereby making the break-in in the biomedical field possible and also to my major sponsor the Swedish Natural Science Research Council and to the Wallenberg Foundation making the new Uppsala nuclear microprobe possible.
References [l] E.J. Underwood
and W. Mertz, in: Trace Elements in Human and Animal Nutrition, 5th ed.. vol. 1, ed. W. Mertz (Academic Press, New York, 1986) p. 1. [2] KM. Hambridge, C.E. Casey and N.F. Krebs, in: Trace Elements in Human and Animal Nutrition, 5th ed.. vol. 2. rd. W. Mertz (Academic Press, New York, 1986) p. 1. [3] G.V. Iyengar. W.E. Kollmer and H.J.M. Bowen, The Elemental Composition of Human Tissues and Body Fluids (Verlag Chemie, Weinheim, 1978). [4] B. Sansoni and G.V. Iyengar, in: Elemental Analysis of Biological Materials: IAEA Technical Report 197 (IAEA, Vienna, 1978) p. 57.
[5] G.V. fyengar and B. Sansoni, in: Elemental Analysis of Biological Materials: IAEA Technical Report 197 (IAEA, Vienna, 1978) p. 73. [6] G.V. Iyengar, in: Trace Elements in Health and Disease. eds. H. Bostriim and N. Ljungstedt (Almqvist & Wiksell, Stockholm, 1985) p, 64. [7] R.D. Vis. The Proton Microprobe: Applications in the Biomedical Field (CRC Press, Boca Raton, 1985). [8] K.G. Malmqvist. Scanning Electron Microsc. III (1986) p. 821. [9] F. Watt and G.W. Grime. in: Principles and Applications of High-Energy Ion Microbeams. eds. F. Watt and G.W. Grime (Adam Hi&r, Bristol. 1987) p. 154. [lo] D.J.T. Vaux, F. Watt and G.W. Grime. in: Principles and Applications of High-Energy Ion Microbeams, eds. F. Watt and G.W. Grime (Adam Hilger. Bristol, 1987) p. 203. [ll] U. Lindh. Nucl. Instr. and Meth. B30 (1987) 404. [12] J.A. Cookson and F.D. Pilling. Phys. Med. Biol. 21 (1976) 115. [13] G.J.F. Legge and A.P. Mazzolini, Nucl. Instr. and Meth. 168 (1980) 563. [14] G.D. Blower, D. Phil. Thesis, University of Oxford (1983). 1151 B. Gonsior, in: Nuclear Analytical Techniques in Medicine. ed. R. Cesareo (Elsevier. Amsterdam. 1988) p, 123. 1161 D. Heck and E. Rokita, IEEE Trans. Nucl. Sci. NS-30 (1983) 1220. [17] P.M. O’Brien and G.J.F. Legge. Biol. Trace Elem. Res. 13 (1987) 159. [IS] K. Themner. KG. Malmqvist, E. Martins, K. Inamura and B.K. Siesjii. Nucl. ins%. and Meth. B22 (1987) 214. [19] E. Martins. K. Inamura, K. Themner, K.G. Malmqvist and B.K. Siesjii. J. Cereb. Blood Flow Metabol. 8 (1988) 531. [20] K. Themner, K.G. Malmqvist and B.K. Siesjo. Nucl. Instr. and Meth. 830 (1988) 424. [21] F. Watt. G.W. Grime. D.N. Jamieson and B. McDonald. Scanning Microsc. Intl.. in press. [22] K. Inamura. E. Martins, K. Themner, S. Tapper, J. Pallon. G. Lovestam, IS. Malmqvist and B. Siesjii, accepted for publication in Brain Res. [23] L.C. Salford, A. Brun. U.A.S. Tapper. E. Swietlicki and K. Malmqvist. submitted. [24] U. Lindh. N.G. Conradi and P. Sourander, Acta Neuropathol. 79 (1989) 153. [25] B. Ahlrot-Westerlund. B. Carlmark, S.-O. Gronquist, E. Johansson, U. Lindh, H. Theorell and K. de Vahl. Nutr. Res. Suppl. I (1985) 442. 1261 U. Lindh. S.-O. Gronquist and A. Lindvall, in: Proc. 11th Congr. on X-ray Optics and Microanal., eds. J.D. Brown and R.H. Packwood (LJniv. Western Ontario, London, Canada, 1987) p. 328. [27] A. Lindvall, G. Friman. S-O. Gronquist, A. Linde and U. Lindh, submitted. [28] U. Lindh and E. Johansson, Biol. Trace Elem. Res. 12 (1987) 351. [29] U. Lindh and E. Johansson. Biol. Trace Elem. Res. 12 (1987) 109. [30] U. Lindh and T. Sunde, submitted. [31] T.A. Gasiewicz and J.C. Smith, B&hem. Biophys. Acta 428 (1976) 113.
VIII. MICROPROBE
APPLICATIONS
464
[32]
[33] [34] [35] [36] [37] [38] [39] [40]
U, Lindh
/ Nuclear probes in biomedicirw
W.E.C. Wacker and B.L. Vallee. J. Biol. Chem. 234 (1959) 3251. H. Zumkley, Biol. Trace Elem. Res. 15 (1988) 139. R. Hlllgren. K. Svensson, E. Johansson and U. Lindh. Arthritis Rheum. 28 (1985) 169. R. HLllgren, K. Svensson. E. Johansson and U. Lindh. J. Lab. Clin. Med. 104 (1984) 893. K. Svensson. R. Hlllgren, E. Johansson and U. Lindh. Inflammation 9 (1985) 189. U. Lindh and E. Johansson, Neurotoxicol. 4 (1983) 177. G. Anne&, E. Johansson and U. Lindh, Acta Paediatr. Stand. 74 (1985) 259. U. Lindh. L. Juntti-Berggren. P.-O. Berggren and B. Hellman, Biomed. Biochim. Acta 44 (1985) 55. L. Juntti-Berggren. U. Lindh, P.-O. Berggren and B. Frankel, Biosci. Rep. 7 (1987) 33.
[41] T. Foucard. M. Gebre-Medhin. K.-H. Gustavson and U. Lindh, submitted. [42] D.L. Massart. B.G.M. Vandeginste. S.N. Deming, Y. Michotte and L. Kaufman. Chemometrics: A Textbook (Elsevier, Amsterdam. 1988). [43] P. Armitage and G. Berry, Statistical Methods in Medical Research, 2nd ed. (Blackwell Scientific Publ.. Oxford. 1987). [44] N. Feltelius. R. HIllgren and U. Lindh. J. Rheumatol. 15 (1988) 308. [45] R. HYllgren. N. Feltelius and U. Lindh. J. Rheumatol. 14 (1987) 548. [46] L. Grodzins. Nucl. Instr. and Meth. 218 (1983) 203. [47] M. Cholewa and G.J.F. Legge, Nucl. Instr. and Meth. B40/41 (1989) 651.
MICRON
and Methods
in Physics
AND SUBMICRON
Research
NUCLEAR
B49 (1990) 451-464
PROBES
451
IN BIOMEDICINE
Nuclear or proton microprobes are now well-established analytical instruments, aith~)u~h the accessibility is rather low. The majority of applications are found in the biomedical field but the number of areas where the nuclear probes are utilised are steadily increasing: good examples are archaeology. geology and microelectronics. A series of typical and exciting applications in biomedicine will be presented and their biological and medical impact discussed.
1. I~~oduction
The exploitation of PIXE and other analytical techbased on the interaction between nuclear projectiles and the atoms and nuclei of the irradiated specimen has now reached a stage of development that allows a fruitful application to biomedical research. The awareness and acceptation of the nuclear microprobes in the biological and medical societies are, however, rather low. The reason for this is probably the low accessibility of instruments. The most prominent feature of nuclear microprobes. in co~lparison with other well-established techniques such as X-ray microanalysis exercised in the scanning electron microscope, is the capability of trace element determination. The designation trace elements developed because early workers were unable to measure their precise concentrations. This designation has remained in popular usage, although analytical methods today allow almost all trace elements in biological objects to be determined with some accuracy and precision. Lacking a logical classification of trace elements, the chemical characterisation of trace elements as those present in concentrations less then 100 ppm is adopted for this presentation. Trace elements, or micronutrients. are intimately involved in physiological function and dysfunction at all levels of organisation. Depending on its action the trace elements has been named either esserz~ia~ or toxic. The feature of being essential, of course, reflects the state of knowledge at a certain point of time. On the other hand. it does not seem logical to establish a group of toxic elements because toxicity is inherent in at1 elements and depends mainly on the concentration. The essentiality has been defined some years ago [l]. A trace element is considered to be essential if it meets the following criteria: niques
0168-583X/90/$03.50 (North-Holland)
8~ Elsevier Science Publishers
B.V
1) it is present in all healthy tissue of all living things, 2) its concentration from one animal to the next is fairly constant. 3) its withdrawal from the body induces reproducibly the same physiological and structural abnormalities, regardless of the species studied, 4) its addition either reverses or prevents these ahnormalities, 5) the abnorRlal~ties induced by deficiencies are always accompanied by specific biochemical changes and 6) these biological changes can be prevented or cured when deficiency is prevented or cured. Trace elements are often bound as important cofaetars in enzymes, the most prominent example being zinc. The importance of zinc as an essential trace elements is evident from the fact that more than 200 enzymes are zinc-dependent 171. Trace elements are necessary for maintenance and regulation of compnrtmentation of cell functions, gene regulation and regulation of membrane functions, although some of the elements, by definition. cannot be regarded as trace elements. The development of life has brought about elaborate, diverse and subtle control mechanisms to provide for trace elements homoeostasjs. The trace elements thus exert profound influence on human health and disease states. The homocostasis and interaction of trace elements with each other and with nonessential trace elements such as heavy metals in biological systems is very complex. In spite of the research efforts spent, detailed knowledge of trace element functions is rather scarce. A lot of information has been gained through the use of radioactive n&ides in cell cultures and in experimental animal models. With the advent of Positron Emission Tomography it has been possible to perform dynamic studies with short-lived radionuclides. The gross concentration of major and trace elements in human tissues VI11. MICROPROBE
APPLICATIONS
452
V. Lmdh / Nuclear probes in biomedicrne
is well characterised by various analytical methods, e.g. [3]. The literature, however, lacks a good compilation of major and trace element concentrations in animal tissues. Through the use of X-ray microanalysis in the scanning electron microscope and the nuclear microprobe, data are being gathered of trace element distributions in different tissues and cells. Trace element importance for the normal biological function and disease processes has hitherto only scarcely been studied. Approaches to study cell-to-cell interaction and communication are similarly scarce. mainly because techniques and instrumentation have been lacking. The adequate application of nuclear microprobes to cellular studies of trace-element biology should, therefore. constitute a valuable source of information. Some organs or organ structures. for example parts of the brain. are extraordinarily difficult to remove cleanly and reproducibly from the surrounding tissue. In addition. it is often necessary to perfuse the tissue to circumvent the influence from remaining blood corpuscles if the objective is to determine total concentrations of trace elements. The nucIear microprobe, in contrast, offers a unique possibility of trace element analysis providing organ and cell distributions. Thus it will be within reach to relate trace element concentration and distribution to function and dysfunction of almost any organismal structure. The medical cell biologist often wants to extrapolate data and conclusions obtained at one level of biological organisation to higher and lower levels. It is necessary, therefore. to carefully sample and prepare the specimens before analysis. This is the most critical part of trace element analysis and has to be performed with utmost expertise. Eventually it puts heavy demands on the analyst and the equipment. A valuable series of papers on the problem has been presented by Iyengar and Sansoni [4-61. The sampling will be dependent on the kind of problem under study. Preparation techniques should optimally preserve the elemental and morphological integrity of the sample. When the appropriate microprobe has been selected with adequate sensitivity and the appropriate spatial resolution the possible beam-induced damages should be monitored and the loss of volatile elements taken into account. The greatest impediment to the practical application of our knowledge of trace elements in human health is the inadequacy of status diagnosis. The beneficial effects of trace element supplementation. like those of other nutrients, depend on the existence of a deficiency: supplements do not further improve an adequate, element-dependent function. Since there are some very recent reviews [7-111 of nuclear microprobe applications to biomedicine the objective of this work is not to present an extensive review of these applications. A small sample of representative and exciting nuclear microprobe approaches to bio-
medical problems will be presented and discussed. The exploitation of elemental data is also dealt with in some depth.
2. Examples of nuclear microprobe applications With a somewhat arbitrarily chosen restriction of a beam intensity of 100 pA in a spot less than or equal to 10 x 10 l_trn’ there are roughly twenty nuclear microprobes over the world involved with biomedicine (table 1). For this presentation, the main applications are separated in: cell biology, neurology. immunology, toxicology. trace-element biology and internal medicine.
The average human and animal cells display a diameter of 5-10 pm in the tissue. Some other cells. for example white blood corpuscles, have diameters in the region lo-20 pm. This living cell comprises a very complicated structure with a lot of organelles of varying size. Most of the existing nuclear microprobes will, therefore, be excluded from detailed elemental mapping
Table 1 Nuclear microprobes used in biomedicine ( I 10 pm’) Location
Size of probe
SUNY. Albany. USA
1.3 x
1.3
pm’
Applications
biology. trace elements Vrije U. Amsterdam. NL 5x5 tXl1’ biology 40% U Birmingham, GB 5x5 [Lrn’ biological samples U Bochum. FRG medicine 50%, 10 pm biology 20% Bordeaux-Gradignan. F 1.8~ 1.8 pm2 biology ( z 1988) Eindhoven U of Tech, NL 10 x 10 pm’ biology 80% U Oregon. Eugene, Or. US 8 ym biology 70% Fame, South Africa 4x4 pm’ biology MPI. Heidelberg, FRG 3x2 pm’ biology 30% Karlsruhe, FRG 2x3 vrn’ medicine 80%. closed 1987 Queen’s U. Kingston. biology 30% Canada 10 Pm biomedicine 50% Lund U of Tech. S 5x5 1”“2 Manitoba, Canada medical and SxSpm’ biophysics U Melbourne. Australia biomedicine 50% 1 iLm U Oxford. GB ilxl pm’ biology 50%. medicine 30% 2 Kernforsch, Rossendorf, biology 10% GDK 6pm Schonland Res C, South Africa
453
U. Lindh / Nuclear probes in biomedicine
of cells. Even the best resolution achieved, = 1 ym. is barely adequate to extract detailed spatial information of the smallest organelles. Nuclear microprobe studies of individual cells include the works of Cookson and Pilling [12], Legge and Mazzolini [13], Blower 1141, Consior [15] and Heck and Rokita [16]. Part of the work on individual cells in our laboratory will be described in subsequent sections. Here we present a special case of elemental mapping (fig. 1) of individual erythrocytes performed with a beam of size 1.7 x 1.7 pm2 with only 35 pA proton current. An area of 25 X 25 pm’ was scanned in a raster pattern. The low current implied total irradiation times of nearly ten hours. In some of the elemental maps it is possible to discern the biconcave shape of the erythrocytes. The central part in, for example, the chlorine map displays lower intensity, thus reflecting the fact that the mass probed is less than in the periphery. The results from the experiment is in good agreement with those of O’Brien and Legge [17]. There is one exception, though, in that the zinc concentrations differ between the studies. The Melbourne group indicated appro~mately 1 pg/g Zn, whereas our study revealed 25-35 pg/g. The difference might be explained by differences in preparation techniques. It should also be added that nuclear microprobe analysis of red blood cells mainly has been used for calibration purposes. The elemental maps in fig. 1 also demonstrate that contamination can be controlled. The structure that is revealed in the aluminium and silicon maps is a contamination. Using bulk analytical methods
would not have been possible to attribute the aluminium and silicon signals to a contamination. Another exciting individual cell study was performed by the Oxford group [9} on the yeast Cundida afbicans. In addition to elemental maps with l-micron resolution, they presented quantitative elemental concentrations of ten elements ranging from sodium to zinc. The study of this pathogenic yeast has been brought into focus for various reasons, one of which is the possibility of a very frequent human disease candidiasis. it
2.2. Neurologv The number of applications to neurology and neurobiology is steadily increasing. Extensive research on trace elements alterations in ischaemic rat brain was carried out by the Lund group [18-201. Total calcium and potassium and some trace elements were measured in the stratum oriens, pyramidale, radiatum and moleculare of the CA1 and CA2 sectors of the hippocampus. using a 20 X 20 pm2 static microbeam. High resolution elemental mapping of honey bee brain was performed by Watt and Grime [9]. Senile plaques from the brain of patients with Alzheimer’s disease were examined by the Oxford group [21]. Aluminium and silicon were consistently found and appeared to be co-localised in the centre of the plaque cores. Fairly high concentrations of particulate titanium were also revealed. The authors present the hypothesis that the titanium in the senile piaque cores originate
Sulphw
Phosphorus ” ~:,..’
.,
ron
%gn&um Fig. 1. Elemental maps of eqthrocytes
Aluminium
Silicon
from a healthy individual. ‘The intensity scales are not equal for all elements. example, is at the 1 pg/g level or less, VIII. MICROPROBE
Manganese,
APPLICATIONS
for
from air-borne pollution entering the organism via olfactory bulb. Recent work by the Lund group include studies calcium accumulation in substantia nigra lesions patients suffering from epilepsy and trace elements astrocytomas [22,23]. The latter paper also includes relevant multivariate statistical approach.
the of in in a
17.2.1. Lead distribution in the cerebelhn The possibility of persisting impairments caused by an increased lead burden on the developing central nervous system has attracted an increasing clinical as well as experimental interest. However, relatively little is known about the distribution of lead in the central nervous system following early low-dose Lead exposure. Many developmental studies dealing with chronic lead exposure have focused on the cerebellum where a haemorrhagic encephalopathy has been reproduced, also in rats not showing concomittant growth retardation. The Studsvik Nuclear Microprobe, now transferred to Uppsala and presently being replaced by a new probe, was used to analyse the distribution of lead in the cerebellum of suckling rats exposed to two different doses of iead. the lower of which does not result in growth retardation 1241. Suckling Sprague-Dawley rats were given intraperitoneal injections with lead nitrate from age 1 to 14 days. The calculated daily dose was 1.8 ug/g body weight (b.w.) or 15.6 kg/g b.w. At 20 days the rats were sacrificed. After vascular perfusion with ammonium acetate, the cerebellar vermis was frozen in propane/ propylene (Gasol”) chilled with liquid nitrogen. Cryostate sections, 15 urn thick, were cut and air-dried on formvar coats. Adjoining sections were stretched on glass and stained with toluidine blue. The resulting elemental map of lead and the micrograph of the stained section are displayed in fig. 2 and the sum spectrum From white matter in one animal from the high-dose group is shown in fig. 3. The elemental map and the nlicro~raph do not show a one-to-one correspondence. This is probably caused by the stretching on glass of the stained section and such a procedure cannot be used for the unstained section. The major finding of this study was the greater accutnulation of lead in the white than in the cortical grey matter. The present data do not give information on whether lead was predominant in axons or in myelin sheeths. The relativeI:: sharp border between cortex and white matter does necessarily mean that accumulation was mainly in the myelin rather than in the axons since both these structural components are diluted when spreading into the cartex. Within the cortex. small differences were observed in lead concentration of the granular and the molecular layer. This may be related to an accumulation in nuclei and mitochondria since cell nuclei are low in number in the molecular layer but
Fig. 2. Lead elemental map of rat cerebellum (upper part) and corresponding micrograph of toluidine-blue stained section (lower part)
Fig. 3. An X-ray sum spectrum from nuclear microprobe analysis of white matter in one of the experimental animals.
numerous in the granular layer, whereas the opposite is the case for mitochondria. The low amounts in the Purkinje cell layer would favour an accumulation in mitochondria since Purkinje cells of the rate cerebellum are relatively widely spaced and the Bergmann glial cells exhibit low activity of oxidative enzymes indicating low number of mitochondria. With a submicron beam some of these questions could be answered. .?.3. Immunology In an extension of the studies performed by the Uppsala group on blood cells isolated from patients suffering from the metal syndrome [25,26], a group of patients with an Epstein-Barr virus associated disease was studied in the same manner [27]. The Epstein-Barr virus (EBV) has been thoroughly studied. It is a lymphotropic virus causing infectious mononucelosis. Chronic illness has been associated with serological evidence of persistent active EBV-infection. The terms Chronic Mononucleosis and Chronic Fatigue Syndrome (CFS) have been used. From venous blood, red and white (neutrophit granulocytes) cells were isolated by centrifugation [ZS] and placed on formvar foils as monolayers. After quench freezing in isopentane chilled with liquid nitrogen and freeze-drying, the cells were analysed in the nuclear microprobe to unveil their individual elemental concentrations. The measurements revealed the presence of mercury in one or more of 100 cells for 81% of the patients, whereas the age- and six-matched controls displayed no measurable mercury. The frequency of mercu~-containing cells is depicted in fig. 4. Our findings of mercury in blood cells from patients with CFS as compared to nothing detectable in controls point to a hitherto not observed relationship in man between abnormal immune responses and the presence in tissues of an immunomodulating heavy metal. Knowing the
:6-E
26-50
Fig. 4. The distribution of mercu~-containing white blood ceils in the CFS-study as detected by the nuclear microprobe. A total of 100 cells was studied.
properties of mercury even in minute quantities, the results are somewhat suggestive as to the possibility of mercury being the cause of part of or all the symptoms. Mercury, any how. has to be considered as an important cofactor for the development of the syndrome. This study clearly shows one of the potentials of nuclear microprobe analysis. Measuring elemental contents in individual cells is, of course, tedious and timeconsuming. Skew distributions of heavy metals could. however, remain undetected if the concentrations had been measured on bulk material because of the very small differences in total concentrations. toxic
-7.4. Toxicolog? The Uppsala group have devoted a lot of efforts to the study of interactions between essential trace elements and toxic heavy metals. As experimental model the Sprague-Dawly rat strain was used. Early experiments with selenium and mercury revealed co-localisation of these elements in periportal hepatocytes and epithelial cells in the proximal tubules in kidneys from animals exposed to both elements. i%o ~stopatholo~tally discernable damages could be detected. The conclusion was that selenium exerted a protective effect against mercury toxicity f29.111. The same conclusion was applicable also when zinc and lead were administered simultaneously 1111. In a follow-up study [30] it was shown with elemental maps (fig. 5) and histopathological studies that selenium has a protective effect against cadmium toxicity. In the cadmium-treated group of rats there were scattered signs. in the liver, of parenchymal cell necrosis and a low degree of Kupfer ceil vacuolation. In addition, degeneration of some hepatocytes was observed. The pathological alterations found in kidney sections comprised scarce signs of degeneration in proximal tubule cells exhibited as exfoliation. in the cadmiumselenium-treated group the signs of parenchymal cell necrosis and vacuolation of Kupfer ceils were not observed. Some Kupfer cells with refractile bodies within their cytoplasm, however, were found. In the kidney section the proximal renal tubular epithelial cells showed no signs of exfoliation but many of them contained pro~nent intranuclear refractiie inclusions. In the elemental maps from the cad~um-treated group it was found that cadmium appeared to accumulate in renal proximal tubule epithelial cells and also in hepatic parenchymal cells and Kupfer cells. When cadmium and selenium were administered in combination. the elements were found co-localised in renal proximal proximal tubule lining cells and in hepatic parenchymal and Kupfer cells. There appeared to be a fairly constant ratio of cadmium-to-selenium in these cells. The mechanism by which selenium protects against VIII. MICROPROBE APPLICATIONS
U. Lindh / Nuclear probes in hiomediclne
456
Cadmium map
Selenium
map
Cadmium
Selenium
map
map
Fig. 5. Elemental maps of a liver (upper part) section and a kidney (lower part) section from a rat in the cadmium-selenium Co-localisation
of the elements
cadmium toxicity probably involves a direct interaction of the two elements. Gasiewicz and Smith [31] showed that when SeOz- and Cd2+ were incubated in plasma containing erythrocytes, a protein of 130000 molecular weight, with cadmium and selenium bound in a 1 : 1 ratio, was formed. The same protein peak was found in gel filtration of plasma that had been incubated with H,Se and Cd’+ without erythrocytes, although erythrocytes were necessary to form the cadmiumselenium complex when selenium was present as SeO? The authors hypothesised that erythrocytes must absorb and release H,Se, which then and metabolise SeO:interacts with Cd2+. After SeOiand CdCl, were injected simultaneously into rats, two peaks representing 330000 and 130000 daltons with a Cd : Se stoichiometry of 1 : 1 were found in the gel filtrates of plasma, showing that a cadmium-selenium complex can also be formed in vivo. 2.5. Trace elements The field of trace-element biology has yet only been opened for the nuclear microprobes but it is certainly
group.
is evident.
well-founded to anticipate that a lot of exciting applications will be seen very soon. The characteristic concentrations and functional forms of trace elements must be maintained within narrow limits if the functional and structural integrity of the tissues is to be safeguarded and growth, health and fertility are to remain unimpaired. The degree of homoeostatic control varies from one element to another, but continued ingestion of diets, or continued exposure to total environments that are severely deficient, imbalanced, or excessively high in a particular trace element, induces changes in the functioning forms, activities, or concentrations of that element in the body tissues and fluids so that they fall below or rise above the permissible limits. In these circumstances biochemical defects develop, physiological functions are affected, and structural disordes may arise in ways which differ with different elements, with the degree and duration of the dietary deficiency or toxicity, and with the age, sex, and species of the animal involved. Most, but not all trace elements act primarily as catalysts in enzyme systems in the cells, where they serve a wide range of functions. Evidence is accumulat-
U. Lmdh / Nuclear probes tn bio~edjcj~e ing that the protein-metal interactions not only enhance the catalytic activity of enzymes but also may increase the stability of the protein moiety to metabolic turnover. The high concentrations of certain elements in nucleic acids [32] may be essential to maintain the structure. As an example of possible fields of nuclear microprobe application some thoughts about selenium metabolism are presented 1331. Selenium is known to be essential for mammals since 1957. It is an integral component of the enzyme glutathione peroxidase (GSH-Px). This enzyme has important tasks in the cellular protecting system against lipid peroxide-initiated damage. Animals suffering from selenium-deficiency diseases display decreased GSH-Px activities in organs. This suggests that peroxidative damage to cells and tissues is responsible for at least some of the symptoms. Although similarly well-defined primary selenium deficiency diseases have hitherto not been observed in humans, many researchers consider it to be a contributing factor in a variety of human pathological conditions (table 2). Albeit the available evidence often is only suggestive it must be taken seriously because marginal selenium deficiency is common in patients as well as clinically healthy subjects and a depletion of selenium body stores may occur during certain therapeutic interventions. 2.6. Infernal medicine Applications in this wide area include inflammatory connective-tissue diseases [34-361, neoplasias [37], hereditary diseases such as Down’s syndrome [38] and diabetes [39,40]. Applications to be discussed here are another hereditary disease, cystic fibrosis, and an inflammatory connective-tissue disease called ankylosing spondylitis or Bechterew’s disease. These two examples chosen will also serve as basis for a discussion of elemental data presentation and effective data handling. 2.6.1. Cystic fibrosis Although the cystic fibrosis (CF) gene is known to be localised on chromosome number 7, its gene product is still not known and the basic defect of the disease therefore remains an enigma. Eight children suffering from cystic fibrosis of varying severity and eight ageand sex-matched healthy children were included in the study. Furthermore, the parents of four of the CF children and eight age- and sex-matched healthy adults were included. Monolayers of individual red cells were prepared as described above. The results of the nuclear microprobe analysis are displayed in fig. 6. A closer look at the sodium and magnesium concentrations of erythrocytes is found in fig. 7. It was found 1411 that the CF children had significantly lower median erythrocyte con~ntrations of Na
457
Table 2 Human diseases for which selenium deficiency be a contributing
factor.
is considered
to
From ref. [33]
Malnutrition kwashiorkor malabsorption syndromes total parental nutrition Diseases requiring specia1 diets phenylketonuria maple-syrup disease Keshan disease Cardiovascular diseases myocardial infarction cardiomyopathy hypertension arteriosclerosis Muscular disorders Pancreatic diseases pancreatitis cystic fibrosis Cancer Alcoholism, cirrhosis Other diseases mucoviscidosis Glanzmann-thrombasteny coeliac disease haemolytic anemia neuronal ceroid lipofuscinosis infertility cataract diabetic retinopathy rheumatoid arthritis macular degeneration multiple sclerosis
and Mg than both the healthy children and the parents of the CF children. The parents of the CF children had significantly lower median concentrations of these elements than the healthy adults. In this study emphasis
Fig. 6. Elemental profiles (Na, Mg, Ct. Ca, Cu and Zn) in erythrocytes
from CF children, control children, children and healthy adults. VIII. MICROPROBE
parents
to CF
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458
a 1
Fig. 7. (a) Na-concentration
of erythrocytes in the CF-study. (b) Mg-concentration
was laid on Na, Mg, Cl, Ca, Cu and Zn. Albeit a lot of difference could be extracted by ordinary univariate nonparametric statistical methods, there was an interest to try to elucidate covariations between the elements. To do so one has to resort to multivariate statistical methods. Cluster analysis [42] was performed on the elemental data from control and CF children, respectively. The results from the control children are displayed in fig. 8, where three clusters have been found; Na + Mg + Cl being together in cluster 1, Ca + Cu in cluster 2 and Zn forms a cluster of its own. The corresponding clustering for the CF children (fig. 9) shows that Na + Mg + Cl constitue one cluster (#l), Ca + Zn another (# 2) and Cu a third (# 3). This is an indication
b
of erythrocytes in the CF-study
of differences in the handling of major and trace elements between the two groups. These covariations would have been very hard to extract using univariate methods. Another way of presenting data from cluster analysis is to construct dendrograms. The results of the hierarchical clustering by the nearest neighbour method are presented as dendrograms in fig. 10. The dendrograms may be more illustrative since they comprise all numbers of clusters in the process. The clustering methods thus constitute very important and powerful means in the design and interpretation of trace-element biological studies. Yet another powerful multivariate method that can be used for maximal group separation is the discrimi-
1
, t
.. i
5.
L
. I_, l
Fig. 8. Plot of clusters of elements measure in red cells of control children in the CF-study.
I__ I.
L
2.
0.
Fig. 9. Plot of clusters of elements measured children in the CF-study.
I
._--.
. .
J s.
in red cells of CF
459
U. Lindh / Nuclear probes in biomedicine Dendrogram
for control
although based on very small groups, indicate that erythrocyte concentrations of Na and Mg may be used to distinguish the CF heterozygotes from the healthy controls. As the concentrations of Na and Mg in the heterozygotes were between those of the CF patients and the healthy groups and did not seem to be influenced by nutritional factors, it is possible that the decreased erythrocyte concentrations of these elements reflect a primary defect of the CF disease.
chikben
Na Mg Cl Ca Cu Zn
w-r-I I Dendrognun Na
hfg
-T-
for CFchikiren Cl
Ca
Cu Zn
‘i Fig. 10. Dendrograms for control and CF children groups in the CF-study.
nant analysis [43]. The four groups involved in the CF study were merged to one. The merged group was analysed by discriminant analysis with respect to the Na- and Mg-concentrations in erythrocytes. The result of the analysis is displayed in fig. 11, where group 1 is the CF children, group 2 the control children, group 3 the parents of the CF children and group 4 the healthy adults. One can discern a separation of group 1. Group 2 and 4 constitute one cluster and group 3 is somewhere in between, i.e. the healthy control individuals separate from the CF children and their parents. These results,
2.62. Ankvlosing spon&litis The alterations of trace element metabolism in association with chronic inflammatory diseases has been a field under study for many decades. During recent years, clinical trace element research has come into focus partly due to the development of antirheumatic therapies which involve the manipulation of certain elements, but also due to the discovery of the central role certain metals have in free radical generation and lysosomal degranulation. So far. most works in chronic inflammatory diseases have concentrated on the study of single elements. However, this approach means certain limitations for the understanding of the complex redistribution of metals in chronic inflammation. In the study of ankylosing spondylitis four groups of individuals participated because of the genetic impact of the disease; it appears only in individuals who are HLA-B27 positive. Therefore, the study comprised patients (HLA-B27+), sexand age-matched controls, HLA-B27relatives and HLA-B27+ relatives.
6.
j_
.L ~~~~~
1
m Dircriminrnt
Fig. 11. Discriminant cerning
function
-1
C1
Mn
1
FI
--I
.2~
Zn
El=lllWlt
I
analysis diagram for the CF-study Na- and Mg-concentration.
L__
con-
Fig.
12. Multiple Box-and-Whisker plot of the granulocyte elemental profile of controls in the Bechterew study. VIII. MICROPROBE
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!
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U. Lindh / Nuclear probes in biomedicine
I
I
Ha
c1
mn
Fr
Zn
Element
Fig. 13. Multiple Box-and-Whisker plot of the granulocyte elemental profile of patients in the Bechterew study.
Because of the elemental data nature Box-andWhisker plots were used for the presentation of the elemental profiles of neutrophil granulocytes. The analytical methods used in nuclear microprobes have detection limits of kg/g or slightly better. Therefore, some measurements of trace elements repeatedly result in not detectable concentrations. Ordinary bar charts consistently do not reflect this situation. To cope with this kind of limitation inherent in the analytical methods, Box-and-Whisker plots were chosen. Figs. 12-15 show the elemental profiles of neutrophil granulocytes from the four groups. Compared with the healthy controls the investigated 1st degree B27+ relatives showed significant accumulation of cellulazl,Mg, Ca, Mn and Fe. Compared with the patients, relatives and controls showed significantly lower values of Mg, Ca, Mn and Fe [44]. Zinc was significantly higher in relatives and controls. Granulocyte Sr was below the detection limit in relatives and controls but measurable in all patients. To study possible covariations between elements in neutrophil granulocytes, principal component analysis was resorted to [42]. Principal component analysis is a useful technique for reducing the number of variables in a data set by finding linear combinations of those variables that explain most of the variability. The principal component analysis can be visualised through biplots for the first two principal components accounting for the largest variance or spread in the data. Figs. 16-19 display such biplots for the control, patient, HLA-B27+ relative and HLA-B27relative groups, respectively. The lines intersecting at the origin (0. 0)
Fig. 14. Multiple elemental profile
Box-and-Whisker plot of the granulocyte of HLA-B27 positive relatives in the Bechterew study.
represent the original variables. The length of each vector is proportional to its contribution to the principal components. The angle between any two is in-
!-L----._.
A
no
CI
4 :
.I
mn
Fe
.-..-LA Zn
Elrmmnt
Fig. 15. Multiple elemental profile
3~x-and-W~sker of HLA-B27
plot of the granuiocyte negative relatives in the
Bechterew study.
U. Lindh / Nuclear probes In bromedicwze
N
_
Component.I
componqlt
Fig. 16. Biplot of the first two principal components trols in the Bechterew study.
versely can
proportional
be concluded
lation
between
relatives.
Zinc
to the correlation that
there
of con-
between
is a similar
them.
lack
It
of corre-
Mn and Zn in the control and HLA-B27and manganese seem to be similarly
1
Fig. 18. Biplot of the first two principal components of HLAB27 positive relatives to the patients in the Bechterew study.
correlated in the patient group and the group with HLA-B27+ relatives. In the same way Ca and Fe are similarly correlated in the patient group and in the
Mt-l
.I.,
FR
-
I
-3.7
-1.7
FI
a.,
._D
componrnt
1.3
1
Fig. 17. Biplot of the first two principal components tients in the Bechterew study.
of pa-
Fig. 19. Biplot of the first two p:incipal components of HLAB27 negative relatives to the patients in the Bechterew study. VIII. MICROPROBE
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U. Lindh / Nuclear probes VI hromedicine
to elucidate the controversial borders between health and disease. manifest or hidden disease in HLA-B27 positive individuals. Such a sensitive tool can also be of value for epidemiological studies concerning the propensity to inflammtory reactions in HLA-B27 positive individuals.
3. Exploitation
I
3
-2
Fig. 20. Discriminant analysis diagram for the Bechterew study concerning Mg. Ca, Mn, Fe and Zn.
group with HLA-B27+ relatives. Calcium and zinc correlate quite well in the control and HLA-B27relative groups. Our study was designed to recruit healthy HLA-B27 positive 1st degree relatives of HLA-B27 positive patients with ankylosing spondylitis. Thus, only relative that considered themselves healthy and had no obvious history of rheumatic disease were included. No radiographic examination was done at the time of inclusion for ethical reasons. These relatives, defined healthy by these subjective criteria, nevertheless, had altered cellular major and trace elements stores, similar to those observed with ankylosing spondylitis [45] in a qualitative way but quantitatively substantially lower. One hypothesis at this point was that the changes were of primary importance for disease susceptibility in HLAB27 positive individuals. The element pattern found in the granulocytes of the patients with ankylosing spondylitis and their relatives can be expected to stimulate the inflammatory activity of the cells. A discriminant analysis of the total study group of ankylosing spondylitis reveals that the patient group (# 1) is clearly separated from the other groups (fig. 20) and that the control group (#2) together with the group of HLA-B27relatives (#4) constitute an inseparable cluster. The HLA-B27+ relatives (#3) is closer to the latter cluster but seem to be separable and with a trend towards the patient group. Sophisticated analyses of major and trace elements in cells may be of signficant value in research in order
of elemental
data
The bar graph is probably the most common chart type for presenting elemental data. It has. however, serious drawbacks. Firstly. if the base level is zero it does not account for the detection limits of the analytical methods; to account for the detection limit it may be necessary to use different base lines for each element. Secondly, the bar chart displays neither range nor variation. The Box-and-Whisker plot meets all the demands on elemental data presentation and, furthermore. it is usually created from nonparametric values for central tendency and variation. Examples of Box-and-Whisker plots are found in figs. 12-15. The central box covers the middle 50% of the data values. between the lower and upper quartiles. The whiskers extend out to the extremes, while the central line is at the median. When unusual values occur far away from the bulk of the data, they are plotted as separate points. The whiskers extend only to those points that are within 1.5 times the interquartile range. One of the main advantages of multielemental analytic methods is that they are unbiased. This means that one can get information on elements that was not primarily sought. Multielemental methods can generate data of lo-15 elements when biological objects are analysed. The effective handling of such large volumes of elemental data calls fore multivariate statistical methods such as principal component analysis, discriminant analysis. cluster analysis and pattern recognition.
4. Caution Analysing very minute volumes in biological tissues causes problems in assessing the accuracy of determination. When probing volumes as small as 10m9 mm3. which corresponds to 1 pg or less. one has to bear in mind that trace element analysis can prove meaning-less. With an analytical sensitivity of 1 pg/g. the absolute mass detection limit approaches lo-” g. Pushing the limits further may imply practical obstacles. For example, 10~tx g of selenium contain some 76000 selenium atoms. Extending the spatial resolution of the nuclear microprobe to 0.1 pm, a goal which is within realistic reach, means that the probed volume can be as small as 10-l’ mm3 corresponding to a mass of 1 fg. This would
imply, if the detection limit can be pushed down to 100 rig/g.. the analysis of 8 selenium atoms. This situation is. of course, absurd. The cross sections will probably not permit such analyses within practical limits. If the lack of material to be analysed with very small probes is one obstacle, another is the possible beam-induced damages when current densities are improved. A current of 100 pA or more in a spot of 0.01-0.25 rJ.m’ implies current densities of at least 400-10000 PA/pm*. It can be questioned if a specimen can withstand such an intense radiation even if rapid beam scanning is employed. Some reassuring results were reviewed by Watt and Grime (91 but they extend only to current densities of 100 pA/l.tm*. Much more work is consequently necessary on the subject of radiation damage before firm conclusions can be reached. The problem of radiation damage has been addressed by Grodzins [46] and the temperature rise in irradiated specimens was recently investigated by Cholewa and Legge [47]. Unfortunately. there is no evident conclusion regarding nuclear probes with spatial resolution of 0.1 pm or better in nuclear microscopy and trace element mapping.
5. Conclusions The conclusions in an earlier review of nuclear microprobe applications in medicine [ll] are in practice still valid: the nuclear microprobe has not yet been fully appreciated by the biological and medical communities. It is now. however, the time to try to show the versatility of the instrument and choose scientifically sound applications. It is a very challenging and promising task for all nuclear microprobers. My sincere thanks go to all colleagues working with nuclear microprobes thereby making the break-in in the biomedical field possible and also to my major sponsor the Swedish Natural Science Research Council and to the Wallenberg Foundation making the new Uppsala nuclear microprobe possible.
References [l] E.J. Underwood
and W. Mertz, in: Trace Elements in Human and Animal Nutrition, 5th ed.. vol. 1, ed. W. Mertz (Academic Press, New York, 1986) p. 1. [2] KM. Hambridge, C.E. Casey and N.F. Krebs, in: Trace Elements in Human and Animal Nutrition, 5th ed.. vol. 2. rd. W. Mertz (Academic Press, New York, 1986) p. 1. [3] G.V. Iyengar. W.E. Kollmer and H.J.M. Bowen, The Elemental Composition of Human Tissues and Body Fluids (Verlag Chemie, Weinheim, 1978). [4] B. Sansoni and G.V. Iyengar, in: Elemental Analysis of Biological Materials: IAEA Technical Report 197 (IAEA, Vienna, 1978) p. 57.
[5] G.V. fyengar and B. Sansoni, in: Elemental Analysis of Biological Materials: IAEA Technical Report 197 (IAEA, Vienna, 1978) p. 73. [6] G.V. Iyengar, in: Trace Elements in Health and Disease. eds. H. Bostriim and N. Ljungstedt (Almqvist & Wiksell, Stockholm, 1985) p, 64. [7] R.D. Vis. The Proton Microprobe: Applications in the Biomedical Field (CRC Press, Boca Raton, 1985). [8] K.G. Malmqvist. Scanning Electron Microsc. III (1986) p. 821. [9] F. Watt and G.W. Grime. in: Principles and Applications of High-Energy Ion Microbeams. eds. F. Watt and G.W. Grime (Adam Hi&r, Bristol. 1987) p. 154. [lo] D.J.T. Vaux, F. Watt and G.W. Grime. in: Principles and Applications of High-Energy Ion Microbeams, eds. F. Watt and G.W. Grime (Adam Hilger. Bristol, 1987) p. 203. [ll] U. Lindh. Nucl. Instr. and Meth. B30 (1987) 404. [12] J.A. Cookson and F.D. Pilling. Phys. Med. Biol. 21 (1976) 115. [13] G.J.F. Legge and A.P. Mazzolini, Nucl. Instr. and Meth. 168 (1980) 563. [14] G.D. Blower, D. Phil. Thesis, University of Oxford (1983). 1151 B. Gonsior, in: Nuclear Analytical Techniques in Medicine. ed. R. Cesareo (Elsevier. Amsterdam. 1988) p, 123. 1161 D. Heck and E. Rokita, IEEE Trans. Nucl. Sci. NS-30 (1983) 1220. [17] P.M. O’Brien and G.J.F. Legge. Biol. Trace Elem. Res. 13 (1987) 159. [IS] K. Themner. KG. Malmqvist, E. Martins, K. Inamura and B.K. Siesjii. Nucl. ins%. and Meth. B22 (1987) 214. [19] E. Martins. K. Inamura, K. Themner, K.G. Malmqvist and B.K. Siesjii. J. Cereb. Blood Flow Metabol. 8 (1988) 531. [20] K. Themner, K.G. Malmqvist and B.K. Siesjo. Nucl. Instr. and Meth. 830 (1988) 424. [21] F. Watt. G.W. Grime. D.N. Jamieson and B. McDonald. Scanning Microsc. Intl.. in press. [22] K. Inamura. E. Martins, K. Themner, S. Tapper, J. Pallon. G. Lovestam, IS. Malmqvist and B. Siesjii, accepted for publication in Brain Res. [23] L.C. Salford, A. Brun. U.A.S. Tapper. E. Swietlicki and K. Malmqvist. submitted. [24] U. Lindh. N.G. Conradi and P. Sourander, Acta Neuropathol. 79 (1989) 153. [25] B. Ahlrot-Westerlund. B. Carlmark, S.-O. Gronquist, E. Johansson, U. Lindh, H. Theorell and K. de Vahl. Nutr. Res. Suppl. I (1985) 442. 1261 U. Lindh. S.-O. Gronquist and A. Lindvall, in: Proc. 11th Congr. on X-ray Optics and Microanal., eds. J.D. Brown and R.H. Packwood (LJniv. Western Ontario, London, Canada, 1987) p. 328. [27] A. Lindvall, G. Friman. S-O. Gronquist, A. Linde and U. Lindh, submitted. [28] U. Lindh and E. Johansson, Biol. Trace Elem. Res. 12 (1987) 351. [29] U. Lindh and E. Johansson. Biol. Trace Elem. Res. 12 (1987) 109. [30] U. Lindh and T. Sunde, submitted. [31] T.A. Gasiewicz and J.C. Smith, B&hem. Biophys. Acta 428 (1976) 113.
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/ Nuclear probes in biomedicirw
W.E.C. Wacker and B.L. Vallee. J. Biol. Chem. 234 (1959) 3251. H. Zumkley, Biol. Trace Elem. Res. 15 (1988) 139. R. Hlllgren. K. Svensson, E. Johansson and U. Lindh. Arthritis Rheum. 28 (1985) 169. R. HLllgren, K. Svensson. E. Johansson and U. Lindh. J. Lab. Clin. Med. 104 (1984) 893. K. Svensson. R. Hlllgren, E. Johansson and U. Lindh. Inflammation 9 (1985) 189. U. Lindh and E. Johansson, Neurotoxicol. 4 (1983) 177. G. Anne&, E. Johansson and U. Lindh, Acta Paediatr. Stand. 74 (1985) 259. U. Lindh. L. Juntti-Berggren. P.-O. Berggren and B. Hellman, Biomed. Biochim. Acta 44 (1985) 55. L. Juntti-Berggren. U. Lindh, P.-O. Berggren and B. Frankel, Biosci. Rep. 7 (1987) 33.
[41] T. Foucard. M. Gebre-Medhin. K.-H. Gustavson and U. Lindh, submitted. [42] D.L. Massart. B.G.M. Vandeginste. S.N. Deming, Y. Michotte and L. Kaufman. Chemometrics: A Textbook (Elsevier, Amsterdam. 1988). [43] P. Armitage and G. Berry, Statistical Methods in Medical Research, 2nd ed. (Blackwell Scientific Publ.. Oxford. 1987). [44] N. Feltelius. R. HIllgren and U. Lindh. J. Rheumatol. 15 (1988) 308. [45] R. HYllgren. N. Feltelius and U. Lindh. J. Rheumatol. 14 (1987) 548. [46] L. Grodzins. Nucl. Instr. and Meth. 218 (1983) 203. [47] M. Cholewa and G.J.F. Legge, Nucl. Instr. and Meth. B40/41 (1989) 651.