International Archives of
Oeulmiuonal
Int Arch Occup Environ Hlth 40, 273-282 (1977)
al
E-iro lltal
Health ©
Springer-Verlag 1977
Evaluation of Different Urinary Proteins Excreted after Occupational Cd Exposure Hansen, Lars 1 , Kjellstr 6 m, Tord 2 , and Vesterberg, Olof1 Department of Chemistry, National Board of Occupational Safety and Health, Fack, S-100 26 Stockholm, Sweden 2 Department of Community Health, University of Auckland, Auckland, New Zealand
Summary Workers in an alkaline battery factory were examined for renal effects after several years exposure to cadmium Total urinary protein was determined using a Biuret method To separate the individual proteins, isoelectric focusing was performed in flat beds of polyacrylamide gel The proteins were then stained and evaluated by densitometry The duration of employment at the factory was used as an indicator of cadmium dose. A dose-effect relationship was found between cadmium and the concentration of some of the urinary proteins The most pronounced relative increases in average concentration in relation to years of cadmium exposure were seen in some zones, e.g those containing albumin and /2-microglobulin, respectively The largest quantitative increase was observed for the zone containing albumin The results indicate that isoelectric focusing is suitable to evaluate effects of cadmium on kidneys. Key words: Cadmium
Exposure
Proteins
Isoelectric focusing
Introduction The toxic hazards connected with occupational exposure to cadmium have been recognized more and more during recent years (Friberg et al , 1974) Much concern has been focused on the extraordinary tendency for accumulation of cadmium in the kidneys with an estimated half time for elimination in the order of decades Above a certain critical concentration, which has been estimated to be about 200 ug/g of cadmium in kidney cortex, increased concentration of proteins in urine can be found This damage is mainly in the proximal tubuli (See Friberg et al , 1974 p 105 for references). Since about 15 years paper electrophoresis has been used in such studies (Piscator, 1962 a, and Kazantzis et al , 1963) The information has been increased by use of gel permeation chromatography, which has e g shown that the proteins are to a high extent of low molecular nature (Piscator 1966 a and b) One such protein is 32 -microglobulin the concentration of which may be elevated more than hundred times after high cadmium exposure over many years (Peterson et al , 1969) Also another low molecular protein, retinol-binding protein, has been proposed for evaluation of cad-
274
L Hans 6 N et al.
mium induced kidney damage (Kanai et al , 1971) To be able to take various preventive measures it is of high interest to detect a malfunction at an early stage To this end and to get more information about the effects of cadmium on the kidneys we have made a study of urinary proteins with the method of isoelectric focusing as described by Vesterberg and Nise (1973) and Vesterberg et al ( 1976) which when used in flat beds of polyacrylamide gels is one of the most high resolving analytical methods for proteins, which allows separation of also very similar proteins (Vesterberg, 1973) In this method the proteins focus at their isoelectric points This physico-chemical characteristic, which can be used for, e g characterisation and recognition of proteins can be made simply by measuring the p H In combination with suitable staining procedures (Vesterberg, 1972, 1973 ; Vesterberg et al , 1977) it is very sensitive, allowing detection of tenths of a jig of proteins Furthermore, many protein samples are run in parallel facilitating comparison and densitometric evaluation. Materials and Methods Urine samples were analysed from 55 male workers with occupational cadmium exposure in a battery factory The workers had been employed in two distinctly different exposure conditions in the assembly plant and the material plant (Kjellstr 6m et al , 1977) These 55 workers comprised all those who took part in a tuberculosis health screening on a particular day They comprised about 20 %of the presently employed cadmium-exposed workers in the factory (Kjellstr 6m et al , 1977) Exposure conditions or earlier detected cadmium effects were not considered in the selection The exposed were classified according to duration of employment in cadmium exposure in the plants and stratified into different groups as indicated in Table 1 At the collection the samples were mixed with 0 4 M phosphate buffer of pH 7 6 (1 part buffer to 10 parts of urine) Total protein was determined according to Piscator (1962 b) ,2-microglobulin was analyzed by using radioimmunoassay with a kit ,Phadebas" (Pharmacia, Uppsala, Sweden) The filtered urine samples were concentrated 20 times in collodium tubes (Membran Filter Gesellschaft, G6ttingen, Germany) with reduced pressure, before separation by isoelectric focusing Flat beds of polyacrylamide gels were used essentially as described earlier by Vesterberg (1973) with T = 3 %,C = 5 %containing sucrose 12 %(w/v), Ampholine 2 8 ml pH 3 5-10, 0 4 ml pH 9-11, 0 2 ml pH 4-6 and 0 2 ml pH 5-7 giving a final average concentration of 2 %(w/v) Samples of 100 1l of concentrated urine were soaked into rectangular (8 x 10 mm) filter paper pieces (Grycksbo, Sweden) and applied close to the cathode Focusing was performed with 400 V as initial and 1000 V as final potential and making the increase so that the wattage never exceeded 40 W The focusing time was 90 minutes. After focusing the proteins were stained with a solution of Coomassie Brilliant Blue R 250, 0 1 % in methanol 27 %mixed with trichloroacetic acid 11 %and sulfosalicylic acid 3 3 %in water (figures are final concentrations) (Vesterberg, 1972) After destaining in ethanol-water-acetic acid (26:66:8) solution the gels were placed on a glass plate and covered with a dialysis membrane and were allowed to dry (Vesterberg, 1973). Estimation of the protein content in the separated bands were then performed by densitometry in a Zeiss spectrophotometer (PMQ II) The wavelength was 600 nm and the slit 3 x 0 02 mm In order to simplify the comparison we have estimated the amount of protein with close isoelectric points by calculating the absorbance areas of the densitogram which has been divided into ,,zones" of 5 mm's length each The zones were numbered from the anode side so that albumin always was found in zone 7 A base line was drawn to eliminate the background noise. The absorbance of the urine protein band containing 2,-microglobulin was compared with the absorbance of standards For a typical result see ref Vesterberg and Nise Fig 6 This gave a semiquantitative measure of urinary 0,-microglobulin based on isoelectric focusing. The concentration of the urine was normalized according to density by using the formula published by Levine and Fahy (1945): a x 23 x 10 ' 3 10
where a = area of a zone after densitometric evaluation and 6 = density of sample
Different Urinary Proteins Excreted
275
Table 1 Description of groups of workers studied Employment duration (years) in cadmium exposure 25 Assembly plant Material plant
13
9
11
14 8
total 47 8 55
Results The concentration of total protein in urine in relation to years of occupational cadmium exposure is shown in Fig 1 There was a tendency for increasing concentration with duration of exposure Judged from the linear regression line there was on the average about 3 times increased concentration after 30-40 years exposure. A typical result of isoelectric focusing of urinary proteins is shown in Fig 2 The total number of separated proteins was large More than 25 bands were seen in cases with long exposure It was extremely difficult and time consuming to compare individual protein bands for all persons Therefore we divided the protein spectrum into 20 zones A densitometric evaluation of the stained protein zones from one person with six years of cadmium exposure is shown, in Fig 3 The staining procedure does not stain all bands with the same intensity on weight basis and even less on molar basis as would be desirable Unfortunately, there exists no such ideal procedure No strict comparison of the estimated amount of protein in one zone with that of another could be made Instead the absorbance of the stained protein in each zone was calculated and compared for evaluation on a group basis Several zones had absorbance below the detection limit and medians are therefore used in the following Fig 4 shows the median absorbance in the 11 zones where most cadmium-induced changes are seen In the zones 2, 5 and 7 the absolute increase of,absorbance with increased exposure duration seemed to be the largest Largest relative increases were seen in zones 5 and 11 These zones contained proteins of which some have earlier been mentioned in connection with cadmium damage. The zones have been shown to contain the following proteins: zone 2, orosomucoid; 5, haptoglobin, a l-antitrypsin and probably also retinol binding protein; 7, albumin and zone 11, 32 -microglobulin (to be published, Hansen and Vesterberg). The median absorbance in zones 2, 5, 7 and 11 tended to increase with exposure duration and were highest in the material plant group except for zone 2 Between the group with < 2 years exposure and > 25 years exposure there were statistically significant increases in absorbance in zones 5, 7 and 11 (Fishers exact median test gave P = 0.02, 0 0006 and 0 002 respectively) The highest relative increase of the median occurred in zone 11 (> 44 times) and zone 5 (> 40 times), whereas the increase was 4 times in zone 7 There was a tendency for a relatively sharp increase in median absorbance of zone 11 (containing 12 -microglobulin) already at 2-10 years exposure (P = 0 2) (Fig 5) However, quantitative specific analysis of urinary 2 -microglobulin did not show such an increase (Fig 6) The increase of urinary 32 -microglobulin between the group with < 2 years exposure and the group with > 25 years exposure (Fig 6) appears similar to the corresponding increase of absorbance of zone 11 (Fig 5).
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years 40 30 20 10 Fig 1 The concentration of total protein in urine (corrected to spec gravity = 1 023) as a function of duration of employment for 40 workers in the Cd-battery assembly factory (For technical reasons total protein was not analysed in all urines ) The linear regression line is indicated O
Fig 2 A photography of a gel after focusing and staining of the protein bands The samples were applied at the cathode side (lower part of the gel) A cm-scale is shown to the left The positions are numbered from left to right The following samples were applied: position 1 and 14 standard 2 protein mixture'; 2-13 and 15-23, urine samples from 7 men with different Cd exposure . 'the standard proteins are indicated by letters as follows a, albumin 1 0 Mg; b, -lactoglobulin (2 bands) 2 5 tmg; c, 32 -microglobulin 0 5 Mg; d, myoglobin (2 bands) 2 5 /Ig and e, cytochrome c 2 5 jug 2 each sample occurs in three adjacent positions: one unconcentrated the second concentrated 20 times and the third concentrated and with addition of 0 5 Mug0, -microglobulin
Different Urinary Proteins Excreted
277
Fig 3 Densitogram of urinary proteins in one sample after separation by isoelectric focusing and staining of the protein bands A photo of the gel strip is shown below with the anode to the left The absorbance scale is on the y-axis Classification into zones and a pH scale on the x-axis The albumin band is in zone number 7
We have studied the possibility to detect tubular malfunction as shown up by increased concentration of f 2-microglobulin by use of isoelectric focusing In Table 2 it can be seen that with a very high predicability it was possible by using isoelectric focusing to find those who had elevation of 12 -microglobulin above the 95-percentile of a normal group (Kjellstr 6 m, 1977). In studies by isoelectric focusing of protein patterns from persons with different diagnosis we have obtained experience allowing us to make qualification in the categories glomerular or tubular proteinuria (Vesterberg and Nise, 1973; Vesterberg et al. 1976) When the concentrations of high-, medium and low molecular proteins were elevated the patterns were called mixed glomerular-tubular An evaluation of the persons encompassed in this study is presented in Table 3 Glomerular disorders are much more common in an ordinary population than tubular disorders The opposite is the case for cadmium exposed as can be seen in Table 3 Furthermore, the prevalence of tubular patterns increased with employment duration. Discussion We found that upon increasing duration of exposure to cadmium the urinary concentration of some low molecular proteins such as 32 -microglobulin and retinol binding protein increase in a fashion related to duration of exposure Furthermore the con-
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Fig 4 The protein content estimated by the total absorbance, A, (arbitrary units) of each zone after focusing and staining of the protein bands Within each group the median value and the 75-percentile are shown for each zone
Symbols: Group a
Median A
75-percentile A
b c
V
d
V
e
*
duration of Cd-work 25 > 25
place assembly plant
material plant
number of men 13 9 11 14 8
centration in urine of some medium molecular weight proteins as orosomucoid (Mw 44 100) (zone 2), albumin (Mw 69 000) (zone 7) and al -antitrypsin (Mw 45 000) (zone 5) also increased Thus for persons with effects of cadmium on the kidneys this results in typical patterns after isoelectric focusing characterized by many bands and elevated concentrations of protein in many of the bands This is in accord with recently published observations of cadmium-induced proteinuria (Bernard et al 1976). Total protein is a very crude measure because it is known that different proteins give very different degree of contribution to the protein value when compared on molar basis Furthermore, certain proteins, mainly low molecular ones, are not properly determined by the method for total protein The increase of total protein (Fig 1) with increased employment duration was therefore less than the increase of individual zones in isoelectric focusing (Fig 4) Still more, there are many conditions that may result in increased excretion of protein in urine, which is thus of low specificity when trying to detect effects of cadmium.
279
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The definition of zones in the focusing patterns made it possible to quantify the increases The scatter of zone absorbance between individuals in the same dose group was large but a similar scatter was found for radioimmuno-assay analysis of 02 -microglobulin in the urines (Fig 6) Even in groups with long duration certain persons do not have a pathological proteinuria The prevalence of tubular proteinuria becomes a matter of where to draw the border line According to the criteria used by Kjellstrom et al (1977) for urinary 32 -microglobulin there were 9 tubular proteinurias among 14 workers in the assembly plant with > 25 years exposure (Fig 6). Eight persons in the same group had tubular proteinuria according to our qualitative assessment of the patterns (Table 3) and 11 of 14 had an absorbance in zone 11 (including 2 -microglobulin) > 2 Zones 11 and 5 showed the greatest absorbance increases with dose The analysis of such zone absorbance increases seems to have a similar sensitivity as quantitative measurement of urinary 2 -microglobulin in picking up cadmium induced tubular proteinuria This latter method has been advocated by Kjellstr 6m and Piscator (1977) for health screenings of cadmium workers. Radioimmunoassay of 12 -microglobulin in urine has the advantages over isoelectric focusing that it is quantitative at normal levels, but it requires a gamma-counter. Isoelectric focusing has the advantages that it gives information about several proteins in urine in one analysis, and that it uses much cheaper equipment for the analysis. Futhermore, about 24 samples can be run in parallel on one gel. When the kidneys are intact there is only a small loss of plasma proteins in urine amounting to about 50 mg/24 hours and normally < 100 mg/24 hours (Cannon 1974, p 431) The reason why the loss is not larger is partly that extensive reabsorption occurs, which takes place mainly in the proximal tubuli It has also been shown that the kidneys catabolize different proteins at different rates (Hardwicke et al , 1970),
280
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Fig 6 Urinary concentration of 2 -microglobulin measured with radioimmunoassay as a function of duration of cadmium exposure The ,,normal" 95-percentile (Kjellstr 6 m et al 1977) at 260 gg/l is indicated o = workers in the assembly plant " " " material = "
Table 2 Comparison of two methods for determination of a critical level of ,2-microglobulin. Samples from each of the 55 persons have been examined independently with Radio-immunoassay (RIA) and Isoelectric focusing (IEF) with RIA < 0,3 mg/l
total > 0,3 mg/l
< 0,3 mg/l
36
2
38
> 0,3 mg/l
3
14
17
39
16
55
with IEF
total
Different Urinary Proteins Excreted
281
Table 3 Kind of kidney effects diagnosed by the protein pattern from men working in two different cadmium plants
effects
Assembly plant number of men with different years of employment 25
Glomerular Glomerular (uncertain) Tubular Tubular (slight) Normal
13
Total
13
Material plant
> 25 1
1
3
7 1
4 1
9
8
5
2
9
11
14
8
which is very important for regulating the life span in the body of protein molecules. An increased leakage of protein does not only mean that a corresponding amount of the protein is lost from the body but this may very well also influence the statistical life span in the body of the molecules of the protein Because of the complicated mechanisms involved at loss, regulation and feed back it is not easy to say what consequences a loss of a certain amount of a protein has for the body At moderate loss per day the effects as far as we know today is rather limited However, the fact that a certain protein appears in increased concentration in urine is an indication that there is some dearrangement of the normal kidney function It is also known that when a cadmium induced malfunction of this kind is pronounced, there is also decreased capacity of the kidneys to retain amino acids, glucose and calcium (see Friberg et al , 1974, pp 103-106 for ref ) Renal tubular damage should therefore be prevented among cadmium-exposed workers and our study showed that isoelectric focusing of urinary proteins is one way of making early diagnosis To prevent too high exposure analysis of cadmium in blood and urine are also helpful as indicated by Friberg et al. 1974 pp 77-79 and Vesterberg and Bergstr 6m, 1977. Acknowledgement This work was supported by grants from The Work Environment Fund in Sweden.
References Bernard, A , Roels, H , Hubermont, G , Buchet, J P , Masson, P L , Lauwerys, R R : Characterization of the Proteinuria in Cadmium-Exposed Workers Int Arch Occup Environ Hlth 38, 19-30 (1976) Bernier, G M , Conrad, M E : Catabolism of human 2 -microglobulin by the rat kidney Am J. Physical 217, 1359-1362 (1969) Cannon, H : in Clinical Chemistry: Principles and Techniques 2nd ed , Henry R J Ed , Hagerstown, Md.: Harper and Row 1974 Friberg, L , Piscator, M , Nordberg, G F , Kjellstr 6 m, T : Cadmium in the Environment, 2nd Ed. Ohio: The Chemical Rubber Co 1974 Hardwicke, J , Cameron, J S , Harrison, J F , Hulme, B , Soothill, J F : Proteinuria, Studied by Clearances of Individual Macromolecules Proteins in Normal and Pathological Urine, pp. 111-152, Basel, New York: Karger 1970
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Kanai, M , Nomoto, S , Sasaoka, S , Naiki, M : Clinical significance of urinary excretion of retinolbinding protein in patients with Itai-Itai disease Prog Symp Chem Physiol Pathol 11, 194-199 ( 1971) Kazantzis, G , Flynn, F V , Spowage, J S, Tott, D G : Renal tubular malfunction and pulmonary emphysema in cadmium pigment workers Q J Med 32, 165-196 (1963) Kjellstr 6 m, T : Accumulation and renal effects of cadmium in man A dose-response study Diss. Karolinska Institutet, Stockholm 1977 Kjellstr 6 m, T , Evrin, P -E , Rahnster, B : Dose-response analysis of cadmium-induced tubular proteinuria Env Res (In press) Kjellstr 6 m, T , Piscator, M : Quantitative analysis of 02 -microglobulin in urine as an indicator of renal tubular damage induced by cadmium Phadedoc Diagnostic Communications, No 1, Parmacia Diagnostics AB, Uppsala, Sweden 1977 Levine, L , Fahy, J P : Evaluation of Urinary Lead Determination J Indust Hyg Tox 27, 217223 (1945) Peterson, P A , Evrin, P -E , Berggard, I : Differentiation of glomerular, tubular and normal proteinuria: Determinations of urinary excretion of , -microglobulin, albumin and total protein. J Clin Invest 48, 1189-1198 ( 1969) Piscator, M : Proteinuria in chronic cadmium poisoning 1 An electrophoretic and chemical study of urinary and serum proteins from workers with chronic cadmium poisoning Arch Env. Hlth 4, 607-621 (1962 a) Piscator, M : Proteinuria in chronic cadmium poisoning 2 The applicability of quantitative and qualitative methods of protein determination for the demonstration of cadmium proteinuria. Arch Env Hlth 5, 325-332 (1962 b) Piscator, M : Proteinuria in chronic cadmium poisoning 3 Electrophoretic and immunoelectrophoretic studies on urinary proteins from cadmium workers with special reference to the excretion of lowmolecular weight proteins Arch Env Hlth 12, 335-344 (1966 a) Piscator, M : Proteinuria in chronic cadmium poisoning 4 Gel filtration and ion exchange chromatography of urinary proteins from cadmium workers Arch Env Hlth 12, 345-359 (1966b) Vesterberg, O : Isoelectic focusing of proteins in polyacrylamide gels Biochim Biophys Acta 257, 11-19 (1972) Vesterberg, O : Isoelectric focusing of proteins in thin layers of polyacrylamide gel Sci Tools 20, 22-29 (1973) Vesterberg, O , Nise, G : Urinary proteins studied by use of isoelectric focusing I Tubular malfunction in association with exposure to cadmium Clin Chem 19, 1179-1183 (1973) Vesterberg, O , Nise, G , Hansen, L : Urinary Proteins in Occupational Exposure to Chemicals and in Diseases J Occup Med 18, 473-476 (1976) Vesterberg, O , Hans 6n, L, Sj6sten, A : Staining of proteins after isoelectric focusing in gels by new procedures Biochem Biophys Acta 491,160-166 (1977) Vesterberg, O , Bergstr 6 m, T : Determination of Cadmium in Blood by Use of Atomic Absorption Spectroscopy with Crucibles and a Rational Procedure for Dry-Ashing Chin Chem 23, 555-559 (1977) Received September 5, 1977 /Accepted October 17, 1977
Oeulmiuonal
Int Arch Occup Environ Hlth 40, 273-282 (1977)
al
E-iro lltal
Health ©
Springer-Verlag 1977
Evaluation of Different Urinary Proteins Excreted after Occupational Cd Exposure Hansen, Lars 1 , Kjellstr 6 m, Tord 2 , and Vesterberg, Olof1 Department of Chemistry, National Board of Occupational Safety and Health, Fack, S-100 26 Stockholm, Sweden 2 Department of Community Health, University of Auckland, Auckland, New Zealand
Summary Workers in an alkaline battery factory were examined for renal effects after several years exposure to cadmium Total urinary protein was determined using a Biuret method To separate the individual proteins, isoelectric focusing was performed in flat beds of polyacrylamide gel The proteins were then stained and evaluated by densitometry The duration of employment at the factory was used as an indicator of cadmium dose. A dose-effect relationship was found between cadmium and the concentration of some of the urinary proteins The most pronounced relative increases in average concentration in relation to years of cadmium exposure were seen in some zones, e.g those containing albumin and /2-microglobulin, respectively The largest quantitative increase was observed for the zone containing albumin The results indicate that isoelectric focusing is suitable to evaluate effects of cadmium on kidneys. Key words: Cadmium
Exposure
Proteins
Isoelectric focusing
Introduction The toxic hazards connected with occupational exposure to cadmium have been recognized more and more during recent years (Friberg et al , 1974) Much concern has been focused on the extraordinary tendency for accumulation of cadmium in the kidneys with an estimated half time for elimination in the order of decades Above a certain critical concentration, which has been estimated to be about 200 ug/g of cadmium in kidney cortex, increased concentration of proteins in urine can be found This damage is mainly in the proximal tubuli (See Friberg et al , 1974 p 105 for references). Since about 15 years paper electrophoresis has been used in such studies (Piscator, 1962 a, and Kazantzis et al , 1963) The information has been increased by use of gel permeation chromatography, which has e g shown that the proteins are to a high extent of low molecular nature (Piscator 1966 a and b) One such protein is 32 -microglobulin the concentration of which may be elevated more than hundred times after high cadmium exposure over many years (Peterson et al , 1969) Also another low molecular protein, retinol-binding protein, has been proposed for evaluation of cad-
274
L Hans 6 N et al.
mium induced kidney damage (Kanai et al , 1971) To be able to take various preventive measures it is of high interest to detect a malfunction at an early stage To this end and to get more information about the effects of cadmium on the kidneys we have made a study of urinary proteins with the method of isoelectric focusing as described by Vesterberg and Nise (1973) and Vesterberg et al ( 1976) which when used in flat beds of polyacrylamide gels is one of the most high resolving analytical methods for proteins, which allows separation of also very similar proteins (Vesterberg, 1973) In this method the proteins focus at their isoelectric points This physico-chemical characteristic, which can be used for, e g characterisation and recognition of proteins can be made simply by measuring the p H In combination with suitable staining procedures (Vesterberg, 1972, 1973 ; Vesterberg et al , 1977) it is very sensitive, allowing detection of tenths of a jig of proteins Furthermore, many protein samples are run in parallel facilitating comparison and densitometric evaluation. Materials and Methods Urine samples were analysed from 55 male workers with occupational cadmium exposure in a battery factory The workers had been employed in two distinctly different exposure conditions in the assembly plant and the material plant (Kjellstr 6m et al , 1977) These 55 workers comprised all those who took part in a tuberculosis health screening on a particular day They comprised about 20 %of the presently employed cadmium-exposed workers in the factory (Kjellstr 6m et al , 1977) Exposure conditions or earlier detected cadmium effects were not considered in the selection The exposed were classified according to duration of employment in cadmium exposure in the plants and stratified into different groups as indicated in Table 1 At the collection the samples were mixed with 0 4 M phosphate buffer of pH 7 6 (1 part buffer to 10 parts of urine) Total protein was determined according to Piscator (1962 b) ,2-microglobulin was analyzed by using radioimmunoassay with a kit ,Phadebas" (Pharmacia, Uppsala, Sweden) The filtered urine samples were concentrated 20 times in collodium tubes (Membran Filter Gesellschaft, G6ttingen, Germany) with reduced pressure, before separation by isoelectric focusing Flat beds of polyacrylamide gels were used essentially as described earlier by Vesterberg (1973) with T = 3 %,C = 5 %containing sucrose 12 %(w/v), Ampholine 2 8 ml pH 3 5-10, 0 4 ml pH 9-11, 0 2 ml pH 4-6 and 0 2 ml pH 5-7 giving a final average concentration of 2 %(w/v) Samples of 100 1l of concentrated urine were soaked into rectangular (8 x 10 mm) filter paper pieces (Grycksbo, Sweden) and applied close to the cathode Focusing was performed with 400 V as initial and 1000 V as final potential and making the increase so that the wattage never exceeded 40 W The focusing time was 90 minutes. After focusing the proteins were stained with a solution of Coomassie Brilliant Blue R 250, 0 1 % in methanol 27 %mixed with trichloroacetic acid 11 %and sulfosalicylic acid 3 3 %in water (figures are final concentrations) (Vesterberg, 1972) After destaining in ethanol-water-acetic acid (26:66:8) solution the gels were placed on a glass plate and covered with a dialysis membrane and were allowed to dry (Vesterberg, 1973). Estimation of the protein content in the separated bands were then performed by densitometry in a Zeiss spectrophotometer (PMQ II) The wavelength was 600 nm and the slit 3 x 0 02 mm In order to simplify the comparison we have estimated the amount of protein with close isoelectric points by calculating the absorbance areas of the densitogram which has been divided into ,,zones" of 5 mm's length each The zones were numbered from the anode side so that albumin always was found in zone 7 A base line was drawn to eliminate the background noise. The absorbance of the urine protein band containing 2,-microglobulin was compared with the absorbance of standards For a typical result see ref Vesterberg and Nise Fig 6 This gave a semiquantitative measure of urinary 0,-microglobulin based on isoelectric focusing. The concentration of the urine was normalized according to density by using the formula published by Levine and Fahy (1945): a x 23 x 10 ' 3 10
where a = area of a zone after densitometric evaluation and 6 = density of sample
Different Urinary Proteins Excreted
275
Table 1 Description of groups of workers studied Employment duration (years) in cadmium exposure 25 Assembly plant Material plant
13
9
11
14 8
total 47 8 55
Results The concentration of total protein in urine in relation to years of occupational cadmium exposure is shown in Fig 1 There was a tendency for increasing concentration with duration of exposure Judged from the linear regression line there was on the average about 3 times increased concentration after 30-40 years exposure. A typical result of isoelectric focusing of urinary proteins is shown in Fig 2 The total number of separated proteins was large More than 25 bands were seen in cases with long exposure It was extremely difficult and time consuming to compare individual protein bands for all persons Therefore we divided the protein spectrum into 20 zones A densitometric evaluation of the stained protein zones from one person with six years of cadmium exposure is shown, in Fig 3 The staining procedure does not stain all bands with the same intensity on weight basis and even less on molar basis as would be desirable Unfortunately, there exists no such ideal procedure No strict comparison of the estimated amount of protein in one zone with that of another could be made Instead the absorbance of the stained protein in each zone was calculated and compared for evaluation on a group basis Several zones had absorbance below the detection limit and medians are therefore used in the following Fig 4 shows the median absorbance in the 11 zones where most cadmium-induced changes are seen In the zones 2, 5 and 7 the absolute increase of,absorbance with increased exposure duration seemed to be the largest Largest relative increases were seen in zones 5 and 11 These zones contained proteins of which some have earlier been mentioned in connection with cadmium damage. The zones have been shown to contain the following proteins: zone 2, orosomucoid; 5, haptoglobin, a l-antitrypsin and probably also retinol binding protein; 7, albumin and zone 11, 32 -microglobulin (to be published, Hansen and Vesterberg). The median absorbance in zones 2, 5, 7 and 11 tended to increase with exposure duration and were highest in the material plant group except for zone 2 Between the group with < 2 years exposure and > 25 years exposure there were statistically significant increases in absorbance in zones 5, 7 and 11 (Fishers exact median test gave P = 0.02, 0 0006 and 0 002 respectively) The highest relative increase of the median occurred in zone 11 (> 44 times) and zone 5 (> 40 times), whereas the increase was 4 times in zone 7 There was a tendency for a relatively sharp increase in median absorbance of zone 11 (containing 12 -microglobulin) already at 2-10 years exposure (P = 0 2) (Fig 5) However, quantitative specific analysis of urinary 2 -microglobulin did not show such an increase (Fig 6) The increase of urinary 32 -microglobulin between the group with < 2 years exposure and the group with > 25 years exposure (Fig 6) appears similar to the corresponding increase of absorbance of zone 11 (Fig 5).
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years 40 30 20 10 Fig 1 The concentration of total protein in urine (corrected to spec gravity = 1 023) as a function of duration of employment for 40 workers in the Cd-battery assembly factory (For technical reasons total protein was not analysed in all urines ) The linear regression line is indicated O
Fig 2 A photography of a gel after focusing and staining of the protein bands The samples were applied at the cathode side (lower part of the gel) A cm-scale is shown to the left The positions are numbered from left to right The following samples were applied: position 1 and 14 standard 2 protein mixture'; 2-13 and 15-23, urine samples from 7 men with different Cd exposure . 'the standard proteins are indicated by letters as follows a, albumin 1 0 Mg; b, -lactoglobulin (2 bands) 2 5 tmg; c, 32 -microglobulin 0 5 Mg; d, myoglobin (2 bands) 2 5 /Ig and e, cytochrome c 2 5 jug 2 each sample occurs in three adjacent positions: one unconcentrated the second concentrated 20 times and the third concentrated and with addition of 0 5 Mug0, -microglobulin
Different Urinary Proteins Excreted
277
Fig 3 Densitogram of urinary proteins in one sample after separation by isoelectric focusing and staining of the protein bands A photo of the gel strip is shown below with the anode to the left The absorbance scale is on the y-axis Classification into zones and a pH scale on the x-axis The albumin band is in zone number 7
We have studied the possibility to detect tubular malfunction as shown up by increased concentration of f 2-microglobulin by use of isoelectric focusing In Table 2 it can be seen that with a very high predicability it was possible by using isoelectric focusing to find those who had elevation of 12 -microglobulin above the 95-percentile of a normal group (Kjellstr 6 m, 1977). In studies by isoelectric focusing of protein patterns from persons with different diagnosis we have obtained experience allowing us to make qualification in the categories glomerular or tubular proteinuria (Vesterberg and Nise, 1973; Vesterberg et al. 1976) When the concentrations of high-, medium and low molecular proteins were elevated the patterns were called mixed glomerular-tubular An evaluation of the persons encompassed in this study is presented in Table 3 Glomerular disorders are much more common in an ordinary population than tubular disorders The opposite is the case for cadmium exposed as can be seen in Table 3 Furthermore, the prevalence of tubular patterns increased with employment duration. Discussion We found that upon increasing duration of exposure to cadmium the urinary concentration of some low molecular proteins such as 32 -microglobulin and retinol binding protein increase in a fashion related to duration of exposure Furthermore the con-
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Fig 4 The protein content estimated by the total absorbance, A, (arbitrary units) of each zone after focusing and staining of the protein bands Within each group the median value and the 75-percentile are shown for each zone
Symbols: Group a
Median A
75-percentile A
b c
V
d
V
e
*
duration of Cd-work 25 > 25
place assembly plant
material plant
number of men 13 9 11 14 8
centration in urine of some medium molecular weight proteins as orosomucoid (Mw 44 100) (zone 2), albumin (Mw 69 000) (zone 7) and al -antitrypsin (Mw 45 000) (zone 5) also increased Thus for persons with effects of cadmium on the kidneys this results in typical patterns after isoelectric focusing characterized by many bands and elevated concentrations of protein in many of the bands This is in accord with recently published observations of cadmium-induced proteinuria (Bernard et al 1976). Total protein is a very crude measure because it is known that different proteins give very different degree of contribution to the protein value when compared on molar basis Furthermore, certain proteins, mainly low molecular ones, are not properly determined by the method for total protein The increase of total protein (Fig 1) with increased employment duration was therefore less than the increase of individual zones in isoelectric focusing (Fig 4) Still more, there are many conditions that may result in increased excretion of protein in urine, which is thus of low specificity when trying to detect effects of cadmium.
279
Different Urinary Proteins Excreted log A
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Fig 5 The protein content estimated by the total absorbance of zones 2, 5, 7 and 11 The medians and quartiles (bars) in arbitrary units were plotted For group symbols see Fig 4
The definition of zones in the focusing patterns made it possible to quantify the increases The scatter of zone absorbance between individuals in the same dose group was large but a similar scatter was found for radioimmuno-assay analysis of 02 -microglobulin in the urines (Fig 6) Even in groups with long duration certain persons do not have a pathological proteinuria The prevalence of tubular proteinuria becomes a matter of where to draw the border line According to the criteria used by Kjellstrom et al (1977) for urinary 32 -microglobulin there were 9 tubular proteinurias among 14 workers in the assembly plant with > 25 years exposure (Fig 6). Eight persons in the same group had tubular proteinuria according to our qualitative assessment of the patterns (Table 3) and 11 of 14 had an absorbance in zone 11 (including 2 -microglobulin) > 2 Zones 11 and 5 showed the greatest absorbance increases with dose The analysis of such zone absorbance increases seems to have a similar sensitivity as quantitative measurement of urinary 2 -microglobulin in picking up cadmium induced tubular proteinuria This latter method has been advocated by Kjellstr 6m and Piscator (1977) for health screenings of cadmium workers. Radioimmunoassay of 12 -microglobulin in urine has the advantages over isoelectric focusing that it is quantitative at normal levels, but it requires a gamma-counter. Isoelectric focusing has the advantages that it gives information about several proteins in urine in one analysis, and that it uses much cheaper equipment for the analysis. Futhermore, about 24 samples can be run in parallel on one gel. When the kidneys are intact there is only a small loss of plasma proteins in urine amounting to about 50 mg/24 hours and normally < 100 mg/24 hours (Cannon 1974, p 431) The reason why the loss is not larger is partly that extensive reabsorption occurs, which takes place mainly in the proximal tubuli It has also been shown that the kidneys catabolize different proteins at different rates (Hardwicke et al , 1970),
280
L Hans 6N et al.
Fig 6 Urinary concentration of 2 -microglobulin measured with radioimmunoassay as a function of duration of cadmium exposure The ,,normal" 95-percentile (Kjellstr 6 m et al 1977) at 260 gg/l is indicated o = workers in the assembly plant " " " material = "
Table 2 Comparison of two methods for determination of a critical level of ,2-microglobulin. Samples from each of the 55 persons have been examined independently with Radio-immunoassay (RIA) and Isoelectric focusing (IEF) with RIA < 0,3 mg/l
total > 0,3 mg/l
< 0,3 mg/l
36
2
38
> 0,3 mg/l
3
14
17
39
16
55
with IEF
total
Different Urinary Proteins Excreted
281
Table 3 Kind of kidney effects diagnosed by the protein pattern from men working in two different cadmium plants
effects
Assembly plant number of men with different years of employment 25
Glomerular Glomerular (uncertain) Tubular Tubular (slight) Normal
13
Total
13
Material plant
> 25 1
1
3
7 1
4 1
9
8
5
2
9
11
14
8
which is very important for regulating the life span in the body of protein molecules. An increased leakage of protein does not only mean that a corresponding amount of the protein is lost from the body but this may very well also influence the statistical life span in the body of the molecules of the protein Because of the complicated mechanisms involved at loss, regulation and feed back it is not easy to say what consequences a loss of a certain amount of a protein has for the body At moderate loss per day the effects as far as we know today is rather limited However, the fact that a certain protein appears in increased concentration in urine is an indication that there is some dearrangement of the normal kidney function It is also known that when a cadmium induced malfunction of this kind is pronounced, there is also decreased capacity of the kidneys to retain amino acids, glucose and calcium (see Friberg et al , 1974, pp 103-106 for ref ) Renal tubular damage should therefore be prevented among cadmium-exposed workers and our study showed that isoelectric focusing of urinary proteins is one way of making early diagnosis To prevent too high exposure analysis of cadmium in blood and urine are also helpful as indicated by Friberg et al. 1974 pp 77-79 and Vesterberg and Bergstr 6m, 1977. Acknowledgement This work was supported by grants from The Work Environment Fund in Sweden.
References Bernard, A , Roels, H , Hubermont, G , Buchet, J P , Masson, P L , Lauwerys, R R : Characterization of the Proteinuria in Cadmium-Exposed Workers Int Arch Occup Environ Hlth 38, 19-30 (1976) Bernier, G M , Conrad, M E : Catabolism of human 2 -microglobulin by the rat kidney Am J. Physical 217, 1359-1362 (1969) Cannon, H : in Clinical Chemistry: Principles and Techniques 2nd ed , Henry R J Ed , Hagerstown, Md.: Harper and Row 1974 Friberg, L , Piscator, M , Nordberg, G F , Kjellstr 6 m, T : Cadmium in the Environment, 2nd Ed. Ohio: The Chemical Rubber Co 1974 Hardwicke, J , Cameron, J S , Harrison, J F , Hulme, B , Soothill, J F : Proteinuria, Studied by Clearances of Individual Macromolecules Proteins in Normal and Pathological Urine, pp. 111-152, Basel, New York: Karger 1970
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Kanai, M , Nomoto, S , Sasaoka, S , Naiki, M : Clinical significance of urinary excretion of retinolbinding protein in patients with Itai-Itai disease Prog Symp Chem Physiol Pathol 11, 194-199 ( 1971) Kazantzis, G , Flynn, F V , Spowage, J S, Tott, D G : Renal tubular malfunction and pulmonary emphysema in cadmium pigment workers Q J Med 32, 165-196 (1963) Kjellstr 6 m, T : Accumulation and renal effects of cadmium in man A dose-response study Diss. Karolinska Institutet, Stockholm 1977 Kjellstr 6 m, T , Evrin, P -E , Rahnster, B : Dose-response analysis of cadmium-induced tubular proteinuria Env Res (In press) Kjellstr 6 m, T , Piscator, M : Quantitative analysis of 02 -microglobulin in urine as an indicator of renal tubular damage induced by cadmium Phadedoc Diagnostic Communications, No 1, Parmacia Diagnostics AB, Uppsala, Sweden 1977 Levine, L , Fahy, J P : Evaluation of Urinary Lead Determination J Indust Hyg Tox 27, 217223 (1945) Peterson, P A , Evrin, P -E , Berggard, I : Differentiation of glomerular, tubular and normal proteinuria: Determinations of urinary excretion of , -microglobulin, albumin and total protein. J Clin Invest 48, 1189-1198 ( 1969) Piscator, M : Proteinuria in chronic cadmium poisoning 1 An electrophoretic and chemical study of urinary and serum proteins from workers with chronic cadmium poisoning Arch Env. Hlth 4, 607-621 (1962 a) Piscator, M : Proteinuria in chronic cadmium poisoning 2 The applicability of quantitative and qualitative methods of protein determination for the demonstration of cadmium proteinuria. Arch Env Hlth 5, 325-332 (1962 b) Piscator, M : Proteinuria in chronic cadmium poisoning 3 Electrophoretic and immunoelectrophoretic studies on urinary proteins from cadmium workers with special reference to the excretion of lowmolecular weight proteins Arch Env Hlth 12, 335-344 (1966 a) Piscator, M : Proteinuria in chronic cadmium poisoning 4 Gel filtration and ion exchange chromatography of urinary proteins from cadmium workers Arch Env Hlth 12, 345-359 (1966b) Vesterberg, O : Isoelectic focusing of proteins in polyacrylamide gels Biochim Biophys Acta 257, 11-19 (1972) Vesterberg, O : Isoelectric focusing of proteins in thin layers of polyacrylamide gel Sci Tools 20, 22-29 (1973) Vesterberg, O , Nise, G : Urinary proteins studied by use of isoelectric focusing I Tubular malfunction in association with exposure to cadmium Clin Chem 19, 1179-1183 (1973) Vesterberg, O , Nise, G , Hansen, L : Urinary Proteins in Occupational Exposure to Chemicals and in Diseases J Occup Med 18, 473-476 (1976) Vesterberg, O , Hans 6n, L, Sj6sten, A : Staining of proteins after isoelectric focusing in gels by new procedures Biochem Biophys Acta 491,160-166 (1977) Vesterberg, O , Bergstr 6 m, T : Determination of Cadmium in Blood by Use of Atomic Absorption Spectroscopy with Crucibles and a Rational Procedure for Dry-Ashing Chin Chem 23, 555-559 (1977) Received September 5, 1977 /Accepted October 17, 1977
