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RENAL EXCRETION OF INDUSTRIAL CHEMICALS*
I. Ub INAET-Lb AD CONCENTRATION a n d Bl o o d -Le a d Cl e a b a n c e
Sh b b ma n S, Pin t o , Hb b v e y B. El k in s a n d Jo h n P. Eg , Jb . Massachusetts Division of Occupational Hygiene, Department of Labor and Industries,
Boston, Massachusetts
OXIC chemicals when absorbed various conditions. Whether this in
Tby the body eventually act on dicates a change in the chemical individual cells and produce nature of the lead-bearing compound
changes which are designated as hthaes not been determined.
particular pathological changes pro
In spite of the doubts raised by our
duced by that chemical. Prom the lack of knowledge of the exact form
concentration in the blood of the which lead takes when present in the
chemical under investigation we may blood stream, we feel that blood lead
infer the approximate concentration values are of greater significance than
to which the tissue cells are subjected, are the values for lead concentration
but there is no way of studying the in excreta. Prom a practical indus
concentration and effect of that sub trial viewpoint it is much easier to get
stance in the individual cell. There a sample of urine from a workman than
fore, we feel that the blood level of a it is to get a sample of blood. There
substance is of great significance since fore, if a urinary test can be devised
it is the closest approach we can make which will show a definite relationship
toward learning the concentration of between blood-lead and urinary-lead
that substance to which the cells are levels, a great deal more significance
subjected. In the case of lead even can be placed on the urinary lead test,
this approach is open to some doubt, than is possible at present.
since by our methods of analysis we Since only 10% of the total lead
are not able to determine whether the excreted by the body is eliminated
lead as determined in the blood is in a through the kidneys, the question can
form that might affect the fixed cells very well be raised as to how significant
of the tissues.
urinary lead determinations may be
Smith, Rathwell, and Mareil (11) (15). It has been shown by practical
have studied the distribution of lead experience based on statistical analysis
between serum, cell, and fibrin frac of 'large amounts of data that some
tions of blood in acute and chronic idea of the amount of lead in the blood
lead poisonings. These studies indi can be determined by this means.
cate that there are changes in the lead However, numerous examples can
content of the several fractions under easily be found where the apparent
* Received for publication Marcia 1,1941. lead concentration in the urine did not
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314 JOURNAL OF INDUSTRIAL .HYGIENE AND TOXICOLOGY [vol. 23, no
coincide with the clinical picture The sample is collected in. a ele:
which the patient presented (12, 13). flask to which 1 mg. of heparin
It is the purpose of this paper to added for each 5 cc. of blood (G).
develop formulas by which a closer The volume of blood is measure
correlation between blood-lead and out into a Yycor evaporating dish an
urinary-lead concentrations can be then placed in a muffle furnace. Th
shown.
temperature is gradually brought t
An a l y t ic a l Me t h o d s
650C. and maintained at this tempera ture for half an hour or until all organi
DETERMINATION OP LEA'D IN URINE materials are destroyed.
To 50 ee. of the sample, 50 cc. of glass-distilled water is `added. After bringing the pH to about 4.5 (brom cresol green) with acetic acid, 5 ce. of saturated ammonium oxalate is added. If necessary the pH is readjusted and 0.5 cc. of 10% calcium chloride is added while swilling. After transfer ring to long test tubes and allowing to
The dish is removed from the fur .naee and 20 cc. of 10% citric acid ant enough 6 normal hydrochloric acid art added to obtain solution on heating. After adding 3 drops of a 70% solution of hydroxylamine hydrochloride, the solution is transferred to a separatory, funnel, extracted, and titrated, as in the determination of lead in urine.
stand overnight, the supernatant liquid is siphoned off. The precipitate is transferred to smaller tubes with glass-distilled water, centrifuged,
The Estimation of Average UrinaryLead Concentrationfrom Spot Samples
washed, and oxidized by the Ross- In a fundamental study of kidney
Lucas method (14).
function. Van Slylce and others have
The solution is transferred to a shown that, at least in the case of urea,
separatory funnel, 5 ce. of 10% citric acid and 0.5 cc. of 25% sodium cyanide are added. After bringing the solu tion to a pH of 8.5 (phenol red), the lead is extracted with dithizone. After
elimination of a substance from the blood through the kidneys into the urine can be expressed by either of the two formulas (1) or (2) depending on the rate of urine formation per minute.
complete extraction, the combined extracts are stripped with 10 cc. of 1 (1) C - ^ X VTor (2)C = |XF
N hydrochloric acid and after washing
the aqueous layer with chloroform, Formula (1) is used when less than
0.5 cc. of 25% cyanide is added to the 2 cc. of urine are excreted per minute;
partially neutralized solution. The formula (2) when more than 2 cc. per
pH is brought to 8.5 (phenol red) and minute are formed. (C) is the clear
the lead is titrated by extraction with ance of the substance under study, (U)
' successive portions of dithizone (equiv is urinary concentration in milligrams
alent to approximately 3y lead per cc.). per cent, (B) is blood concentration in
DETERMINATION OP LEAD IN BLOOD
milligrams per cent, and (V) is the volume of urine excreted per minute
Syringes and needles are treated in (4, 5).
the manner suggested by Willoughby The term "clearance" has been de
and Wilkins (8).
fined as designating the volume of
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RENAL EXCRETION OP CHEMICALS. I.
315
blood which 1 minute's excretion , of for lead content, and the period of
.urine suffices to clear of the substance collection varied from 12 to 44 hours.
under. ..investigation. Since all the The ordinary urinalyses (including
blood flowing through the kidneys is microscopic examination) were done
only partially cleared during its pas and no subjects wele used in these
sage through these organs, the volume experiments if it was thought the kid
expressed is virtual rather than real. neys were abnormal. When the
"Clearance" may also be defined as results of lead excretion of the indi
the minimum- volume of blood re vidual samples during any test period
quired to furnish the quantity of are expressed as milligrams per liter
substance excreted in the urine in 1 (we prefer gamma per 100 cc. of mine)
minute's time (2).
quite a variation from the average lead
An attempt was first made to apply content of the entire pooled sample is
this formula to lead excretion in the to be found. Barnes has shown this
urine. Kehoe has shown that during a to vary from 82% to --40% (10).
24 hour period the blood-lead is Our results verified these figures.
constant if no strenuous attempts are Since we knew the time periods repre
made to change it (7). Smith, Rath- sented by each individual sample of
mell and Mardl have shown that any test period, we were able to
nourishment and violent exercise do calculate the lead concentration of
not affect the blood-lead level of nor anyindividual sample by the expres
mal individuals (11). Therefore we sion U V in which U = urinary
based the first of our experiments on lead concentration in y/100 cc. and
the premise that the blood-lead levels V = volume of urine excreted per
would remain fairly constant for at minute. As stated above, if the
least 24 hours. If we may assume that volume per minute was less than 2 cc.
the rate of excretion of lead through per minute, the square root formula
the kidneys is constant, (this assump was used. If greater than 2 cc. per
tion was made only to test .the validity minute, the figure was used directly
of the rest of the hypothesis, as no ' as in the expression U X V. Prom a
proof of such an assumption has been study of eight series of samples it is
shown as yet), then in the formula seen that calculation by the formula
C=
V, the expression U y/~Y yielded less erratic results than expres
S3 sion of results simply as gamma per
should also be a constant. If any two 100 cc. In table 1 are shown the
components of such an equation are scattering about the mean when results
fixed, the third part must of necessity are expressed by the two methods.
he constant also.
In almost every instance it will be seen
Tire urine in each case was collected that the standard deviation of the
from individuals who were recovering calculated concentration is smaller
from lead poisoning. The acute epi- than is that of concentration expressed
' ulc had occurred from 2 weeks to 3 as "gamma per 100 cc, The standard
months prior to the urine sample deviation expressed as percentage of
reilcction. Each individual sample the mean is also shown. Barnes (10)
during a test period was collected showed very clearly that the expression
' 'Ornately and analyzed individually of results of lead excretion as gamma
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316 JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [vol. ,,0
per hour was less subject to variation standard deviation was 1G.S% of t),.j than expression as milligrams per liter, mean, in Subject B, 18.3%, and j;i In his 3 subjects having excessive lead Subject C, 29%. The lead conccutra-
TABLE 1
'
Ex t e n t o p Sc a t t e r o p Ur in ar y -Le a d Co n c e n t r a t io n s a b o u t t h e Me a n w h e n De t e r min e d b y Sp o t Sa mpl e s
The standard deviations from the mean when results are expressed as -y/100 cc., caloulai cG by the formula U-\/V, or determined as y/hr. are shown.. The standard deviation is also shown as percentage of the mean.
285-290 291-295 320-326 378-384 397-400 . 423429 430-435 436441
CALCULATING RESULTS AS
y/100 CC*
Std. dev.
Std. dev. expressed as %
of mean
7.4 12.8 2.8 8.66 7.87 9.25 ' 6.27 6.9
63.0 37.5 12.3 28.8 .29.0 29.0 29.0 37.4
CALCULATING RESULTS BY
WVFORMULA
Btd. dev.
Std. dev. expressed as %
of mean
3.85 8.97 2.42
3.76 6.56 8.95 6.04 4.9
42.0 36.0 8.4 17.0 25.0 '34.0 31.0 23.8
CALCULATING RESULTS AS a s y/aB.
Std. dev.
Std. dev. expressed a* %
of mean
1.44
4.23 3.7 4.2 5.5 6.95 7.37 4.53
3S.O 38.0 33.0 36.0 41.0 51.0 64.0 30.0
TABLE 2
Ex t r e me s o p Ur in a r y -Le a d Co n c en t r a t io n De v ia t io n s p r o m t h e Me a n Wh en Ex p r e s s e d as y /100 c c . a n d Wh e n Ca l c u l a t e d b y t h e Fo r mu l a.-y/lGO cc. X-\/V
The ratio between the two means is also shown
BASSES NO .
SAMS
LBAD CONCENTRATION OP INDIVIDUAL
y/100SAMPLES EXPRESSED AS
CC.
PERIOD OF
COLLEC TIONS
Highest
Lowest
Mean. (actual)
CALCULATED LEAD CONTENT
y/100 cc. XVT
Highest in period y/IGO cc.
Lowest in period *y/10& cc.
Mesa (cal culated)
RATIO
ACTUAL MEAN
CALCULATED MEAN
hours
285-290 21.5
25.0
5.0 11.75 15.5
4.6 9.23
1.27
291-295 39.0
51.0
21.0 34.2
39.2
14.0 24.8
1.38
.1
320-326 28.5
27.0
18.0 22.8
23.6
17.2 20.4
1.12
378-384 44.0
40.0
16.0 31.2
26.1
17.0 22.2
1.40
397400 12.0
52.0
24.2 27.2
30.6
18.0 25.6
1.06
423429 34.0
44.4
21.0 31.8
44.4
13.5 27.0
1.18
430435 33.3
27.8
10.6 21.8
26.6
8.6 19.4
1.12
Mean...........................................................................................................................
1.22
Standard deviation.
0.12 *
11.
exposure, we have calculated the tion of the samples shown in table 1 standard deviation of the gamma per has also been calculated as gamma of hour exeretion. In Subject A the lead excreted per hour. The standard
>ycg.*^ 'j
vKvnftw-
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RENAL EXCRETION OF CHEMICALS. I.
317
deviation of each series has been deter mined and also expressed as percentage ,,f Ihe mean. From this comparison U can be seen that calculating results tiv the formula UVV shows less scatter
ume of urine excreted was less than 1 cc. per minute. The individuals whose urines were examined were carrying on normal light activities, and it has been our general experience that work-
V/IOOCC. 50"
)
i'. fV- ' .
IV- . : :
8AM 6PM 4AM 2PM
4AM
1-30-41
1-31-41 -
TIME - HOURS
Fig . 1. Graphic representation of the variations found when expressing results of
urinaiy lead concentration" y/100 ec. and by calculation from the formula -y/100 ce. XVV. The two means have been superimposed.
TABLE 3 De t a il e d Da t a o n In d iv id u a l Sa mp l e s Co n s t it u t in g On e Co l l e c t io n Pe r io d
SAMPLE NO..
VOL.
Vv o l ./min .
7/100 CO.
DEVIATION FBOU UflAK
uvf
DEVIATION FSOMUBAM
CC.
378 345 0.98 . 26
-5.2
25.4
+2.6
379
233
0.985
22
-9.2
21.6 .
-1.2
880
160
0.56
40
+8.8
22.4
-0.4
381
238
0.65
40
+8.8
26.0
+3.2
382
167
0.87
30
-1.2
26.1
+3.3
383
135
1.06
16
-15.2
17.0
-5.8
384
139
0.46 *
37
+5.8
17.0
-5.8
Mean____
31.2 22.8
about the mean than does calculating men engaged in their usual tasks ex results as either 7/IOO cc. or 7/hr. Crete on the average less than 1 cc. of
Table 2 shows the extremes of urine per minute. Consequently, lead urinary-lead concentrations for each concentration _values calculated as test period when expressed as 7/IOO .7/IOO cc. XVV will usually be lower cc., or 7/IOO cc. XVV. It so happens than the concentration when exthat in all the series studied, the vol- pressed as 7/IOO ec. The ratio Actual
TV
IV
5
w DUP050312574
318 . JOURNAL OP INDUSTRIAL HYGIENE AND TOXICOLOGY [ml. as, no,
Mean: Calculated Mean was found to be 1.22 0.12. This must be borne in mind in the interpretation of results.
In figure 1 a typical example of the
two methods of expressing urinarylead concentration is shown. The mean of the calculated values has been superimposed on the mean, of the plain gamma per 100 cc. figures in order to Bhow the extent of variation about both means. Table 3 gives in detail the data from which figure 1 was constructed.
Calculation of lead concentration by the formula does not invariably give a result closer to the mean of the total sample than does expression of results as simply y/100 ee. In our total of 47 individual samples studied, the calculation of results by the formula was closer to the mean in 32 instances than was the. expression of results as y/100 cc. Expressing re sults of the individual samples as y/100 cc. was closer to the mean of the total sample in 15 instances.
Blood-lead Clearance
An attempt was then made to apply the accepted formulas for blood clear ance to the excretion of lead. Again it should be stated that this was done on an empirical bads, and the calcula tions used because the formulas seemed to apply to the problem at hand. The
formula C =
was used in which
X)
the symbols have the same significance
as above.
There is a constant relationship
between amount of kidney tissue and
body surface area. Therefore, in order
to make all clearance figures com
parable they should be corrected to the
figure for an "ideal man." The ideal
man has a body surface area of 1.73 sq. m. From the height and weight of an individual the surface area may be obtained from a table such as is shown by Osgood (9). This correction factor
1.73 ....................... a--ct--ua7l--s-u--r;f-a-c--e---a-r-e--a is determined ,' and the urine volume per minute mul tiplied by it before the square root of that figure is taken. In our series the effect of not making this correction was to raise the calculated clearance value by 6.5% in the case of a man 6 ft. 2 in. tall and weighing 205 lbs. At the other extreme was an individual who weighed 130 lbs. and was 5 ft. 2 inches tall. Here the effect of not making the correction was to lower the clear ance value by 2.8%. All other cor rections were less than these two and raise the question as to whether it is worthwhile making such calculations. In the interest of exactitude we felt the calculation was justified.
All subjects studied for clearance values were exposed to lead in battery companies and the degree of exposure was high. This accounts for the high blood-lead values observed, and should not be considered as being due to faulty blood-lead analyses. We feel fortunate in obtaining as varied bloodlead levels as we did, since this allows us to study clearance figures over quite a range of blood-lead concentrations. Table 4 shows the fundamental data from which some blood clearance values were calculated. Figure 2 shows the relationship of blood-lead levels and clearance values. From this graph it is seen that there is a slight tendency for the clearance values to fall as the blood-lead level is elevated over about 80 y%. If the greatest part of the lead in the urine
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RENAL EXCRETION OP CHEMICALS. I.
319
is excreted through the kidney tubules that, substance, the clearance value such a result is to be expected. The falls. This does not mean that the
TABLE 4 . Ty p ic a l , Da t a f r o m Wh ic h So me Bl o o d Cl e a r an c e Fig u r e s We r e De r iv e d
401 402 403 404 405 406 407 403 409 410 411 412 413 414
UnXXOS VOLUME
TIME OF UH1NB SAMPLE
DBOTART LEAD CONCENTB-ATIOK
BLOOD LEAD TION
1.73 SURFACE AREA
CLEARANCE
,, uVv* CB
69
141 102 22
75 75
263
293
73
68
134 138
92 39
min.
185
ISO
145' 150 160 160 135
210
190 170 165 185
ISO
130
yflOO cc.
52 40
34
42 34 42 15 24 33 37
20
17 45 23
y/iOO CO.
84
23 67 60 56 77
95 60 91
101
74 56
101
105
1.09 0.955 0.38 1.05 0.99 0.98 0.89 0.91
1.11
0.93 0.955 0.94 0.95 0.805
0.33
0.12
0.40
0.28 0.42 0.37
0.21
0.45
0.24
0.22 0.24
0.25 0.34
0.16
Mean........
0.292
Standard deviation
0.102
a of mean
0.027
BLOOD-LEAD (GAMMA PER 100 CC.) Fig . 2. Relationship between blood-lead concentrations and blood clearance rates
tubular excretory mechanism has an upper limit (1) and as the blood level of any substance is elevated beyond the capacity of the tubules to excrete
actual amount of the substance ex creted is any less, however.
The average clearance, figure of all samples determined was 0.284 0.103.
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320 . JOURNAL OF INDUSTRIAL HYGIENE AND TOXICOLOGY [rob S3, no. 7
In. other words, even in the ideal man lead'level estimated to be 80 7/IOO ce.
the blood-lead clearance value is a would indicate that the individual so
range, more property speaking, than affected was unduly exposed to lead
a constant. More data should be ac and precautions should be taken
quired from patients especially with against more exposure.
higher blood-lead levels than we have studied in order to study the clearance
Su mma r y a n d Co n c l u s io n s
values over a greater blood-lead range. 1. In calculating the lead concen
tration of urine, the formula U\/V
Relationship Between Blood-Lead Levels should be used when the volume of
and Urinary-Lead Concentration
urine excreted is less than 2 cc. per
From Tabic 4 and additional data minute. TJ = urinary lead concen
the average clearance value was found tration in gamma per 100 cc., V =
to be 0.28. If this average value for volume of urine excreted per minute.
(C) is used as a constant in the for- 2. The concentration of lead in
mula C
it should be possible
urine has been compared when results are expressed as 7/IOO ec., y/100 cc.
to estimate the blood-lead concentra XW, and 7/hr. Of these three
tion from the urinary lead concentra methods the use of the formula 7/IOO
tion, providing the period of time ce. XW showed less scatter about
during which the urine was collected, the mean than did the other two
is known. This would be expressed by methods.
the formula B _ UVY C'
3. The 'clearance of blood-lead through the kidneys is expressed by
Since (C) is not exactly a constant, the blood-lead level as determined by
the formula C = B
or C = JtJ
this procedure is just an approximar Blood lead values are expressed as
tion. However, it is a useful approxi gamma per 100 ce. of blood.
mation since it serves to depict the 4. The clearance for lead has been,
approximate concentration of lead to determined to be 0.284 0.103.
which the body tissues of a given 5. It is possible to determine ap
individual are subjected. In the range proximately the blood-lead level by
of blood-fead values below 100 7/IOO ce. of blood such figures are useful
the formula (1) B --
.
U>o
from a clinical point of view. When
the blood-lead level is much higher We wish to express our gratitudeto
than 100 7/IOO cc. of blood, definite Dr. Joseph C. Aub for his continued
symptoms of lead poisoning appear interest and helpful criticisms in con
(3). However, as an example, a blood- nection with this study.
BIBLIOGRAPHY
1. Sh an n o n , J. A.: Renal tubular excre tion. Physiol. Rev., IS, 63-83 (1939).
2. Smit h , H. W.: The physiology of the kidney. Oxford University Press, New York, 1937.
3. To mps et t , S. L., a n d An d er s o n , A. B.: Lead poisoning: lead content of the
blood and excreta. Lancet, 1, 559631 (1939). 4. Pe t e r s , J. P., a n d Va n Sl y k e, D. D.:
v-1,".2171 DUP050312577
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RENAL EXCRETION OF CHEMICALS. I.
321
Quantitative clinical chemistry, Vol. I. Interpretations. Williams & Wilkins Co., Baltimore, 1937 {p. 345). 5. Vas t Sl y x e , D. IJ., St il l ma n E., Mo l l e r , E., Eh r ic h , W., Mc In t o s h , J. F., Lb it er , L., Mac Ka y , E. M., Ha n n o n , R. R., Mo o s e, N. S., a n d Jo h n s t o n , C.: Observations on the course of the different types of Bright's disease, and on the resultant changes in renal anatomy. Medicine, 9, 257 (1930). 6. Ay l w ak d , F. X., Ma in iy a h in g , R. S., a n d Wil k in s o n , J. F.: Effects of some preservatives on stored blood. Lan cet, 1, 685-687 (1940). 7. Ke h o b, R. A., Ch o l a k , J., Hu b b a r d , D. M., Ba mea c h , K., Mc Na b y , R. R., a n d St o r y , R. V.: Experimental studies on the ingestion of lead com pounds. Th is J., 22, 381-400 (1940). S. Wil l o u g h b y , C. E., a n d Wil k in s , E. S.: The lead content of human blood. J. Biol. Chem., Wh 639-657 (1938). 9. Os g o o d , E. E.: A textbook of labora tory diagnosis. P. Blakiston's Son & Co., Inc., Philadelphia, 1935 (pp. 326,
386). 10. Bar n es , E. C.: Possibilities of control
of lead exposure by examining less
than 24 hour urine samples. Th is J., 21, 464-468 (1939). 11. Smit h , F. L., Ra t h me l l , T. K., a n d Ma r c il , G. B.: The early diagnosis of acute and latent plumbism. Am. J. Clin. Path., 8, 471-514 (1938). . 12. Dr bes s en , W. C.: Report of survey of lead hazard in the storage battery industry. National Battery Manu facturers Association. Report of in dustrial hygiene sessions of the 15th annual convention, October 24-25, 1939, (pp. 19-34). National Battery Manufacturers Association, Inc., Akron, Ohio, 1940. 13. Keh o b, R. A.: Discussion of recent studies with summary of analytical results correlated with chemical data and suggested methods of plant con trol. Ibid., (pp. 51-59). 14. Ross, J. R., an d Lu c as , C. C.: A new method for the determination of minute amounts of lead in mine. J.
Biol. Chern., Ill, 285-297 (1935). 15. Au b, J. C., Fa ik b a l l , L. T,, Min o t ,
A. S., a n d Re z n ix o f f , P.: Lead
poisoning. Williams & Wilkins Co.,
Baltimore, 1926, (p. 122).
I
(
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