Increased urinary beta 2-microglobulin in cadmium exposure: dose-effect relationship and biological significance of beta 2-microglobulin.

A study on the general population from both cadmium-polluted and control areas and on cadmium alloy workers indicates that beta 2-MG in urine is very closely correlated with aging, but it indicates an association with cadmium exposure. However, the age factor is stronger than cadmium exposre in both polluted and control areas among persons without clinical proteinuria. On the other hand, cadmium exposure is most likely correlated with beta 2-MG even in nonpolluted areas. Thus it seems that there is no noneffect level of cadmium dose in affecting the elevation of beta 2-MG in urine. beta 2-MG in serum indicated a very close correlation with cadmium in blood among cadmium alloy workers. This may suggest that an increase of beta 2-MG in both blood and urine in an early stage of cadmium exposure is caused by the increased level of beta 2-MG in blood, which may be a result of stimulation due to cadmium, but not necessarily by the clinical dysfunction of reabsorption of beta 2-MG in the renal.


Introduction
Since itai-itai disease began to be suspected as being related to high cadmium exposure via food, particularly rice, more than ten areas in Japan have been officially designated as being cadmiumpolluted. In these areas, some rice samples were found to contain more than 1.0 ppm cadmium. The inhabitants have undergone health examinations conducted by the national or local authorities over a period of several years. The health examinations included qualitative and quantitative determinations of protein in urine and, recently, of 82-microglobulin (J32-MG). The results as reported in numerous studies have been summarized and reviewed in two monographs (1,2). A higher prevalence of increased f82-microglobulin in urine among the inhabitants in these polluted areas has been reported. However, it is known that increased 82-microglobulin in urine is also related to the aging process (3,4). Some studies also discuss the dose-response relationship between f82-microglobulin in urine and cadmium exposure in the general environment (3,5), but in these studies, the dose is not very accurate in quantitative terms because of the length of exposure which covers a period of decades. During this period cadmium concentrations in rice fluctuated greatly; furthermore, the age factor also influences the dose-response relationship between cadmium exposure and 132microglobulinuria. For these reasons, there has been considerable discussion on the dose-response relationship as well as the biological significance of f32-microglobulinuria.
The dose-response relationship between cadmium concentration in the air of the working environment and proteinuria in cadmium workers has been discussed by various investigators (1,2,6). However, the dose-response relationship between cadmium exposure and f82-microglobulin in cadmium-exposed workers is not well documented.

February 1979
The relationship between 82-microglobulinuria and renal function, specifically the reabsorption in the renal tubuli, has also not been elucidated.
In this paper, cadmium concentration in feces or the total daily excretion of cadmium in feces was used as an indicator of dose to examine the doseeffect relationship between cadmium exposure and 32-microglobulin. In examining this relationship, partial correlation coefficients were calculated in order to investigate the influence of age on this dose-effect relationship.
In the second part of this paper, the increase of ,82-microglobulin in serum of cadmium workers was investigated in relation to increase of cadmium concentration in blood. The relationship between p82microglobulin or cadmium in urine and renal reabsorption function was also examined.

Materials and Methods
Three cadmium-polluted areas and three control areas in Japan were selected for the first part of this study. A total of 216 men, 50 to 70 years of age, who are clinically healthy (in particular, without proteinuria or glucosuria) were the subjects for the study, with about 30 to 40 men from each area. The total daily feces and spot urine samples were collected. Cadmium in feces was analyzed by atomic absorption spectrometry after wet ashing and DDTC-MIBK extraction. Cadmium in urine was measured directly by flameless atomic absorption spectrometry. f82-Microglobulin in urine was measured by the radioimmunoassay method.
For the second part of the study, 51 cadmium workers were selected from the rosters of two silver cadmium alloy factories. Cadmium concentration and 82-microglobulin in urine were measured, the former by atomic absorption spectrometry after DDTC-MIBK extraction and the latter by radioimmunoassay. In one factory, 18 workers were subjects not only for determinations of cadmium in urine, but also cadmium in blood. %TRP and %TR,82-MG measurement was possible in only 15 workers.    mium in feces was 149 ,pg in the polluted area and about 102 ,tg in the control area; thus the difference is in fact not very great. Cadmium concentration in feces (CdF) in polluted areas ( Fig. 1) is consistently higher than in the control areas. However, one control area indicated a high CdF with a large standard deviation. Figure 2 shows the daily cadmium excretion in feces; the general trends are similar to those of Figure 1. In one area, the difference between the polluted and control area in Ishikawa Prefecture is very small. The polluted area in Nagasaki Prefecture indicated highest level for cadmium excretion in feces. Cadmium concentration in urine ( Fig. 3) also reached higher levels in polluted areas. However, the control area in Ishikawa Prefecture indicated almost the same level as the polluted area in Akita Prefecture. Thus from fecal and urine data, it may be concluded that in the control area in Ishikawa Prefecture, cadmium exposure is somewhat higher than the control areas in other prefectures.   higher 82-MG than the control area in Nagasaki Prefecture. This again suggests a higher cadmium exposure in that control area. As shown in Figure 5 (t32-MG concentration in urine by age and area), f32-MG increases by age in both polluted and control areas. ,82-MG in urine by daily cadmium excretion and by age is presented in Figure 6. The subjects from the polluted and control areas were combined. The three regression lines were drawn according to grade of cadmium exposure. From 0 to 40 ,ug/day cadmium excretion in feces is noted by a circle; 60 to 120 ,ug/day is indicated by a circle with a dot in the center, and 150 tug/day and above is indicated by a black circle. All three regression lines increase with age, but the line for 150 ,ug/day and over indicates the highest 82-MG. However, there is not much difference between the other two regression lines, which also indicate a tendency toward a higher ,82-MG with age and higher cadmium exposure. The correlation between,82-MG and cadmium in urine was significant, as shown in Figure 7. However, the correlation coefficient is somewhat lower than that between ,82-MG and CdF.    Cadmium in Blood in Cadmium Workers Figure 8 indicates an association between ,82-MG in serum and cadmium concentration in blood of 18 cadmium workers from a silver cadmium alloy factory. As shown in Figure 8, there is a high significant correlation (r = 0.76) between the two factors.

Excretion of Cadmium
This suggests that f82-MG in blood increases with increased cadmium in blood. Figure 9, includes four plots, showing the association between %TRP, %TR,82-MG, cadmium exposure, and cadmium concentration in urine of 15 workers. As shown in these figures, renal tubular function, represented by %TRP or %TRp2-MG, is not lowered by increased cadmium exposure in this case. The highest cadmium excretion in urine, however, was only 25 ,ug/l.

Discussion
Over the years, a number of papers have been published on proteinuria among cadmium workers and it has been well established as an early sign of chronic cadmium poisoning (1,2). However, the quantitative determination of 82-MG, which is the main component in proteinuria due to cadmium poisoning in man, has not been well documented, especially in terms of dose-response relationships. Some reports on inhabitants from cadmiumpolluted areas in Japan indicated a clear-cut doseeffect relationship between, for example, years of residence in a polluted area and increased excretion of 82-MG (3,5). However, some other reports have failed to observe a higher rate of increased ,82-MG in cadmium-polluted areas compared to control areas (2). Furthermore, some reports (3,4) reported a close association between increase of 82-MG and age.
In the reports published up to the present, there are two problems: one is the need for more exact determination of dose or exposure than length of years of residence in a polluted area; the other is to separate the two factors of cadmium exposure and age by more sophisticated statistical analysis. The first part of this study was performed to help solve these problems.
Increase of 82-MG in urine at an early stage of cadmium exposure has been demonstrated to be more sensitive than proteinuria by sulfosalicylic acid (7). However, the clinical significance of increased ,82-MG in urine at an early stage of exposure remains a question, specifically in relation to clinical renal tubular dysfunction. The second part of this paper is concerned with the mechanism of the increase of 82-MG before the development of clinical renal tubular dysfunction.
In the first part of this study, cadmium concen-tration or daily cadmium excretion of feces was used as the indicator of cadmium exposure in polluted areas where inhabitants are exposed to excess cadmium via food, particularly rice. It would seem that cadmium in feces could reflect the present condition of cadmium exposure via food. However, because of the control measures against cadmium pollution and excess intake of cadmium taken by the government, cadmium in feces measured in the present study may not necessarily represent the grade of past exposure. Further, since feces samples were collected over 24-hr periods, they may not necessarily represent average cadmium excretion over a long period in an individual. Nevertheless, it can be safely stated that daily excretion of cadmium is the best direct indicator of exposure in practical terms.
In fact, the present study did reveal an association between cadmium in feces and ,2-MG in urine.
But at the same time, there was an even higher partial correlation coefficient between age and 82-MG when cadmium in feces was fixed. This was even more clear in the control areas. In the control areas, however, an almost significant correlation between cadmium in feces and 82-MG was observed when age was fixed. These findings clearly indicate that both cadmium in feces and age are closely associated with /32-MG in a population without quantitative proteinuria. In such a population, age is more strongly associated with,82-MG than cadmium exposure. However, the grade of exposure seemed to be associated with, 32-MG increase even among a population with a "normal" exposure to cadmium via food. This may well suggest that there is no noneffect level of cadmium exposure for increase of pl2-MG. It is also important to point out, based on the findings in this study, that in a study on f32-MG on the general population, the age factor should not be ignored. For instance, when length of residence is used as an index of exposure, the age factor should be fixed in statistical analysis.
In the second part of this study, the investigation of cadmium alloy workers, a high association was observed between cadmium in blood and ,82-MG in serum. This may suggest that cadmium is a direct stimulant for the production of f82-MG in blood. In fact, Harada (8) recently reported a similar result. In the same study, Harada also observed an increase of (82-MG in urine only three months after exposure to cadmium among cadmium workers. Furthermore, no associations between the grade of exposure to cadmium or cadmium concentration in urine, up to 25 ,ug/l. cadmium, and %TRP or %TRf32-MG were recognized among cadmium alloy workers. This indicates that these workers do not suffer clinical dysfunction of renal tubular reab-Environmental Health Perspectives sorption, in spite of increased,82-MG in urine.
According to these findings, it is difficult to assume the development of clinical renal tubular dysfunction which leads to an elevated 182-MG in urine at an early stage of cadmium exposure. Thus it seems that an increase of 82-MG at an early stage of exposure is caused by a mechanism other than renal tubular dysfunction, most likely increased ,82-MG in blood, which results in an increase of /82-MG in urine.

Conclusion
According to the present study on the general population from both cadmium-polluted and control areas and on cadmium alloy workers, the following conclusions were drawn.
,82-MG in urine is very closely correlated with aging, but it indicates an association with cadmium exposure. However, the age factor is stronger than cadmium exposure in both polluted and control areas among persons without clinical proteinuria. On the other hand, cadmium exposure is most likely correlated with 82-MG even in nonpolluted areas. Thus it seems that there is no noneffect level of cadmium dose in affecting the elevation of 82-MG in urine.
,82-MG in serum indicated a very close correlation with cadmium in blood among cadmium alloy workers. This may suggest that an increase of 82-MG in both blood and urine in an early stage of cadmium exposure is caused by the increased level of ,82-MG in blood, which may be a result of stimulation due to cadmium, but not necessarily by the clinical dysfunction of reabsorption of /82-MG in the renal tubules.