Review of trace elements in blood, serum and urine for the Czech and Slovak populations and critical evaluation of their possible use as reference values

the Science of the Total Environment The Scienceof the Total Environment 166(1995) 211-234

Review of trace elements in blood, serum and urine for the Czech and Slovak populations and critical evaluation of their possible use as reference values J. K&era*“, bInstitute

dEuropean

V. Benckob, E. Sabbioni”, M.T. Van der Venned

a Czech Ecological Institute, NAA Laboratory, CZ-250 68 Re.? near Prague, Czech Republic of Hygiene and Epidemiology, 1st Medical Faculty, Charles University, StudniEkoua 7, CZ-120 00 Prague 2, Czech Republic ‘European Commission, Environment Institute, Joint Research Centre, Ispra, I-21020 Varese, Italy Commission, Directorate General for Employment, Industrial Relations and Social Affairs (DG VI, Health and Safety Directorate, L-2920, Luxembourg, Luxembourg

Received 28 May 1994;accepted7 July 1994

Abstract

The availability of accurate trace element reference valuesin human tissuesrepresentsan important indicator to the health status of the general population and occupational groups exposed to trace elements. The EURO TERVIHT project (Trace Element Reference Values in Human Tissues)aims to establishand compare trace elementreference valuesin tissuesfrom inhabitants of the European countriesasbaselinevaluesfor clinical/toxicological assessment studies[3]. In this context, one of the first stepsconsideredis the critical evaluation (state of the art) of existing literature on trace element reference values in blood, serumand urine in the general population of each European country. This paper reviewsthe Czech and Slovak situation by assessing studiescarried out in these countries for Al, As, Cd, Co, Cr, Cu, F, Mn, Hg, Ni, Pb, Rb, SC,Se, V and Zn in blood, serum and urine. These studiesshowthat most of the data available do not meet criteria designedrecently for deriving reference intervals, especiallyregarding the number of subjects,the age of population samplestudiesaswell as the useof appropriate samplingtechniquesand quality assuranceprocedures.Elementswhich present the highestpotential risk for health in Czech and Slovak populations and for which reference values should be urgently establishedare: Cd, Hg, Pb (major pollutants); As, Cr, Ni (carcinogenic metals); Al, F, Mn, Tl, V (released into the environment by coal combustionand other industrial activities); Pt (increasinguse of Pt catalyst in petrol-driven automobiles);essential trace elementssuch as I, Se and Zn for which a deficiency in Czech and Slovak populations was detected or is suspected. Trace elements,human tissue; Trace elements,body fluids; Trace elements,reference values; Blood; Urine; Serum; Plasma Keywords:

*Corresponding author,NuclearPhysics Institute,Academyof Sciences of the CzechRepublic, CZ-250 Republic. 004%9697/95/$09.50 0 1995ElsevierScienceBV. All rightsreserved. SSDI

0048-9697(95)04425-M

68 keg near Prague,

Czech

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1. Introduction In recent years, there has been an increased awareness in the European Union (EU) countries about the health effects of toxic and other metals in relation to nutritional disorders, environmental or occupational exposure, pathological status and medical therapy [l-5]. Several toxic metals, such as As, Cd, Hg, Pb and Ni, have been released in large quantities into the environment [6] in industrial areas, and occupational exposure to these metals occurs in various industries. Large population groups may be exposed to toxic metals from the polluted environment and limited groups of workers are exposed at their workplace. Thus, exposure to these metals, and other environmental pollutants, are considered as severe environmental pollution and occupational hygiene problems with possible short- and long-term adverse effects on human health. Compared to Western Europe, the above mentioned problems were relatively underestimated or at least partially neglected in most Central and Eastern European countries for many years. Hence, more pronounced long-term adverse effects on human health could be expected in these countries. In the assessment of health risk arising from environmental, occupational or accidental exposure to toxic metals [7], there is an increasing use of biological monitoring of metal concentrations in human tissues and body fluids [8,9]. For this purpose, knowledge of accurate reference intervals (baseline values) of trace elements in human tissues and body fluids is of paramount importance in order to make possible a comparison with trace metal levels in the corresponding tissues and body fluids of unexposed general population 131. In addition, the accurate trace element reference values in the general population form the basis of setting legal limits of exposure to trace elements for the protection of public and occupational health [lo] or may provide the scientific basis for biomedical research in trace element-related diseases [5]. Obviously, the element reference values may be influenced by several factors, especially for non-homeostatically controlled trace elements, e.g. geographical factors

166 (1995) 211-234

including life-style and dietary habits. In this context, literature on trace element values in blood, serum and urine for the Czech and Slovak populations are reviewed and a critical evaluation made on the possible use of existing data for deriving trace element reference intervals. 2. Introductory

remarks and abbreviations

The International Federation of Clinical Chemists (IFCC) has recommended a series of definitions and recommendations [ll-141 which were also adopted in a recent paper on critical evaluation of reference values for the Danish population [5]. To prevent confusion due to using different definitions of ‘reference values’ and ‘reference intervals’, the above mentioned [ll-141 definitions and recommendations and those recently suggested for another international project (TRACY project) which aims at deriving trace element reference values from the critical evaluation of the published information concerning elements in human tissues and fluids [15] are used in this work. These terms are briefly explained here for the sake of completeness. A reference value is the result of the measurement of a trace element in a biological sample from one subject in the reference population. A reference interval is the interval which covers 95% of the reference values in the reference population. Concerning statistical treatment of reference values, and estimation and presentation of reference intervals, the following parameters should be available to provide an adequate description of the population: -

-

Results of statistical tests for normal distribution of the reference values. Since the distribution is frequently skewed, transformation of the reference values is necessary (e.g. logarithmic transformation) to obtain a normal distribution; Mean and median to give an impression of the non-centrality of the distribution.

The reference interval can then be evaluated as an interval encompassing 95% of a normal (or

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transformed to normal) population, i.e. mean + 2 standard deviations (SD.), or as a non-parametric 95% reference interval of any type of distribution independent of its type, i.e. the interval between the 25th and 97.5th percentiles, based on at least 120 reference values. The reference intervals should preferentially be established for defined sub-populations such as geographical populations (urban, rural regions), demographic sub-populations (sex and age dependence), and lifestyle sub-populations (smokers, alcohol drinkers, specific food, frequent fish eaters, etc.). Minoia et al. [2] indicated that it may be useful to distinguish between ‘reference intervals’ based on a large number of subjects and ‘indicative intervals’ based on less than 120 subjects to provide some useful information on the order of magnitude of reference values in the studied population. A standardized method for the calculation and presentation of indicative intervals based on the calculation of valid confidence and tolerance intervals for the description of the reference population is in preparation [16]. Poulsen et al. [5] pointed out that presenting one-sided tolerance intervals can be considered advantageous when a fraction of samples analyzed has an element concentration below the limit of detection. Another way of presenting reference values consists of an evaluation of limit values which are given either as the mean + 2 S.D. for a normal (or transformed to normal) population or as the 95th percentile of any type of distribution independent of its type [15]. Reference values must be based on results of measurements with proven accuracy. The accurate results can only be obtained if strict adherence to quality assurance principles (QA) is pursued. QA consists of two different, but overlapping procedures - quality control and quality assessment [17]. In both procedures, uncertified, home-made or commercially available control materials, internal reference materials (IRM), reference materials (RM), certified and standard reference materials (CRM and SRM, respectively, the latter term being used for RM certified by U.S. National Institute of Standards and Tech-

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nology), intercomparison studies and agreement within principally different methods are indispensable parts of analytical activities for proving accuracy (i.e. precision and trueness) of results. All values of trace elements in blood, serum are expressed as nmol/l, if not otherwise stated, while values of trace elements in urine are expressed as nmol/l or nmol/mmol creatinine. The status of reference values in blood, serum and urine of the Czech and Slovak populations is reported for a number of elements reviewed in the following sections. In this review, literature data available on toxic and/or essential trace elements since 1977 have been scrutinized and data on major elements such as Ca, Cl, Fe, Mg, P, K, Na and S are not included. In the text and tables, the following abbreviations are used: For biological matrices: B, blood; S, serum; P, plasma; U, urine. For analytical techniques: AAS, atomic absorption spectrometry (without specification); F-AA& flame AAS; ETA-AAS, AAS with electrothermal atomization in graphite furnace; HG-AAS, AAS with hydride generation; CV-AAS, AAS using cold vapour technique; TMA, trace mercury analyzer (single purpose atomic absorption spectrometer); ISE, ion-selective electrode; INAA, instrumental neutron activation analysis; RNAA, neutron activation analysis with radiochemical separation; COL, calorimetry. For population description: N, number of subjects; W, women; M, men; G, girls; B, boys. For result presentation: AM, arithmetic mean; GM, geometric mean; S.D., standard deviation; G.S.D., geometric standard deviation; M, median. 3. Status of Czech and Slovak reference values for trace elements in blood, serum and urine 3.1. Aluminium

In a study on dialyzed patients, S-Al was measured in groups of patients and of healthy control subjects. For the control groups, ETA-AAS on pyrographite cuvettes treated with ZrOCl, [18] provided values below the limit of determination (< 1300 nmol/l). Apparently a lower limit of determination of S-Al was achieved in a study aimed at an evaluation of serum levels for Al, Cu,

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Cr, Mn, Se and Zn in 19 healthy subjects 12 males, seven women of average age 33 years (range 21-49 years) in Central Slovakia using ETA-AAS (except for Zn which was determined by F-&AS) by DlhopolEek and Laurincovi [19]. In this study, blood was collected into polypropylene vials precleaned by washing in bidistilled water, leaching in Suprapure 5% HNO, (Suprapur Merck) for 6 h and rinsing with deionized water. Serum was separated after 1 h and stored prior to analysis at - 10°C. Arithmetic mean f S.D. of S-Al was found to be 83 f 52 nmol/l [19] which compares well with recently reported S-Al and P-Al levels in the range of 37-185 nmol/l in other countries [20]. However, due to the limited number of subjects and missing proof of accuracy of the Al determination, no attempt is made to evaluate S-Al reference or indicative values. 3.2. Arsenic

Arsenic content in hair and urine was determined by a calorimetric method, using silverdiethyldithiocarbamate [21], and in blood by RNAA with proven accuracy (by control analysis of RM Bowen’s Kale) in one of the authors’ (JK) laboratory. Groups of lo-year-old boys, each numbering 20-25 individuals, were tested residing at various distances from a power plant in Central Slovakia using local coal of high arsenic content and in a control group [22]. From hair analysis, it could be inferred that a group of boys living 36 km from the emission source could be considered as unexposed (denoted as group 0 in reference [22]) and the results of this study and the control group from Prague (denoted as group P in reference [22]) are given in Table 1. The

values found are somewhat lower compared with median values of U-As and B-As of 267 and 67 nmol/l, respectively, which have been evaluated by Iyengar and Woittiez [20] as possible reference values. The U-As values presented here should not be used for deriving reference or indicative intervals, because no proof of accuracy was given. The B-As values can only be considered as tentative indicative values for lo-year-old boys living in industrial regions of Central Slovakia and Central Bohemia (groups 0 and P, respectively). The limits of B-As of 37.7 and 49.1 nmol/l (mean + 2 S.D.) can be proposed for the former and latter subpopulations, respectively. To establish reference (or indicative) intervals for As, a larger number of samples from adults should be analyzed and As intake from their diet should also be studied, because besides regional differences, S-As and U-As levels are subject to short-term effects of intake [20,23,24]. For instance, elevated levels were reported after high consumption of fish [23]. From this point of view, inhabitants of both Central Slovakia and Bohemia can be regarded as infrequent fish-eaters. 3.3. Cadmium

Several studies were carried out on Cd and Ni health effects in occupationally exposed workers in a Cd-Ni storage battery plant [25-321. The Cd content in urine, serum or blood was also determined in four control groups. The first control group for the U-Cd determination was formed of 54 workers in a research institute located about 30 km from the plant [25]. For the second control group consisting of 20 persons, no details were

Table 1 Arsenic in urine and whole blood of IO-year-old boysa Tissue

U B

Method

COL RNAA “Calculated from [22]. bLiving in industrialized

N

20 24 10 10

Groupb

0 P 0 P

As content (nmol/l) AM

SD.

GM

Range

G.S.D.

107 142 18.3 23.7

169 200 9.7 12.7

< 32 <32 6.3 6.3

-

481 474 40.3 47.8

area of Central Slovakia (Group 0) and Central Bohemia (Group PI.

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published [26], while the third control group was formed of 30 healthy persons, mostly women, who lived about 10 km from the plant and worked as health care assistants. The average age in the third control group was 37.7 years, range 24-52 years, and the group contained eight moderate smokers (average 7.5 cigarettes per day) [27-311. In the fourth control group consisting of 10 women, non-smokers, of the age 22-40 years (median 32 years), administrative workers from another factory in the same districts as the storage battery plant, U-Cd and S-Cd levels were determined by RNAA with proven accuracy at the ng level [32]. Values were below the limit of detection (3) which were, however, about one order of magnitude lower than those measured by the AAS techniques (Table 2). Concentrations of Cd, Hg, and Pb in mothers and their newborns were studied by the element determination in maternal erythrocytes and plasma in placenta, and in erythrocytes and plasma of umbilical cord blood in 50 pregnant women resident in an urban industrial area and

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in a semirural area in Slovakia without much industrial activity [33]. The results for the latter control group are given in Table 2. Mean age of the mothers from the control group was 23.9 years, 60% were smokers, but according to the clinical data, none continued smoking during pregnancy. For comparison, the biological samples were analyzed with the same techniques in two different laboratories in the former Czechoslovakia. However, the results of the comparison were not reported [33]. On comparing the arithmetic means, medians, and geometric means, inference can be made that the cadmium results in the study were rather log-normally than normally distributed. Recently, a large scale study, MONICA (MONItoring of trends and determinants in CArdiovascular diseases) [34,35] was organized by the WHO. In the framework of this study, Cd, Hg, and Pb are being determined in blood of inhabitants in several districts of the Czech Republic. The mean Cd values measured in 611 inhabitants (300 men and 311 women) in the districts of

Table 2 Cadmium in urine and blood Tissue

Method

Urine Urine Urine

AAS AAS

Urine Serum Serum Blood MBEb MBPC CBEd CBPe Blood

RNAA RNAA ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS

N

54 20 30

F, ETA-AAS

10 10 30 30 50 50 50 50 611 300 M 311 W

M, men; W, women. anmol/mmol creatinine. bMaternal blood erythrocytes. ‘Maternal blood plasma. dCord blood erythrocytes. eCord blood plasma.

Cd content (nmol/l) AM

S.D.

262 249 37.4 700.1a <7.1 < 4.4 26.9 52.5 54.3 40.9 52.5 51.6 8.3 10.1 6.5

105 84 29.4 314.7a 10.5 31.1 48.9 34.7 50.7 46.3 11.3 13.0 9.0

Reference Range

M

GM

-

-

-

-

< 0.2-9.70 < 0.2-9.70 < 0.2-9.40

44.5 35.6 35.6 44.5 4.1 5.0 3.3

36.5 32.0 34.7 37.4 4.0 4.7 3.4

G.S.D.

3.4 3.7 3.1

25 26 27-31 30,31 32 32 28,29 30 33 33 33 33 36 36 36

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Prague-East (sub-urban region) and Jindi-ichfiv Hradec (South Bohemia, not industrialized, rural region) are given in Table 2. The range of B-Cd values amounts to < 1.8-86.3 nmol/l and the close agreement of medians with geometric means both in the whole group and the subgroups of men and women indicate log-normal distribution of B-Cd values. Accuracy of the results was proved by analysis of reference serum Seronom 904 (Nycomed, Norway) and comparative analyses by RNAA in one of the authors’ (J.K.) laboratory. Very good agreement was obtained between the assigned Seronom 904 and measured values and also between the ETA-AAS and RNAA results for several blood samples [36]. The geometric mean of B-Cd values [361 compares well with those values determined in USA [37], Belgium, Japan, India, Peru [38], and Denmark 151, while somewhat lower mean values were determined in Sweden, Yugoslavia, Israel, China, and Mexico [39]. The effect of smoking on B-Cd levels was also examined in this part of the MONICA study and the results obtained are summarized in Table 3 (without distinguishing the sex of the subjects). Spearman correlation coefficient, r, was calculated for these data and the value of r = 0.666 (P = 0.01) suggests a high association of B-Cd with the number of cigarettes smoked per day. Obviously, after completing statistical evaluation of the results of the study [36], separately for smokers and non-smokers, women and men, these values can be used for evaluating reference B-Cd intervals for the Czech population, because a sufficient number of subjects were examined, their personal data and lifestyle are well documented, and the accuracy of the results was proven. At present, the 95% reference interval of < 0.2-8.0 nmol/l with a mean value of 3.6 nmol/l of B-Cd for non-smokers without distinguishing sex is available. The accuracy of other B-Cd or S-Cd values determined in a considerably smaller number of subjects [28-301 was not tested, neither was the distribution of these results, so that their use for deriving reference values cannot be recommended. The same applies for U-Cd values. Moreover, the U-Cd values determined by ETAAAS seem to be about five times higher than those evaluated by Iyengar and Woittiez [20] as reference values.

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Table 3 The effect of cigarette smoking on blood cadmium levels Cigarettes per day

N

Non-smokers Less than 10 10-20 More than 20

406 57 91 55

B-Cd (nmol/l) AM

S.D.

3.6 11.7 18.7 22.7

4.2 10.1 12.7 18.9

3.4. Cesium

S-Cs was determined by INAA in 109 samples obtained from men aged 35-70 years from Central Bohemia. Normally distributed results yielded an arithmetic mean k S.D. of 8.1 f 2.3 nmol/l as calculated from the results given in @g/g dry weight [43] using a conversion factor of 1 pug/g = 684.0 nmol/l. Although CRM IAEA H-4 Animal Muscle was analyzed as a control sample, no proof of accuracy was obtained, because there is no certified Cs value in this CRM. Thus, the results obtained should not be used for deriving reference or indicative intervals for S-Cs. 3.5. Chromium

Chromium values in urine, serum, erythrocytes, and whole blood of occupationally non-exposed persons were studied in two regions of Slovakia [40]. The results achieved by ETA-AAS are summarized in Table 4. No details on sampling and sample preparation procedures were given, or information on subjects and quality assurance of the analyses, although it is well known that avoidance of sample contamination is of utmost importance especially for the accurate determination of chromium in blood and its components [41,42]. Although there are several metallurgical plants in the Orava region, which may contribute to increased chromium levels in the environment, sample contamination and/or analytical bias seem to be the most likely explanation for the high values reported. About 3-4 times lower S-Cr values were determined using the same analytical techniques by DlhopolEek and Laurincova 1191 in subjects from another Slovak region (Table 4). S-Cr levels were also determined, together with levels of other elements, by INAA in samples

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Table 4 Chromium values in urine, serum, erythrocytes and whole blood Tissue

U U S S RBC” RBCa B B S S

Method

ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS INAA

N

17 26 10 26 10 8 10 8 19 109 MS

Region

Turiec Orava Turiec Orava Turiec Orava Turiec Orava B. Bystrica C. Bohemia

Cr content (nmol/l) AM

SD.

17 34 27 42 59 43 82 108 9 568

6 21 16 35 35 22 22 26 2 381

Reference M

GM

G.S.D.

-

489

2.19

447

40 40 40 40 40 40 40 40 19 43

M, men. “Red blood cells.

from about 120 men aged 35-70 years from Central Bohemia [43]. In this study, blood was collected using a stainless steel needle. Although, the first 25 ml of blood was not taken for elemental analysis, this apparently did not prevent contamination by Cr or contamination occurred on subsequent treatment (freeze-drying, pelletizing prior to irradiation), because a geometric mean of log-normally distributed S-Cr results amounted to 554 nmol/l as calculated from the results given in lug/g dry weight using a conversion factor of 1 pg/g = 1747.9 nmol/l. This value critically exceeds evaluated S-Cr values in a TRACY project in the range of l-3 nmol/l[441 by more than two orders of magnitude. Since no proof of accuracy was given in the above mentioned papers and the samples analyzed were probably contaminated on sampling or sample handling, no attempt should be made to use the reported values for deriving reference or indicative intervals of S-Cr. This also applies to U-Cr values, because they exceed significantly the critically evaluated ranges of 2-5 nmol/l of U-Cr in the TRACY project [44], and because they were determined in the same laboratory that reported unrealistically high values of S-Cr, B-Cr and Cr in erythrocytes [40]. 3.6. Cobalt S-Co was determined by INAA in 109 samples obtained from men from Central Bohemia in the study [43] mentioned in the sections on Cs and Cr. A median value for unevenly distributed re-

sults amounted to 15.0 nmol/l (recalculated from the results in pg/g dry weight using a conversion factor of 1 pg/g = 1542.4 nmol/l) which exceeds the reference median values proposed by Iyengar and Woittiez [20] about five times. The S-Co value reported in [43] should not be used for deriving reference or indicative intervals for reasons already given in the section on chromium (probable contamination on sampling or sample handling, no proof of accuracy). 3.7. Copper S-Cu and U-Cu levels were measured by F-AAS in samples from 10 healthy men aged 17-35 years after a l-h physical load on a bicycle ergometer and at rest (control measurement). The levels did not differ significantly in both measurements and the values at rest (AM + S.D.) were 20.08 & 2.29 pmol/l and 4.0 * 1.1 Fmol/l for S-Cu and U-Cu, respectively [45]. Recently, S-Cu levels were determined in 19 healthy subjects by ETA-AAS and an AM + S.D. of 14.54 + 1.83 pmol/l was found [191. Although the S-Cu results are within the range of reference values of 12.6-20.5 pmol/l proposed by Iyengar and Woittiez [20], the accuracy of the analyses was not tested and thus no reference or indicative values are proposed. 3.8. Fluorine In a model example of fluoride excretion [46], U-fluoride (UF) concentrations were determined

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by ion selective electrode in four subjects with no fluoride supplementation. The values obtained were 28.1-51.4 pmol/l. A study on ingestion of fluorine compounds (NaF, CaF,) showed a sharp increase in UF concentration after oral administration of 2 mg of NaF in a week and a quick return to UF values prior to the start of administration when fluoride supplementation ceased. No increase in UF was observed after oral administration of 4 mg of CaF, in 1 week. Average UF values determined by ion selective electrode in many urine samples from four volunteers varied in the range of 53.4-72.3 pmol/l prior to the start of the fluoride compound administration, after the end of administration and during the CaF, administration [47]. In a study on welders exposed to manganese- and fluoride-rich dust and/or vapours, fluoride ions were determined in urine of a control group of 189 unspecified persons using ion selective electrode [48]. Only the arithmetic mean _+ S.D. of 33 + 14 pmol/l was reported. These UF levels fall well within the range of values of 16-53 pmol/l reported in other countries [20]. However, the element levels are still considered to be poorly documented [20]. No attempt should be made to use the above mentioned values to derive reference values, because proof of accuracy and required information on subjects examined are lacking. 3.9. Manganese

There are only a few, mostly older, papers on U-Mn and S-Mn values in Czech and Slovak populations. U-Mn levels determined in the early 70s by F-AAS in 19 healthy workers in a Prague research institute non-exposed to the element

yielded an arithmetic mean of 314.8 nmol/l 1491. Using an improved F-AAS method with manganese preconcentration by ion-exchange chromatography, U-Mn was measured in an unspecified group of 60 persons and an arithmetic mean + S.D. of 24 + 15 nmol/l was evaluated [48]. In Slovakia, manganese was determined by F-AAS in urine, serum, and hair of 60 workers occupationally exposed to manganese in a plant producing ferromanganese alloys and in a control group consisting of 30 patients in a Clinic of Occupational Health aged between 22 and 64 years (average 46 years), non-exposed to manganese [50]. Significantly higher manganese levels were found in all tissues of exposed workers compared to those in the control group (Table 5). The U-Mn levels in urine reported from both republics, except for those found in [48], exceed the reference values evaluated by Iyengar and Woittiez [20] by more than one order of magnitude and the S-Mn level reported in [50] exceeds the critically evaluated values in [20,41] by two orders of magnitude. No details were given on avoidance of possible contamination during sample collection and analysis, which is a crucial factor for obtaining accurate manganese values in biological materials, especially in blood and its components [41]. Hence, the results of the studies [49,50] should be suspected as significantly elevated due to possible contamination [41,42] and their use for deriving reference values should be avoided. The U-Mn values found in [48] compare reasonably well with the reference median value of 11 nmol/l (range 9.1-178 nmol/l) suggested by Iyengar and Woittiez [20]. They originate from a relatively large number of subjects, unfortunately without any specification and without any proof of accuracy.

Table 5 Manganese values in urine and serum Tissue

u U u s S

N

19 60 30 30 19

Technique

F-AAS F-AAS F-AAS F-AAS ETA-AAS

Mn content (nmol/l)

Reference

AM

SD.

315 24 321 1194 11

15 175 389 3

48 47 49 49 19

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Thus, no attempt is made to evaluate these U-Mn reference values. Recently, S-Mn levels were determined in 19 healthy subjects in Slovakia and an arithmetic mean +_ S.D. of 11 + 3 nmol/l was found [19] which compare well with the critically evaluated values [20,41]. Here again, information is lacking for the type of needle used for sampling and for the accuracy of the analyses and invalidates these results for deriving indicative or reference intervals. 3.10. Mercury

An average B-Hg value of 93.7 nmol/l was determined by CV-AAS in an undefined control group in a study on the exposed population in a plant producing mercury [51]. Mercury mobilization from organisms of exposed and control persons using Unitiol (sodium 2,3-dimercaptopropane-1-sulphonate, a Russian analogue of BAL) was studied to justify the use of the preparation for the treatment of mercury poisoning [52]. U-Hg in 10 control persons prior to the start of the Unitiol administration yielded a median value of 8.2 nmol/l. For seed mordant processing, chemi-

219

cal mordants such as Agronal which contains phenylmercurichloride as the active agent are mainly used in Czech Republic. The level of chromosome aberrations of peripheral blood lymphocytes and B-Hg and U-Hg levels were studied in workers exposed to Agronal and in a control group consisting of 40 persons without any specification. Using CV-AAS, arithmetic mean values of 25 nmol/l and 3 nmol/l were found for U-Hg and B-Hg levels, respectively, in the control group [531. Mercury values found in the Slovak study [33] in mothers and their newborn are also shown in Table 6. In another recent study in Slovakia, B-Hg values were measured in lo-year-old boys using a single purpose AAS with mercury preconcentration and with separation by amalgamation and subsequent mercury evaporation (Trace Mercury Analyzer TMA-254, Labora, Czech Republic). The median value was 9.0 nmol/l, and range 4.0-16.4 nmol/l [54]. Within the framework of the MONICA project, B-Hg was determined using the TMA-254 device in the blood of 611 inhabitants in the suburban

Table 6 Mercury values in urine, blood, etythrocytes and plasma Tissue

U U B B MBEa MBPb CBE’ CBPd B Be ;: B’

Method

cv-AAS

CV-AAS cv-AAS cv-AAS cv-AAS cv-AAS cv-AAS cv-AAS TMA-254 TMA-254 TMA-254 TMA-254 TMA-254

M, men; W, women. aMatemal blood erythrocytes. bMaternal blood plasma. cCord blood etythrocytes. dCord blood plasma. eDistrict Prague-East. ‘District Jindiichfiv Hradec.

N

10 40 ? 40 50 50 50 50 30 153 M 141 w 147 M 170 w

Hg content (nmol/l)

Reference

AM

SD.

M

11.7 3 93.7 25 27.4 15.0 21.9 12.0 10.1 5.48 4.04 5.83 3.29

10.3 0

-

25 8.5 7.0 7.5 5.0 3.8 6.38 5.73 8.08 4.39

8.2

27.4 15.0 19.9 10.0 9.0 -

GM 9.6 25.9 13.5 20.9 11.0 -

G.S.D. 1.79 -

52 53 51 53 33 33 33 33 54 55 55 55 55

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district Prague-East and the rural district JindfichSv Hradec. In both districts, somewhat lower values were found in women than in men. However, the differences were not statistically significant (Table 6). The accuracy of the results was confirmed by the control mercury determination of Seronom 904 reference material (arithmetic mean f S.D. found from six replicate analyses of 15.5 + 3.8 nmol/l compared reasonably well with the declared value of 19.9 nmol/l [55]). Undoubtedly, upon completing this extensive study and its statistical evaluation, the results can form a basis for deriving B-Hg reference intervals for the Czech population. At present, only the tentative reference limit of 16.9 nmol/l (mean + 2 S.D.> is proposed based on pooling the B-Hg values in Table 6 by calculating the weighed arithmetic mean and S.D. according to [56]. In a review of ‘normal’ concentrations of B-Hg in various countries, mean values in the range of 1.5-822 nmol/l were reported [56]. The wide scatter of B-Hg values reflects the dietary intake of Hg, because Hg readily binds to red blood cells and thus the highest B-Hg levels are found in populations consuming large quantities of fish and other sea foods with a high content of Hg [20,56]. Low B-Hg values in the Czech population

correspond to low sea-fish eating graphic location.

in this geo-

3.11. Nickel U-Ni and S-Ni concentrations were determined by F-AAS in two control groups of about 30 children aged 10 f 1 year living about 1 km (group Cl) and 14-17 km (group C2) from a nickel production plant [57]. Measurable U-Ni concentrations were found in all subjects examined, while for S-Ni analysis, three samples were pooled in both groups and still 41% and 62.5% of analyses yielded values below the limit of detection (443 nmol/l) in group Cl and C2, respectively, (Table 7) due to the small amount of blood collected (5 ml). Using the same F-AAS method, U-Ni was determined in 24-h samples of urine from 19 healthy men, workers in a research institute (average age 35 years, range 23-47 years), and in serum obtained from 18 randomly selected blood donors of a similar age structure [581. Results are presented in Table 7. Other authors [59] employed an ETA-AAS method with nickel preseparation by extraction with dimethylglyoxime in chloroform for the element determination in urine and serum obtained from 20 unexposed persons (10 medical workers of average age 39 years,

Table 7 Nickel levels in urine and serum Tissue

U U U U U U S s S S s S

Technique

F-AA’S F-AAS F-AAS ETA-AAS F-AAS ETA-AAS F-AAS F-AAS F-AAS ERA-AAS ETA-AAS ETA-AAS

N

27’ 32d 19 20 30 19 7&C 8b.d

18 20 19 19

Ni content (nmol/l)

718 497 375 49.4 42.5 61.3 1226 988 460 35.8 49.4 27

a41% values below the detection limit. b62.5% values below the detection limit. ‘Children living 1 km from a nickel production plant. dChildren living 14-17 km from a nickel production plant.

Reference SD.

M

261 303 119 25.5 25.5 42.6 -

-

221 23.8 32.4 18

56.2 39.2

57 57 58 59,60 27 61-63 57 57 58 59,60 61-63 19

J. Ku&m et al. /Sci. Total Environ. 166 (1995) 211-234

range 28-51 years, 10 patients in a Clinic of Occupational Health aged 37-64 years, average 51 years). This study [59,60] yielded more than one order of magnitude lower values compared to previous ones [57,58] (Table 71 presumably due to the removal of interferences and unspecific absorption in the AAS determination. The ETAAAS method developed was also used for S-Ni and U-Ni determination in 19 control administrative workers in a plant producing nickel compounds [61-631 of an age approximately matching that of occupationally exposed workers (average 46 years, range 27-59 years). Very similar values were found as in the last mentioned study [59,601 (Table 7). About the same U-Ni values were also found by F-AM in a control group of 30 occupationally non-exposed persons (average age 37.7 years, range 24-52 years) living about 10 km from a Cd-Ni storage battery plant [27]. The lowest S-Ni value of 27 ? 18 nmol/l (arithmetic mean + S.D.) was determined by DlhopolEek et al. [19] in 19 subjects from Central Slovakia. The nickel values from the 70s were apparently obtained using an inadequate F-AAS method. For more recent values, proof of accuracy is missing, blood samples were collected using steel needles, and blood and urine samples were not handled in ultraclean laboratories with appropriate contamination control so that the risk of sample contamination, which seems to be the critical factor for accurate determination of U-Ni and especially of S-Ni [20], cannot be excluded. The levels of U-Ni reported for small groups of the Czech and Slovak population are at the upper end of those mentioned in the review by Iyengar and Woittiez [20] (median 22 nmol/l, range 5.1-78.4 nmol/l) or those critically evaluated in the TRACY project [64] (17-51 nmol/l), while for S-Ni these differences are much higher, because in the review [20], a median of 3.4 nmol/l (range < 0.9-22 nmol/l) is given and even lower values (< 3.4 nmol/O are proposed as the most reliable reference values for S-Ni in the critical evaluation in the TRACY project [64]. For the above given reasons, no reference or indicative reference values for U-Ni or S-Ni in the Czech and Slovak population can be proposed.

121

3.12. Lead

U-Pb and B-Pb levels were determined in several studies, because there are known cases of both occupational and non-occupational exposure to lead, the latter being reported for instance in families using lead-containing ceramic dishes [65,66], poisoning by lead-pigment [67,68], mouth-handling of lead shot [69], and due to gastro intestinal poisoning [701, etc. B-Pb concentrations were determined in a non-specified control group of children living in an area where lead levels in the air did not exceed a value of 0.7 /*.g/m3. Using an undescribed AAS method, values in the range of 584-1021 nmol/l were found [71]. In another study, B-Pb levels were investigated by F-AAS in the venous blood of 20 boys aged between 7-14 years. Here again, only the range of blood lead values between 241-589 nmol/l was reported [72]. In a study on 230 occupationally exposed workers, B-Pb levels were determined by ETA-AAS in 44 control men with an average age of 34 years, workers in a wood processing plant. The mean value and S.D. yielded 800 &- 270 nmol/l [73]. The prevalence of respiratory diseases was studied in children in an area with about one order of magnitude higher levels of atmospheric lead, due to pollution from a lead-waste processing plant, compared to a control, rural region where only limited lead emissions from road traffic occurred. B-Pb levels in the children from both groups were determined by F-AAS twice a year. The results obtained for the control group were significantly lower than in the exposed group [74] (Table 8). The health impact of exposure to atmospheric Pb in children was also studied by other authors [75]. B-Pb levels repeatedly determined by ETA-AAS in this study in 125 control children aged between lo-13 years were about 1200 nmol/l (Table 8). Biochemical indicators of kidney damage were followed in 10 men chronically exposed to Pb in the working environment in a chemical plant and in 11 control men working in administration in the same factory matched by age, way of life and other demographic factors [76]. The average age of controls was 48 years, range 31-57 years, the group contained two smokers and one man admitted regular drinking of more than 0.5 1 of

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Table 8 Blood lead levels in children and adults Method F-AAS F-AAS ETA-AAS F-AAS F-AAS F-AAS F-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS ETA-AAS

N ? 20 44 39 G” 38 Ba 38 Gb 34 Bb 68 B 68 B 70 B 73 B 54 G 55 G 50 G 53 G 11 266 138 M 128 W

Pb content (nmol/l) AM S.D.

Reference

800 854 1018 1023 1062 1289 1260 1289 1235 1226 1178 1274 1139 1430 278 344 206

M

Range 584-1021 241-589

270 256 256 285 273 188 159 183 164 275 183 309 227 270 140 154 73

GM -

434-1737 676-1737 627-2027 483-1737 1100-2170 68-1230 109-1230 68-468

249 319 201

-

G.S.D. -

250 317 193

1.57 1.50 1.43

71 72 73 74 74 74 74 75 75 75 75 75 75 75 75 76 77 77 77

G, girls; B, boys; M, men; W, women. aAutumn (October) sampling. bSpring (April) sampling.

beer daily. The arithmetic mean f SD. of B-Pb determined by ETA-AAS was 1430 + 270 nmol/l. The most extensive study of B-Pb levels in adults from the Czech population is being carried out in the framework of the MONICA project [34,35]. Until now, only results from the suburban region Prague East are available, where blood Pb levels in 266 inhabitants (138 men and 128 women) were determined by ETA-A&S [77]. These results are given in Table 8 for each sex group and also for the whole population. The mean values compare well with those recently reported from Denmark and Sweden. However, they are somewhat lower compared to those found in other European countries such as Belgium, England, Switzerland, France and Italy [78]. The mean values of the study [77] are also markedly lower and the distribution of the results is narrower compared to other results reported in Table 8 for various groups of the Czech population. Several factors can be considered to explain the differences observed. The time factor (a decrease in blood Pb levels in many European countries [78]

due to the reduction of lead in petrol) can be neglected, because the values reported in the studies [73,77] were determined in samples collected in approximately the same period (late 8Os, early 90s). The suburban area of Prague-East is apparently not less environmentally contaminated or characterized by a lower lead intake from diet than other regions where control samples in the studies [71-761 were collected. The most important differences between the studies [71-761 and [77] seems to be due to the fact that samples were prepared for analysis and analyzed in an ultratrace laboratory which meets 100 Class criteria, with a high standard of good laboratory practice, and with adequate adherence to quality assurance principles. Thus, the results of the MONICA study for B-Pb, as with results for cadmium and mercury, can form the basis for establishing reference B-Pb values in the Czech population, after the study has been completed and statistically evaluated. The other results for blood Pb given in Table 8 should not be used for the above mentioned purpose, because of the limited number of

J. Ku&a

et al. / Sci. Total Environ.

samples analyzed, no attempt was made to prove the accuracy of these results, and mainly because contamination on sample handling and/or analysis cannot be excluded. At present, a tentative reference interval (mean k 2 S.D.) of 109-951 and 68-352 nmol/l for men and women, respectively can be proposed based on the results of the study [77]. 3.13. Rubidium

S-Rb values were determined by INAA in samples from 62 men aged 35-70 years from Central Bohemia [43]. Arithmetic mean + S.D. of normally distributed results yielded 1.80 + 0.40 pmol/l as calculated from the results given in pg/g dry weight [43] using a conversion factor of 1 pmol/l = 1.0636 pg/g. The accuracy of Rb determination was proven by concurrent analysis of RM IAEA H-4 Animal Muscle and the mean value for S-Rb found compares well with that of 2.34 pmol/l (range 1.76-6.55 pmol/l) evaluated by Iyengar and Woittiez [20] as a possible reference value and with those reviewed by Versieck and Cornelis [41]. Thus, an indicative interval (mean 5 2 S.D.> of 1.00-2.60 pmol/l can be proposed for S-Rb. 3.14. Scandium

S-SC levels were also measured by INAA in the above mentioned study [43] in samples from 109 men from Central Bohemia. From log-normally distributed results, a geometric mean and geometric standard deviation of 2.62 and 1.98 nmol/l, respectively, were evaluated from the results given in pg/g dry weight using a conversion factor of 1 nmol/l = 2022.0 pg/g. The mean value is in agreement with the upper limit of 22.4 nmol/l of S-SC mentioned by Iyengar and Woittiez [20]. However, proof of the accuracy of the SC determination by INAA is missing, because no certified value for SC is available in RM IAEA H-4 Animal Muscle which was simultaneously analyzed for quality assurance purposes. Thus, no indicative value of S-SC is proposed. 3.15. Selenium

Since the late 5Os, selenium has been recognized to be an essential trace element in mam-

I66 (1995)

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223

mals [79]. Low selenium intake by man has been associated with Keshan disease, an endemic cardiomyopathy that occurs in certain areas of China [80]. Moreover, the results of several epidemiological studies documented an inverse relationship between selenium status and risk of cancer [81-861 and ischemic heart disease [871. Whole blood and serum (plasma) selenium levels are generally considered appropriate markers of selenium status in man under equilibrium conditions. Therefore, several studies were carried out on selenium levels in blood and serum aimed at evaluating the selenium status in various parts of the Czech Republic. B-Se was determined by HG-AAS and INAA as a control method in 287 healthy persons, voluntary blood donors aged 20-45 years, from five Czech regions. The evaluated arithmetic mean k S.D. of 975 + 228 nmol/l (range 469-1811 nmol/l) placed Bohemia fourth in the world in 1987 regarding low B-Se [88]. In conjunction with the second round of the WHO-coordinated MONICA project [34,35], a random 1% adult population sample stratified by age and sex was selected in six Czech districts for S-Se determination [891. One of the population samples from the Benesov district, Central Bohemia had already been evaluated [90]. Blood samples were taken from 367 healthy persons 25-64 years old (191 women and 176 men) after an overnight fast and serum was separated by centrifugation without any addition of anticoagulants. Using ETA-AA& Se was determined in the examined serum samples and in the Versieck’s Freeze Dried Human Serum Second Generation reference material. The arithmetic mean value -t S.D. of 1.11 + 0.109 pg/g dry wt. determined in five replicates compared well with the certified value of 1.05 pg/g for the latter material. An extremely wide range of S-Se values was found in the whole population sample (< 253-3749 nmol/l) as well as in each sex or age category studied. Arithmetic mean + S.D. of S-Se calculated after logarithmic transformation of the data was 937 + 38 nmol/l for the whole population sample, 912 ‘I: 62 nmol/l for men and 962 f 62 nmol/l for women. There was no significant correlation between serum selenium and sex or age or smoking status of the probants. Thus, again a

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low Se status, below the European average, was found for the Central Bohemian population. Several INAA studies were also performed to examine the Se status in the Czech Republic. S-Se values in 109 men aged 35-70 years from Central Bohemia yielded a normal distribution with an arithmetic mean + S.D. of 814 f 161 nmol/l [47]. In another INAA study [91], S-Se was determined in 65 men aged between 36-49 years living in three regions of the Czech Republic (urban, Prague; rural highland, the Vsetin district; and rural lowland, the Znojmo district). The overall arithmetic mean of serum selenium from three districts was 675 nmol/l with a range of 253-1178 nmol/l, the values for the particular

166 (1995) 211-234

districts being shown in Table 9. This study was later extended to determine S-Se in 235 Prague inhabitants, 55 boys aged 6-13 years, 63 men aged 18-65 years, 59 girls and 58 women of the same age structure as for boys and men, respectively [92,93]. Results for the whole Prague group and for the particular subgroups are given in Table 9. The S-Se values evaluated in the studies [91-931 in pg/g dry weight were recalculated to nmol/l using a conversion factor of 1 nmol/l = 1151.9 pg/g. For quality assurance purposes, RMs Versieck’s serum and IAEA Animal muscle H-4 reference materials were analyzed in the studies [47,91-931 and good agreement with the certified values proved the accuracy of the results. The

Table 9 Selenium levels in blood serum and urine of adults Tissue

B S S S S S S S S S S S S U U U U U U U U

Method

ETA-AAS ETA-AAS ETA-AAS ETA-AAS INAA INAA INAA INAA INAA INAA INAA INAA INAA Fluor. Fluor. Fluor. Fluor. Fluor. Fluor. Fluor. Fluor.

N

287 367 176 M 191 w 109 M 21b 22c 20* 235 55’ 63s 59h 58’ 32b 37C 32* 370 88’ 769 99h 107’

Se content (nmol/l)

Reference

AM

SD.

M

GM

Range

975 93Y 912a 962a 814 79Ze 645e 564e 794 713 855 705 896 119 113 93 115 116 105 127 111

228 3ga 62a 62a 161 145e 140e looe 203 138 231 124 219 46 40 35 62 53 52 73 64

-

798 771 701 822 695 876 100 104 91 112 95

469-1811 < 253-3749

800 772 714 831 698 850 104 102 94 113 104

M, men; W, women. a Evaluated after logarithmic transformation. bThe Prague district. ‘The Vsetk district. *The Znojmo district. eRecalculated from dry weight in pg/g using a conversion factor nmol/l = 1151.9 pg/g. ‘Boys 6-13 years. gMen 18-65 years. hGirls 6-13 years. i Women 18-65 years.

-

-

254-2065 417-1207 254-1557 474-1269 602-2065

8.7-509 27-238 8.7-306 31-509 30-372

88 90 90 90 47 91 91 91 92,93 92,93 92,93 92,93 92,93 91 91 91 92,93 92,93 92,93 92,93 92,93

J. KuEera et al. / Sri. Total Environ.

S-Se values in the studies [47,91-931 followed a normal distribution and were markedly lower. However, more consistent levels were found in children compared with adults with no significant (a = 0.05) differences between the sex categories. The B-Se and S-Se data are suitable for deriving reference intervals, because a sufficient number of samples was analyzed and the accuracy of the analyses was confirmed using different analytical methods (AAS X INAA) or by concurrent analyses of suitable reference materials or by both methods. In general, the very low Se status in the Czech Republic follows from blood and serum analysis. Significant differences in S-Se were found in various districts of the Czech Republic and age differences in selenium intake in Prague inhabitants were also detected. Therefore, the S-Se reference values should be geographically and age stratified in detail which will be possible after completing the analyses being carried out in the framework of the MONICA project. At present, tentative reference or indicative intervals for S-Se are proposed which are calculated from weighted arithmetic means and standard deviations according to [56] separately for men and women, boys and girls from Prague and Central Bohemia based on the results of the studies [47,90-931. The tentative reference and indicative values are listed in Table 12. In the studies [91-931, U-Se was also determined using a fluorimetric method to assess the dietary selenium intake. The overall AM of U-Se in single void (morning) urine samples of inhabitants of the three above specified Czech districts [91] was found to be 110 nmol/l with the range of values of 23.7-245 nmol/l. Arithmetic means + S.D. for U-Se for the particular districts are shown in Table 9. U-Se levels in 370 Prague inhabitants determined by the same analytical methods yielded an overall AM + S.D. of 115 + 62 nmol/l [92,93] and the mean values for the particular sex and age subgroups are given in Table 9. The U-Se values determined seem to suggest a low selenium intake from the diet and support the results of B-Se analysis which demonstrate a low selenium status of inhabitants of the Czech Republic. Since the accuracy of the U-Se determination was tested by control analysis of

166 (1995) 21 l-234

225

Lyphocheck Urine samples and no significant differences were found for U-Se levels in two age categories of Prague inhabitants (men and women), a combined reference limit of 239 nmol/l is proposed for all categories studied (Tables 9,12X 3.16. Vanadium

In the early 70s vanadium was reported to be an essential trace element for chick and rat [94,95] and this finding has lead to speculations as to whether the element could also be essential for man. Both aspects seem to be still an open question [96]. Several adverse health effects in occupationally exposed workers (on boiler cleaning, vanadium pentoxide production, in metallurgical processes involving production of vanadium containing vapours which condense to form respirable aerosols, etc.), mainly to the respiratory tract were detected [97] and possible vanadium toxicity needs further research. Until now, largely divergent ‘normal’ values have been reported for B-V and S-V which vary from 0.59 to 15700 nmol/l, with a somewhat lower range being reported for U-V, 4.7-609 nmol/l. Analytical difficulties have been suggested to be the main reason for the existing discrepancies [98,99]. Recently, B-V and U-V levels and V levels in hair were determined by RNAA with proven accuracy (by concurrent analysis of several reference materials containing low levels of vanadium) in workers exposed at a vanadium pentoxide-producing plant, children potentially exposed to vanadium from the polluted environment in the vicinity of the plant and in control groups of children and adults [99,100]. The former control group consisted of 15 boys and two girls aged 9-13 years living in a rural region. The control group of adults for B-V analysis was formed by seven men and four women aged 25-67 years, workers at a research institute, who lived in an urban area. No significant differences were found between B-V levels in children and adults. Another control group of adults for U-V analysis, workers at another research institute, consisted of 12 men and nine women of age 22-63 years, who lived in two urban areas. The results for control groups are summarized in Table 10. The values

226

J. KuEera et al. /Sci. Total Environ. 166 (1995) 211-234

Table 10 Vanadium levels in blood of children and adults and in urine of adults Tissue

B B U

Method

RNAA RNAA RNAA

N

178 lib 2F

V content (nmol/l)

Reference

AM

SD.

M

GM

Range

1.06 1.14 4.38

0.94 0.39 1.88

0.82 1.10 4.16

0.88 1.08 3.99

0.47-4.44 0.63-1.86 1.30-9.60

99 99 100

a15 boys and two girls aged 9-13 years. bSeven men and four women aged 25-67 years. ‘12 men and nine women aged 22-63 years.

given in Table 10 are at the lowest end of the reported values [98,100]. There seems to be increasing evidence that the lowest B-V, S-V and U-V levels can be considered typical for occupationally and environmentally non-exposed populations. However, geographical differences may exist, because vanadium is released in large quantities into the environment by burning fossil fuels [6], mainly in aerosols resulting from firing heavy oil [loll, and because inhalation is one of important routes of V intake [98]. The values of B-V presented in Table 10 were pooled according to [56] and an indicative interval of 0.55-3.24 nmol/l was derived which should be considered typical for inhabitants of rural and urban regions, which are not very much polluted by vanadium from oil combustion. The same applies for the derived indicative interval of 1.35-7.50 of U-V (Table 12). 3,lZ Zinc

Zinc as an essential trace element is indispensable for the function of various mammalian enzymes and its concentration in various tissues and body fluids is homeostatically regulated. Zinc concentration in blood and serum is thus expected to fluctuate within rather narrow limits. The same may be expected for urine, because the main excretion route for zinc from mammalian organisms is through feaces and not through urine. Zinc deficiency in man is exhibited mainly by growth retardation and sexual maturity retardation. Zn toxicity is not very common, the most dangerous form being inhalation of vapours or fine powder of metallic Zn or ZnO which can lead to irritation of the respiratory tract and fever (smelters’ or metallic fever) [1021.

S-Zn was determined by AAS in 30 blood donors (23 men, seven women, average age 24 years) which formed a control group in a study on vitamin A and Zn concentrations in the serum of patients in the polyuric state of chronic renal failure with and without dialyzation treatment [103]. The S-Zn levels found in the controls are given in Table 11. In a methodological study [104] on Zn determination by F-AA& S-Zn was determined in 41 healthy persons (23 men, 18 women), P-Zn in 46 healthy persons (28 men, 18 women), and U-Zn (24-h samples) in 33 healthy persons (17 men, 16 women). No other specification of the subjects was given [1041. The following values in pmol/l (AM f S.D.) were determined in the studied groups: S-Zn, 15.74 f 2.30 (N = 41); P-Zn, 14.79 + 1.46 (N = 46); U-Zn, 3.99 f 2.79 (N = 33). The separate values for men and women are shown in Table 11. Patients in the terminal phase of chronic renal insufficiency and in kidney failure exhibited a significant decrease in S-Zn compared to a control group of 33 healthy volunteers, 17 men, 16 women, average age 30.7 years, range 19-54 years. The values found by F-AAS in the control group yielded 16.12 + 2.84 pmol/l [105], decreased S-Zn was also found by F-AAS in patients with chronic inflammatory and neoplastic liver disease [106]. In this study, a control group was formed by 50 healthy blood donors (25 men aged 35 f 8 years, 25 women aged 37 f 10 years) and the AM f S.D. for S-Zn was 16.2 f 1.5 pmol/l. A relationship between S-Zn and U-Zn was studied using F-AM in women occupationally exposed to Zn in a metallurgy plant and in a control group of 117 women

J. K&era Table 11 Zinc levels Tissue

in serum, Method

plasma

et al. / Sci. Total Environ.

F-AAS F-AAS F-AAS F-AAS F-AAS F-AAS F-AAS F-AAS F-AAS RNAA INAA F-PAS F-AAS F-AAS F-AAS F-AAS F-AAS F-AAS RNAA

211-234

221

and urine N

Zn content

30 23 M 18 w 33 50 112 10 46 ? 10 w 108 28 M 18W 20 17 M 16 W 116 46 low

Reference

( ~mol/l)

AM S S s S S S S S S S S P P P U u U U u

I66 (1995)

SD.

17.06 16.25 15.08 16.12 16.2 16.51 20.81 13.7 16.98

0.41 1.95 2.60 2.84 1.5 6.16 3.95 2.7 1.53 -

13.37 15.08 14.25 16.0 5.06 2.85 7.3 7.3 -

2.17 1.50 1.23 1.5 2.93 2.10 2.1 3.6 4.55

M

Range

-

-

-

14.30 13.21 -

-

10.86-18.35 2.14-9.02

103 104 104 105 106 107 109 110 111 32 43 104 104 109 104 104 108 110 32

M, men; W, women.

living in the vicinity of the plant in northern Moravia [107]. No significant correlation was found in either of the groups studied. The Kolmogorov-Smirnov test showed that the distribution of S-Zn and U-Zn values departed from a normal distribution in both groups, being log-normal in the control group. In spite of this fact, the arithmetic mean and S.D. were calculated from non-transformed results and amounted to 16.51 f 6.16 pmol/l and 7.29 + 5.43 pmol/l for S-Zn and U-Zn, respectively. S-Zn and U-Zn levels were also studied in 10 healthy men aged 17-35 years at rest (control measurement) and after a l-h physical load on a bicycle ergometer [108]. The S-Zn and U-Zn levels determined by F-AAS did not differ significantly in both measurements and the values at rest were 20.81 + 3.95 pmol/l and 7.3 +_ 2.1 pmol/l, respectively. Significantly lower P-Zn levels were found by F-AAS in the third trimester of pregnancy compared to the levels during the second trimester of pregnancy and to the levels in a control group of 20 nongravid women with average age of 28 years (range 21-35 years) which amounted to 16.0 + 1.5

pmol/l [109]. The metabolism of Zn, i.e. S-Zn and U-Zn levels were also studied in insulin-dependent diabetes in a group of diabetics with normal renal functions, with chronic renal insufficiency as a result of diabetic nephropathy, in non-diabetic subjects with chronic renal insufficiency, and in a control group of 46 healthy volunteers (28 men, 18 women, average age 32) without any clinical signs of zinc-deficiency and without permanent medication [llO]. The levels of S-Zn and U-Zn (24-h samples) in the control group determined by F-AAS were 13.7 + 2.7 pmol/l and 7.3 + 3.6 pmol/l, respectively [llO]. In a study of the biochemical parameters of osteoporosis, S-Zn was determined by F-AAS in an unspecified control group and an AM + SD. of 16.98 + 1.53 pmol/l was reported [ill]. In a study of biochemical parameters of workers occupationally exposed to Cd, previously mentioned in the Cd-section (Section 3.3.) [32], S-Zn and U-Zn levels were also determined by RNAA with proven accuracy at various Zn levels (as can be inferred from the results of control analyses of various biological RMs, namely NIST SRM-1577

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Bovine Liver, RM Bowen’s Kale, IAEA RMs Milk Powder A-11 and Horse Kidney H-8) in a control group of 10 women, aged 22-40 years (median 32 years), non-smokers, administrative workers in a plant where no exposure to either Cd or Zn occurs. Median values of S-Zn and U-Zn of 14.30 pmol/l and 4.05 pmol/l, respectively were found, and ranges of the values are shown in Table 11. S-Zn was also determined by INAA in 108 men aged 35-70 years from Central Bohemia. Normally distributed results yielded an AM f S.D. of 13.37 & 2.17 pmol/l [43]. The accuracy of the results was checked by control analysis of RM IAEA Animal Muscle H-4. S-Zn and U-Zn levels are considerably higher compared to the majority of elements discussed in this work and the Zn determination either by F-AAS, INAA or RNAA is not associated with severe analytical difficulties. Thus, reasonably consistent data were found for S-Zn (Table 11). However, the accuracy of the Zn determinations still has to be proved to establish the element reference intervals. Unfortunately, this has not been done in the studies [103-1111, except for the values determined by KuEera et al. [32] and Kvicala and Havelka 1431. These values compare well with reference values of S-Zn evaluated by Iyengar and Woittiez [20] (median 14.22 pmol/l, range 10.70-18.35 pmol/l>. Although, the data obtained by non-verified analytical procedures by other authors [103-1111 are not in conflict with those obtained by procedures with proven accuracy, it is thought preferable to derive an indicative S-Zn interval of 9.23-17.73 pmol/l using the latter data only by calculating a weighted AM and S.D. according to [56] from the studies [32,43]. Since U-Zn values with proven accuracy were measured in only 10 women [32], no attempt has been made to evaluate a U-Zn indicative interval or limit without further research. 4. Conclusions

Most of the data reported in this paper were obtained by analyzing blood, serum and urine samples of subjects from control groups in studies

166 (1995) 211-234

on various occupational, environmental or biomedical problems for over the last 20 years. For this reason, most of the data usually do not meet the criteria designed recently to derive reference intervals [15], especially number of subjects and age structure of population samples studied. The only exception is the MONICA project which has been recently started [34,35] to search for the reasons for the increased incidence of cardiovascular diseases in the Czech population. In addition, some of the reported Czech and Slovak studies were performed in the late 70s or early 80s when it was difficult to meet all the criteria for appropriate sampling techniques and quality assurance procedures. For instance, plastic catheters or siliconized needles required for contamination-free sampling of blood, dust-free laboratories or clean flow boxes, and high purity chemicals were not readily available, mostly for financial reasons. The same applies to the very limited availability of appropriate reference materials for proving the accuracy of analysis. Many of the results listed in this paper were obtained in local hygiene stations or at the National Institute of Public Health, Prague (formerly Institute of Hygiene and Epidemiology) in which atomic absorption spectrometry has usually been the only analytical technique available for elemental analysis. Comparative analyses employing other analytical techniques especially suitable for low-level element analysis, such as NAA, were rather exceptions, again mainly due to budgetary limitations. Detailed statistical examination of the results obtained was also rather rare in the older papers. On the other hand, the results of the recent MONICA study and some results obtained by NAA clearly demonstrate that if adequate technical conditions are available, which means in practice sufficient funding and its appropriate use, there is a good analytical potential for producing accurate results from trace and ultratrace element analysis in our countries. As a result, only a limited number of tentative reference or indicative intervals or limits could be evaluated which are summarized in Table 12. Moreover, other

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Table 12 Tentative reference/indicative interval (or limit) values of trace elements in blood, serum and urine of Czech and Slovak populations as derived from a critical analysis of literature Element

Tissue

N

Element content (nmol/l, unless otherwise stated) Mean

B B

18.3 23.7 3.6 4.62

37.7 49.1 < 0.2-8.0 16.8

B S S S S S U B U S

138 62 348 249 55 59 370 28 21 118

344 1.80 871 947 713 705 115 0.96 4.38 13.48

109-951 1.00-2.60 623-1119 749-1145 437-989 457-953 239 0.55-3.24 1.35-7.50 9.23-17.73

s S

Cd Hg Pb Rbb Se

V Znb

Interval (or limit) value?

10 10 406 611

As

Remarks

lo-year-old boys, Central Slovakia lo-year-old boys, Prague Men and women, non-smokers Men and women from two regions (rural and suburban) Men and women from suburban region Men, Central Bohemia Men, Prague and Central Bohemia Women, Central Bohemia 6- to 13-year-old boys, Prague 6- to 13-year-old girls, Prague Prague inhabitants Children and adults Adults of urban region Men and women, Central Bohemia

aReference interval (or limit) values when N > 120; Indicative interval (or limit) values when N < 120 [2]. For details see Section, ‘Introductory remarks and abbreviations’. bwmol/l.

data on levels of the elements Al, As, Cu, F, and Zn in blood, serum or urine were obtained which are probably correct and compare well with those from other countries. However, since proof of accuracy of analysis were not given in the respective studies, these data could be rigorously used for evaluation of the above mentioned intervals or limits. It should be also pointed out that for an exposure assessment in occupational settings in our countries, biological limits were applied for a variety of biomarkers including those of toxic metals like Cd, Pb and Hg. This practice is rooted in the long-lasting tradition of using biomarkers of human exposure to toxic metals in the former Czechoslovakia. 5. Needs for future research

More systematic and large scale studies are obviously needed to establish reference values of several trace elements in human blood, serum

and urine in the Czech and Slovak populations. Such studies require a well designed strategy which involves an adequate sampling plan selection for the reference population, the control of preanalytical factors, strict adherence to quality assurance principles (e.g. regular monitoring of accuracy of analytical methods by control analyses of test samples, appropriate CRMs and/or participation in intercomparative analyses at national and international scales), the use of facilities equipped for ultratrace element analyses and appropriate statistical treatment of the data [3]. The accurate determination of several elements such as Co, Cr, Mn, and Ni in blood of serum requires contamination-free sampling devices, other elements such as Al, Cd, Pb, etc. are prone to contamination if samples of human tissues and/or body fluids are not handled in special laboratories for ultratrace element analysis. Therefore, determination of the above mentioned elements should only be carried out by teams and in laboratories specialized for this purpose if valid

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data are to be obtained to be used for establishing reference values. The elements which present the highest potential risk for health in the Czech and Slovak population do not include only Cd, Hg and Pb, as in any other countries. Other elements of special concern in the Czech Republic and Slovakia are carcinogenic metals such as As, Ni, Cr and other toxic (Tl) or potentially toxic elements such as Al, F, Mn, V, as result of their large release into the environment by coal combustion and other industrial activities (Al, steel, V,O, production). Attention should also be paid to Pt, Pd and Rh because the increased use of Pt catalysts in petrol-driven automobiles which could lead to the increased discharge of the three metals into the environment. It also seems very important to study in more detail reference values of several essential trace elements for which their deficiency has already been detected (e.g. Se> or which is suspected (e.g. I or Zn> to provide the scientific basis for research into the effect of deficiency of such elements on the health of populations with different body burdens of carcinogenic trace elements or organic pollutants. In addition, since the establishment of trace element reference values in human tissues requires harmonization and standardization of methodologies, new experimental activities in each country must be coordinated at an international level. In this context, the EURO TERVIHT project of the EU [112] represents an important landmark. Acknowledgements Financial contributions for this work received from the Environmental Institute, JRC Ispra, (No. LIS EG 920 77 341D) and the Grant Agency of the Czech Republic (No. 313/93/0108) are greatly acknowledged. References [I]

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