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ABSTRACT
A COMPARATIVE STUDY OF LABORATORY

METHODS FOR DETECTING LEAD TOXICOSIS

BY

Norman J. Gatzemeyer.

Blood and.tissues from lead poisoned rabbits were utilized to compare
various laboratory tests and procedures routinely used as aids in.the
diagnosis of lead toxicosis. The 2 most common methods to determine lead
exposure in.the living animal include chemical analysis of the blood for

_ lead concentration and examination of stained blood smears for immature,
abnormal erythrocytes .

Good statistical correlation was noted between blood lead levels and
lead dosage. This correlation was determined by 2 dithizone lead methods.
The trichloroacetic acid method,.as suggested by Hammond et a1. (1956) was
the more accurate, simpler and faster test.

Basophilic stippling and polychromatophilic staining of erythrocytes
in Wright's-stained smears increased significantly following ingestion
of lead. Animals receiving lead had a wide range in the number of.
stippled cells counted. This would limit its use as a single diagnostic
tool. There was a consistent increase in the number of polychromatophilic
erythrocytes; therefore, this could be considered a more reliable test.

The gravimetric lead-chromate method for tissue lead levels had

little correlation between its results and known amounts of lead ingested.

Norman J . Gat zemeyer
Examination of special stained tissue sections, sodium rhodizonate,
Ziehl-Neelsen acid-fast stain for kidney inclusion bodies, and bone sec-
tions treated with hydrogen sulfide failed to aid in identifying lead

poisoned animals.

A COMPARATIVE STUDY OF LABORATORY
METHODS FOR DETECTING LEAD TOXIOOSIS
3?

yfl
Norman J? Gatzemeyer

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1971

Dedicated to
Mary

and the family

11

ACKNOWLEDGEMENTS

This study was made possible by the encouragement and financial
assistance of the Michigan Department of Agriculture. Great apprecia~
tion and thanks are accorded to them, especially to Director B. Dale
Ball and Dr. George Whitehead.

I am extremely grateful to Dr. K. K. Keahey, my major professor,
for his advice, counsel, and guidance in conducting the project.

I wish to express my indebtedness to Dr. C. C. Merrill, Chairman,
and the entire staff of the Department of Pathology for providing the
opportunity and encouragement to pursue this course of study.

The author wishes to thank Mr. Arlo Pickens, Michigan Department
of Agriculture chemist and toxicologist, for his assistance in determin-
ing the blood lead levels.

Appreciation is extended to Dr. Delbert L. Whitenack-for his-aid in
analyzing the statistical material.

Special thanks go to my wife Mary, who has been so understanding

and helpful to me throughout my years as a student.

iii

INTRODUCTION . . . . . .

OBJECTIVES O O O O O O 0

LITERATURE REVIEW.

Historical. . . .

TABLE OF

Industrial Lead Toxicosis .

Infants and Lead Toxicosis.

Animals and Lead Toxicosis.

Lead Determinations . . . .

MATERIALS AND METHODS. .

Experimental Animals. . . .

Animal Care . . .

Source and Method

Sample Collection .

Experiments .
RESULTS. . .
Trial 1 . . .
Trial 2 . . . . .
Trial 3 . . . . .
Trial 4 . . .
Trial 5 . . . . .
Trial 6 . . . . .

Trial 7 . . . . .

CONTENTS

of Producing Toxicosis.

iv

Page

12
12
12
13
13
15
17
19
25
25
34
34
34

34

Page
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
SUMMARY. . . . . . . . . . . . . . . . . . .'. . . . . . . . . . . . 40
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Table

10

11

12

13

LIST OF TABLES

Weight changes of rabbits given lead. . . . . . . . . .

Weight changes of control rabbits . . . . . . . . . . . . .

Total number of basophilic stippled cells per 10,000

erythrocytes counted in lead treated animals. . . . . . .

Total number of polychromatophilic erythrocytes per 10,000

erythrocytes counted in lead treated animals. . . . . . . .

Basophilic stippling as affected by repeated bleeding .

Polychromatophilic erythrocyte count as affected by
repeated bleeding . . . . . . . . . . . . . . . . . . .

Hemoglobin values as affected by repeated bleeding
(gms/loo mls) s s s s s s s s s s s s s s s s o s s s s

Hematocrit values as affected by repeated bleeding
(percent) . . . . . . . . . . . . . . . . . . . . . . .

Hemoglobin values for each animal at various total
dosage levels (gm./100 ml.) . . . . . . . . . . . . . .

Hematocrit values for each animal at various dosage
levels (percent). .‘. . . . . . . . . . . . . . . . . .

A comparison of hemoglobin (gm./100 ml.) and hematocrit
values (percent). . . . . . . . . . . . . . . . . . . .

Comparison of 2 dithizone methods for blood lead level
determinations (ppmo. . . . . . . . . . . . . . . . . .

Lead levels in liver and kidney composite samples
(gravimetric lead-chromate method). . . . . . . . . . .

vi

Page
18

18

20

23

26

26

27

27

29

30

31

32

36

Figure

LIST OF FIGURES

Plastic tube and plunger used as balling gun. . . . .

Photomicrograph of Wright's-stained blood smear with

basophilic stippling. . . . . . . . . . . . . . . . .

Average number of stippled and polychromatophilic

erythrocytes per 10,000 erythrocytes counted. . . . .

Photomicrograph of Wright's-stained blood smear with

polychromatophilic erythrocytes . . . . . . . . .

Average hemoglobin and hematocrit change. . . . .

Average blood lead levels figured in days prior to death.

vii

Page
14

21

22

24
28

35

INTRODUCTION

Lead toxicosis has been, and still is, considered a common and
serious disease in both man and animals. Acute lead toxicosis usually
does not cause great difficulty in diagnosis. However, the most persistent
problem lies in the inability to determine poisoning early enough in the
disease process to effectively initiate treatment.

Throughout the years many types of tests have been used or advo-
cated for use in lead determinations. Differences in test animals (both,
species and individual), route of administration and dose levels are but
3 variables that make it difficult to evaluate laboratory findings

effectively.

OBJECTIVES

The objectives of this research were to compare several types of
laboratory procedures for lead determinations in animal tissues as
reported in the literature. New Zealand White rabbits were chosen as
the experimental animal because they could be easily dosed orally with
known levels of lead and were sufficiently large for blood and tissue
collections and subsequent determinations.

The specific aims of this research:

1. To evaluate several laboratory tests and.procedures, described
in the literature, for lead determinations.

2. To observe gross and microscopic lesions of lead toxicosis
and to compare these with lead levels as revealed by selected clinical
pathology tests.

3. To determine‘whether these laboratory tests are.effective and

accurate in the diagnosis of lead toxicosis.

LITERATURE REVIEW

A review of the literature reveals that much has been‘written on
lead and its toxic effects. Reviews by Blansdorf (1922), Hutton (1923),
Key (1924) and Aub et al. (1926) described some of the work on lead
toxicosis. The symptoms of plumbism.have been known since ancient times,
but it was not until the early part of the 19th century that experimental
investigations on lead and its toxic effects were begun. These investi-
gators understood and described the correlation between lead and so-called
"metallic colic" of lead miners and smelters. Other writers in historical
books on medicine have written on palsy, colic and other symptoms follow-

ing the ingestion of lead.

Historical
The history of lead and its toxic properties was well known and

recognized as early as the 2nd century B.C. Nikander's description of
the signs of poisoning, as seen in workers mining and refining lead,
included constipation, abdominal pain, and pallor (Aub et al., 1926;
Gilfillan, 1965; Morgan at al., 1966). Alderson (1852) quoted
Dioscorides as saying,

"the drinking of litharge (red lead) causes

oppression to the stomach, belly and intestines,

with intense wringing pains; sometimes it even

wounds the intestines by its severe pressure, it

suppresses the urine, while the body swells and

acquires an unsightly leaden hue."
Hippocrates, 370 B.C., was the first to associate certain clinical signs
with an exposure to lead (Waldron, 1966). The ease in refining lead

3

4
from.natural ore, along with its malleability and.resistance to corrosion,
lent itself easily to a widespread use by man. The practice of lining
cooking and drinking vessels with lead, the construction of plumbing
systems in the homes of the wealthy and the sweetening of wine, as well
as many other purposes, have recently been incriminated as one cause of
the deterioration of the Roman civilization (Gilfillan, 1965). Water
contaminated by lead in Amsterdam and cider contaminated by lead in Devon.
appeared to cause epidemics of poisoning during the 16th century (Morgan
et al., 1966).

In the 17th and 18th centuries, lead was used in medicine, especially
for its action upon the blood. Lead was found to be hemostatic and,
because of its power to coagulate albuminous tissue, was used in thei
treatment of ulcers. Lead was also used for the treatment of fevers and
as a drug to combat malaria (Legge and Goodby, 1912). In 1839, the first
detailed treatise describing clinical features of lead poisoning was.
published (Tanquerel des Planches, 1839). By the end of the 19th century
there was an increase in use of lead as well as literature describing
lead poisoning. At about this time pioneer investigators such as Legge,
Teleky, Lehmann, Aub, Minot, Reznikoff and Fairhall initiated independent

studies on the toxic effects and metabolism of lead.

Industrial Lead Toxicosis
One of the earliest indications that a health problem existed in the
people working in the lead industry, in general, and the lead mines of
England in particular can be determined from an 1842 subcommission report
of the Children's Employment Commission.‘ This commission not only was~
concerned about.wages but also with miners' health. In a letter to the

Weardale lead miners, they wrote,”

5

"by moving only twenty miles lower down into the

coal country, a young man might nearly double his

income and have the prospect of adding many years

to his life." (Raistrick and Jennings, 1965)
This commission also commented on the state of poverty of the miners and
concluded that the chief cause of pauperism was due to the large number
of widows and orphans. The husbands of many of these widows had fallen
victim to the "miner's complaint," which materially shortened the lives
of the men and made the miners old men at the age of 50.

The harmful effects of lead in the painting and varnishing trades
was the subject for a 38-page paper by Koelsch in 1921 given to the
German Painters Guild. In this lecture, he detailed symptoms, modes of
entrance into the body, and individual susceptibility. He also presented
statistics in regard to the frequency of poisoning in various localities:
of Germany. Teleky (1922) described the dangers of using leadébased
paints. This article was the result of world-wide attention given to
the lead paint industry following World War I.

In 1912, cases of lead toxicosis in workers of smelting and refining
plants in the United States numbered 1667 out of a total of 7400 men, or
approximately 22%, while in England the rate of lead toxicosis for that
year was.l.8%. For this same.year, statistics from.Germany revealed an.
incidence of 10.5% employee lead poisoning and one report from a large.
Austrian plant reported that 92 of their total work force were poisoned
(U. S. Bureau of Labor Statistics Bull. 141, 1912).

Burnham (1923), in his study on industrial lead poisoning and its
prevention in factories, presented statistics showing the reduction of

reported cases of lead poisoning between 1900 and 1920 in England.. A

total of 1058 cases with 38 deaths was seen in 1900, while in 1920 only

6
243 cases were reported with 23 deaths. He further stated that in the
Uhited States during a 9~year period (1911 to 1920) about 2000 cases of.
chronic lead poisoning in humans was seen annually.

In a survey conducted by Pratt (1913) comparing European and American
industries that were sources of lead poisoning to the workers, it was.
found that great variations of the problem existed. For example, companies
that made dinnerware in Great Britain and Germany had a high incidence of
lead toxicosis, while in America no lead poisoning was noted. According
to Pratt, this was due to the use of lead in tin plating in Europe, while
in America enamel dinnerware was used. He also concluded that workers
hand-making files in Europe had more lead poisoning than workers using
machines in America.

Edsall (cited by Hamilton, 1919), comparing plumbism in the dyeing
and calico printing trades, found a high level of lead poisoning in
m employees of companies in Great Britain; however, he found no evidence

of lead poisoning in the textile industry of Philadelphia.

Infants and Lead Toxicosis

Although industrial lead poisoning was recognized for many years in
adults, few references are found concerning lead toxicosis in infants
prior to 1945. Here the disease prevailed mainly in infants aged 1 to 4
and was reportedly the most common type of fatal poisoning in children
(Perlstein and Attala, 1966). The source of poisoning usually was flaking
paint or crumbling plaster, especially in slum areas where property upkeep
was poor.-

In infants, lead poisoning was usually quite severe. Abdomdnal pain,
constipation, diarrhea, vomiting, and lethargy were commonly seen and,

in more prolonged cases of lead ingestion, central nervous system

7
disturbances were noted. Even with modern forms of therapy the mortality
rate was 22 to 65% (Coffin et aZ., 1966). In a study in the Chicago area,
Perlstein and Attala (1966) reported_that-39Z of 425 cases revealed per-
sistent neurological after-effects of lead poisoning ranging from profound
mental retardation to optic atrOphy and grand mal.‘

Legge and Goodby (1912), Aub et a1. (1926), Bradley et a1. (1956), and
Waldron (1966) reported basophilic stippling of RBCs, a mild hypochromic
anemia with many.immature red blood cells, proteinuria, glycosuria, and
amino-aciduria, as some common laboratOry findings in lead poisoning of

children.

Animals and Lead Toxicosis

Domesticated animals accidentally poisoned by lead included horses,
dogs, cattle, sheep, swine, and cats (Zook et al., 1969). Experimental
lead poisoning has been carried out using all "common laboratory animals."
The rabbit and the rat were the 2 most common small animals used, while
in the large domestic animals cattle and sheep were the animals of choice
(Blaxter, 1950; Allcroft, 1951).

The horse was the 1st animal reported to be subject to lead poison-
ing. These reports of poisoning were usually from lead mining and smelt-
ing areas where this animal was used as the major source of power. Haring
(1915) reported the lead caused a paralysis of the laryngeal nerves causing
failure of normal abduction of the laryngeal cartilages during inspira-
tion, hence the term "roarers." In this case death was usually caused by
foreign-body pneumonia due to failure of the epiglottis to close. The 2nd
area where accidental lead poisoning was seen concerned fruit orchards
where lead arsenate was commonly used as a pesticide. Today lead poison-

ing in horses is reported far less than in earlier years, due to the more

8
effective means of removing lead from smelter fumes and the use of the
newer pesticides.

Although the horse was the first animal reported with lead toxicosis,
in recent years lead poisoning has been reported as.a disease of cattle
almost exclusively. This was primarily due to the nature of cattle
smelling, licking or chewing foreign objects. The source of the lead
usually was found to be lead-based paints, plasters, greases or old
storage batteries (Hammond et al., 1956).

A wealth of data is available concerning experimental lead poison-
ing in the dog (0phuls, 1907; Horwitt and Cowgill, 1937; Calvery et al.,
1938; Finner and Calvery, 1939; Fauts and Page, 1942); however, examina-
tion of available literature concerning accidental lead poisonings revealed
few reported cases. Recently one investigator diagnosed and described
lead poisoning in 60 dogs at the Angell Memorial Animal Hospital (Zook et
al., 1969). Other accidental lead poisonings in the United States include
reports by Bond and Rubin (1949) and Berry (1966), while Scott (1963)
reported on 28 cases of lead poisoning in dogs which occurred at Broken
Hill, Northern Rhodesia, and Dodd and Staples (1956) reported on lead

poisoning in New Zealand.

Lead Determinations

A search of the literature revealed that the chemical methods most
frequently used for lead determinations were: (1) the colorimetric sulfide
uethod, (2) gravimetric or volumetric sulfate, molybdate and chromate methods;
and (3) the electrolytic method. Fourcroy and Hahnemann were the first
investigators to propose the use.of hydrochloric acid saturated with hydrogen
sulfide as a test for lead (Friend, 1917). Pelouze (cited by Fairhall,
1922), first proposed a colorimetric method for the determination of small

amounts of lead. He converted the lead into sulfide and compared the

9
intensity of the brownish coloration produced against standard lead solu-
tions similarly treated.

A number of objections have been defined in the colorimetric methods
of estimating lead as the sulfide. These included acid concentration,
amount and character of the salts and the size of the lead particles. Any
one of these variables could change the color intensity and would thereby
give imprOper values (Fairhall, 1922).

The sulfate method was advocated for the analysis of larger amounts
of lead. A number of substances such as ammonium chloride, tartrate,
acetate, the alkaline chlorides and caustic alkalis increased the solu-
bility of lead sulfate which produced low results. Potassium salts often
gave high results due to the precipitation of a potassium and lead complex.
This complex was practically impossible to remove. This method was not
recommended for biological tissues because of the false high and.low
values (Fox, 1909).

Lead may be determined either gravimetrically or volumetrically as
the molybdate since lead molybdate is extremely insoluble and lends itself
readily to either process (Alexander, 1893; Weiser, 1916). Using ammonium
molybdate as a precipitant, Eegriwe reported that the sensitivity was
1:800,000; however, because ammonium molybdate gave insoluble precipi-
tates with both the calcium salts and phosphates in tissues, this method
was not recommended for biological material (Fairhall, 1922).

The electrolytic process for lead determination has been subjected
to much investigation (Frankel, 1893; Exner, 1903; Snoden, 1906; Patten,
1917; Denis and Minot, 1919). The difficulties of manipulation of the
equipment plus the low results obtained when phosphates are present or
when using sulfuric acid in the test solutions has resulted in its disuse

for lead determination in biological materials (Patten, 1920).

10

The chromate method of lead determination proposed by Diehl has been
modified by several investigators (Crookes, 1894; Cushman and Hayes-
Campbell, 1895; Pope, 1896; Seeker and Clayton, 1915; Fairhall, 1922) in
an attempt to prevent the lower results obtained in the presence of sodium
acetate. Cox (cited by Fairhall, 1922) reported that by using acetic
acid to convert the basic salt into the normal salt, one could precipitate
all the lead present and thereby prevent the low, inaccurate analysis.
Scott (1939) presented a simplified chromate method for lead determination
that has since been used as a standard test in some laboratories.

Other tests for the estimation of lead, using urine, feces or tissues,
included one by Finner and Calvery (1939). They described a method that
consisted of placing bones in a.10Z formaldehyde solution saturated with
hydrogen sulfide. Pieces of the long bones were placed in the solution
for up to 3 weeks and, if-lead were present, it combined with the sulfide
and black particles could be seen following treatment with formic acid.

In 1942, Bambach and Burkey described a micromethod of lead determina—
tion in tissues using the chemical, dithizone. This was an adaptation of
the titrimetric method that was used by Horwith and Cowgill (1937).
Hammond et a1. (1956) further modified this test by replacing the ashing
procedure with an extraction method using trichloroacetic acid.

Feigl and Suter (1924) described a chemical method for the simul-
taneous demonstration of barium, strontium and lead. Later this was
adapted for use in tissue sections and was presented for use by Thompson
(1966). This method used the chemical, sodium rhodizonate, to stain
tissue sections. Four duplicate sections of the tissue to be examined
were prepared and stained separately. Changing the pH of the staining

solutions allowed a differentiation of the contaminated metals.

10

The chromate method of lead determination proposed by Diehl has been
modified by several investigators (Crookes, 1894; Cushman and Hayes-
Campbell, 1895; Pope, 1896; Seeker and Clayton, 1915; Fairhall, 1922) in
an attempt to prevent the lower results obtained in the presence of sodium
acetate. Cox (cited by Fairhall, 1922) reported that by using.acetic
acid to convert the basic salt into the normal salt, one could precipitate
all the lead present and thereby prevent the low, inaccurate analysis.
Scott (1939) presented a simplified chromate method for lead determination
that has since been used as a standard test in some laboratories.

Other tests for the estimation of lead, using urine, feces or tissues,
included one by Finner and Calvery (1939). They described a method that
consisted of placing bones in a 10% formaldehyde solution saturatedeith
hydrogen sulfide. Pieces of the long bones were placed in the solution
for up to 3 weeks and, if lead were present, it combined with the sulfide
and black particles could be seen following treatment with formic acid.

In 1942, Bambach and Burkey described a micromethod of lead determina-
tion in tissues using the chemical, dithizone. This was an adaptation of
the titrimetric method that was used by Horwith and Cowgill (1937).
Hammond et al. (1956) further modified this test by replacing the ashing
procedure with an extraction method using trichloroacetic acid.

Feigl and Suter (1924) described a chemical method for the simul-
taneous demonstration of barium, strontium and lead. Later this was
adapted for use in tissue sections and was presented for use by Thompson
(1966). This method used the chemical, sodium rhodizonate, to stain
tissue sections. Four duplicate sections of the tissue to be examined
were prepared and stained separately. Changing the pH of the staining

solutions allowed a differentiation of the contaminated metals.

11

The atomic absorption spectrophotometry method to determine small
amounts of metals was suggested by Walsh (1955). Since that time investi-
gators have improved on both the instrumentation and extraction methods.
Simple procedures using the raw sample have been proposed for lead detection
(Slavin et aZ., 1964), while more elaborate methods required ashing or
extraction (Willis, 1962; Berman, 1964; Kopito and Schwochman, 1967).

Garrod (1892) reported that lead inhibited heme synthesis from
glycine. Because of this inhibition, 2 intermediate stages, delta amino
levulinic acid and coproporphyrin III, were found in the urine. Recently
the levels of the 2 intermediate stages in the urine were employed as a
basis for determining lead toxicosis (Benson and Chisolm, 1960; Chisolm,
1964; Balbo et al., 1965; Ellis, 1966; Bruin and Hoolboom, 1967).

Clinical pathology determinations for lead toxicosis include the
examination of blood smears stained with Wright's stain for the presence
of basophilic stippling of erythrocytes (Key, 1924; Finner.and Calvery,
1939; Mitchell, 1940; look et al., 1970). Slight anemia, as evidenced by
a reduction in PCV and hemoglobin values, has been reported in hemograms

from lead poisoning suspects (Zook et al., 1969).

 

F‘
“

MATERIALS AND METHODS'

Experimental Animals

Fifteen apparently healthy, 8- to lO-month-old female New Zealand
White rabbits were used in this study.‘ This experimental animal was
chosen because it (1) was large enough to permit collection of blood and
tissues needed for analytical work; (2) was easily obtained at a low cost;
and (3) has known susceptibility to heavy metal poisoning. The experiment
was designed to cause death in 21 days, and any rabbits still alive would
be killed and necropsied at that time.

By random selection the rabbits were divided into 2 groups. Group
1 consisted of 10 animals identified as Nos. 1 to 10. Group 2 consisted 0f
the remaining 5 rabbits, to serve as controls, and were identified as

Nbs. 11 to 15.

Animal Care-
The rabbits were maintained in individual, wire-bottomed, stainless
steel rabbit cages in the Center for Laboratory Animal Resources (CLAR)
facilities at Michigan State University. They were fed free choice a

standard rabbit pellet ration.* Water was provided ad Zibitum.

 

*John A. Van Den Bosch Co., Zealand, Mich.

12

13
Source and Method of Toxicosis

Reagent grade lead carbonate (2 PbCoan(0H)2)* MQW. 774.7 was used
as the toxicologic agent and was supplied by the Michigan.Department of
Agriculture Regulatory Laboratory. A standard analytical balance was used
to weigh 125 mg. of lead carbonate calculated to be 100 mg. pure.lead,
for each animal's daily dose and was placed into No. 00** gelatin capsules.
One rabbit at a time was held on a table and, with the aid of a short
piece of plastic tubing and plunger (Figure l), the capsule containing the
lead was placed as far back in the mouth as possible. The animal was kept
on the table and observed until chewing and swallowing were completed and

then was returned to its cage.

Sample Collection
To establish normal hemoglobin, packed cell volume and blood lead
levels, blood samples were taken from each animal before the beginning of
the experiment. A rabbit board was used to facilitate the holding of the
animals. Blood was collected by cardiac puncture.with the needle inserted
caudolateral to the xiphoid cartilage toward the location of the heart.

Heparin***

solution was used as the anticoagulant in the 10 cc. plastic
syringes and 2-inch, 18 gauge needles. A minimum of 10 ml. of blood was
collected every 48 hours from the treated animals during the experimental
period and placed in plastic heparinized vials for individual analyses.

A second study was initiated using another group of 6 rabbits in an

attempt to determine what effect the removal of 10 ml, of blood every 48

 

*J. T. Baker Chemical Co., Phillipsburg, N.J.
**Parke, Davis & Company, Detroit, Mich.

***American Hospital Supply Corp., Evanston, Ill.

14

 

 

Figure 1. Plastic tube and plunger used as
balling gun.

15
hours would have on the hemoglobin, packed cell volume, and morphology of
the Wright's-stained blood smears.

Tissue collections were taken at the time of death or as soon as
possible following death. Tissues collected from all animals included
the following: (1) a portion of the right and left lateral lobe of the
liver; (2) right and left kidney; (3) duodenum; (4) jejunum; (5) ileum;
(6) brain; (7) heart; and (8) right and left femur. Following necropsy,
collected tissues were divided into 2 groups. One portion was fixed in
a 10% buffered formalin solution for sectioning and appropriately stained
for histopathologic examination. The 2nd portion was refrigerated until

chemical analyses were performed for lead.

Experiments

During the 21 days of the experiment, the 5 nontreated controls and
the 10 rabbits given the daily dose of lead were maintained in the
same room under identical conditions. In each trial a comparison was
made between controls and poisoned animals. The trials are summarized
as follows:

1. Two blood smears were made at the time of bleeding of each animal
and stained by Wright's method (Benjamin, 1961) for stippled cell
determinations. .

2. Hemoglobin values were determined by the cyanmethemoglobin
method (Benjamin, 1961). The microhematocrit method (Benjamin, 1961)
was used for the packed cell volume (PCV) values.

3. Blood lead levels were determined by 2 different dithizone
(diphenylthiocarbazone) methods. Method I (Hammond et al., 1956) described a
trichloroacetic acid solution to precipitate the blood proteins. This

step replaced ashing of the sample. The lead was extracted using dithizone

l6
reagent and the lead level was determined by visual comparison to a group
of color blocks. Method 11 involved the ashing.of the sample in a muffle
furnace, extracting the lead using dithizone reagent and titrating against
a standard lead solution (Hurwitt and Cowgill, 1937).

4. Lead levels in the-tissues were determined by the lead chromate
method as described by Scott (1939).

5. Bone lead levels were determined by a method used by Finner and
Calvery (1939). One femur from each animal was fixed for 3 weeks in a solution
of formaldehyde diluted 1:10 with distilled water and saturated with hydrogen.
sulfide. Following fixation, bone was treated with formic acid; The other
femur was fixed in buffered 10% formalin. Both femurs were decalcified
using Cal-Ex decalcifying solution,* sectioned and stained with hematoxylin
and eosin. A.histopathologic comparison was made between.the 2 fixation
methods and the control animals.

6.‘ Special stains. Kidney sections were stained using the Ziehl-
Neelsen acid-fast method far inclusion body determinations. Kidney and
liver sections were stained using the sodium rhodizonate** method for lead
(Thompson, 1966).

7. A histopathologic comparison was made between hematoxylin and

eosin-stained sections of major organs from poisoned and control animals.

 

*No. 3-391, Fisher Scientific Co., Fairlawn, N.J.

**Fisher Scientific Co., Fairlawn, N.J.

RESULTS

The rabbits were weighed prior to and again at the end of the experi-
mental period, or following death, to determine weight changes. The 10
rabbits that were given lead had an average weight loss of 0.015 kg.-
(Table 1), while there was an average weight gain of 0.254 kg. in the
5 control animals (Table 2).

Signs attributable to lead toxicosis were observed in only 1 rabbit.
In this animal there were signs of ataxia, incoordination and mental
derangement. This rabbit repeatedly charged the walls of the cage_and
had high-pitched crying sounds with open-mouthed breathing. The-above
signs lasted only a short period of time and were followed by death.

All rabbits given lead had roughened hair coats, paleness of the
mucous membranes, and signs of dehydration. Terminal diarrhea was evi-
denced by the soiled hair coat of the hindquarters in all but 2 animals.
In contrast, control rabbits appeared normal in all respects.

Upon death, an autopsy was performed on every animal. The-first to
die, rabbit No. 1, was pregnant. The 4 approximately 25-day-old fetuses
were removed from the uterus for tissue lead determination. subsequent
chemical examination of the composite sample (all tissue of the 4)
revealed a tissue lead level of 44.8 ppm. Pathologic changes in
rabbit No. 1 included a pale yellowish liver, a thickened edematous peri-
cardium, and a few small, circumscribed white spots on the surface of the

kidney. No other lesions were seen.

17

18

Table 1. Weight changes of rabbits given lead

— — , "

 

 

 

 

Animal Pretrial Wt. Final Wt. Total Lead
No. (kg.) (kg.) Net Change Given (mg.)
1 4.20 4.12 -0.08 800
2 4.10 4.01 -0.09‘ 1500
3 3.50 3.50 -- 1700
4 3.54 3.50 -0.04 1700
5 2.62 2.74 +0.12 1900
6 3.12 2.98 —0.14 1700
7 4.00 4.14 +0.14 1900
8 3.30 3.24 -0.06 900
9 2.80 2.80 —-- 1900
10 __3_:_9_4 ___3_._9_4_ --- 1700

Totals 35.12 34.97
35.12 16 34.97 - 0.015 kg. average loss

 

Table 2. Weight changes of control rabbits

 

 

 

 

-----r* a ‘L_
Animal Pretrial Wt. Final Wt.
No. (kg.) (kg.) Net Change

11 3.50 3.80 +0.30

12 3.40‘ 3.51 +0.11

13 4.22 4.55 +0.33

14 4.12 4.20 +0.08

15 _§;§§_ 3.98 +0.44

Totals 18.78 20.04
20 . 04 - 18 . 78 I O .254 kg, average gain

 

 

l9

Necropsy revealed Animal No. 8 had anacute pneumonia. All areas of
the lungs were solidified and full of a yellowish, tenacious material.
Microbiologic examination of portions of the lung identified the causative
organism as Pasteurella multocida. No other gross lesions were noted.

Examination of the remaining 8 poisoned animals and the 5 controls
did not reveal any gross lesions, except in the area of the heart. Some
pericardial thickening with an increase in fibrous connective tissue and

edema around the heart was noted in these animals.

Trial 1

Examination of the Wright's-stained blood smears revealed anisocy-
tosis, poikilocytosis, and hypochromia of the red blood cells in lead-
poisoned animals. Approximately 10,000 red blood cells were examined on
each of 53 blood smears.‘ Forty-four of the smears had an average of 39
basophilic stippled "reticular" and/or "punctate" red blood cells (Table
3, Figure 2). Nine of the smears did not have red blood cells with baso-
philic stippling. A sharp increase in erythrocytes with basophilic
stippling was seen when the total cumulative dose of lead exceeded 1400
mg. per animal (Figure 3).

Blood smears were made and examined from all animals, prior to the
experiment, and from.the 5 control animals before they were killed at
the termination of the experiment, to establish normal values. Only 1
stippled cell was observed in these slides.

Examination of-the blood smears revealed the number of polychromato-
philic erythrocytes were noticeably increased with each additional level
of lead (Table 4, Figure 4), with an average of 232 polychromatophilic

erythrocytes in the 53 smears.

20

 

 

 

Table 3. Total number of basophilic stippled cells per 10,000 erythro-
cytes counted in lead treated animals
JI
Pretrial Final
Animal Examina- Examina- Cumulative Lead Intake (mg.)
No. tion tion 200 700 900 1100 1400 1600 1800
l O 1 118**
2 0 23 20 17 50 64**
3 0 18 14 0 20 10**
4 0 0 2 18 12 10 44**
5 0 2 2 22 28 --- 292 469***
6 0 0 0 5 7 14 154**
7 0 4 10 4 2 4 3 7***
8 0 2 2 2M:
9 0 0 0 2 0 5 16 64***
10 0 0 0 14 10 20 134**
11* 0 0
12* 0 0
13* 0 0
14* 0 1
15* 0 0
Mean 0.05 5 17 8 14 18 107 180
j;S.E 2.74 11.46 2.82 6.28 7.91 49.1. 178
P < 0.05 .01 .01 .01 .01 .01 .01

 

*Control animals .
**Last sample taken prior to death.

***Last sample taken prior to euthanasia.

21

 

Figure 2. Photomicrograph of Wright's-stained blood
smear with basophilic stippling. Notice both "punctate"
and "reticular" forms of stippling. x 1500.

22

 

 

350
-- stippled cells (lead treated)
___. polychromatophilic cells (lead treated)
325 o-o-o stippled cells (bleeding effect)
._,_A Polychromatophilic cells
(bleeding effect)
300
275 \
250
225
200
Abnormal
Cells
175
l
/
150 I
I
/
125 /
/
l
100 I
I
/"°\, ,
75 /. \.‘-.l
./'\ ° / \\
p/ '\3 g/
50 / \.’ /
0’.’. /
//. ,, ’—" I
25 ’,/. I / ’ I
" ———————— fiae,
L email/0-.-.
1 2 3 4 5 6 7 8 9 10 ll 12 13 14 15 16 17 18
Days

Figure 3. Average number of stippled and polychromatophilic erythro-
cytes per 10,000 erythrocytes counted. Lead treated animals received
100 mg. lead per day.‘

23

Table 4. Total number of polychromatophilic erythrocytes per 10,000
erythrocytes counted in lead treated animals

 

Pretrial Final

 

 

Animal Examina- Examina- Cumulative Lead Intake'(gg:)
No. tion tion 200 700 900 1100 1400 1600 1800
1 21 168 258*
2 70 234 156 150 152 156**
3 54 238 344 290 278 146**
4 28 136 272 252 254 222 206**
5 16 41 160 274 456 --- 446 802***
6 22 30 195 146 ~ 236 398 326**
7 40 47 166 141 234 206 200 121***
8 17 32 148 142**
9 19 87 84 123 208 270 234 84***
10 44 200 464 417 470 514 292**
11* 29 78
12* 34 58
13* 16 24
14* 47 77
15* 36 49
Mean 39 121 225 215 286 273 284 336
i_S. E. 4.51 26.7 11.3 33.2 39.5 51.2 38.1 233.5
P < .001 .001 .001 .001 .001 .001 .001

 

*Cont rol animals .
**Last sample taken prior to death.

***Last sample taken prior to euthanasia.

24

 

 

Figure 4. Photomicrograph of Wright's-stained-blood
smear with polychromatophilic erythrocytes. x 1500.

25
The greatest number of polychromatophilic erythrocytes seen in blood
from untreated animals was 78. The average number of polychromatophilic
cells counted per smear is illustrated (Figure 3).

Six additional animals were bled to determine the effect of blood
loss on the number of abnormal red blood cells present in Wright's-stained
smears and on hemoglobin and hematocrit (Tables 5, 6, 7, and 8, Figures
3 and 5). The total number of cells with basophilic stippling in this
study ranged from no stippled cells to a high of 8 cells with stippling.
While the average.number of polychromatophilic erythrocytes gradually
increased with repeated bleeding from a low of 13 to a high of 84, the
total number counted did not approach the numbers noted in the animals
given lead.

No appreciable change in hemoglobin or hematocrit values due to bleed-

ing could-be seen‘when compared to initial readings from dhe same animals.

Trial 2
Hemoglobin and hematocrit values from lead poisoned animals indi-
cated mild anemia (Tables 9 and 10, Figure 5).
An average of the last PCV and Hb values taken before death on the
poisoned animals compared to the average pretrial reading of the same
animals revealed a reduction of 21.2% hematocrit and a 20% reduction in

hemoglobin (Table 11).

Trial 3
A comparison of~2 methods to determine blood lead levels using
dithizone are presented in Table 12. In general, good correlation was
noted between the 2 methods.' Initial levels prior to poisoning indicated
normal lead values in the rabbit blood ranged from 0.16 to 0.23 ppm‘with

an average of 0.204 ppm. Examination of the average blood lead levels

26

Table 5. Basophilic stippling as affected by repeated bleeding

 

 

Bleedings (48-hour interval)

 

 

Animal No. 1 2 3 4 5 6 7 8 9
16 0 o 4 1 4 3 2 0 o
17 0 - 0 1 - o 1 o 4
18 1 0 0 o 2 1 o 2 o
19 0 0 8 s 0 0 1 o 0
20 0 0 0 0 0 0 o 0 0
21 0 - 0 0 7 0 4 5 0
Mean 0.2 0.0 2.0 1.2 2.3 0.2 1.2 1.2 0.7
is. E. .20 .0 1.36 1.28 1.33 .54 .62 .83 .66
P < N.S. N.S.' N.S. N.S. N.S.' N.S. N.S.- N.S.

 

Table 6. PolychromatOphilic erythrocyte count as affected by repeated

 

 

 

 

bleeding
Eleedingg_(48-hour-interval)

Animal No. 1. 2 3 4 5 6 7 8 9
l6 8 10 51 48 55 54 71 48 62
17 16 - 38 31 - 41 78 116 111
18 24 23 44 56 87 51 62> 56 27
19 13 30 88 72 67 79 34. 44 58
20 12 5 15 23 44 36 80 90 103
21 4 - 9 68 91 89 130 78 50

Mean l3 17 41 48 68 58 84 72 68
S. E. 2.81 5.75 11.6 8.06 9.06 8.67 9.69 11.5 13.2

< NOS 6 0025 0001 .001 .001 0001 0001 0001

 

27

Table 7. Hemoglobin values as affected by repeated bleeding (gm/100 ml.)

Bleeding; (48-hour interval)

 

 

Animal No. 1 2 3 4 5 6 7 8 9
16 11.7 10.8 12.0 10.8 9.7 10.8 10.3 10.0 12.5
17 12.0 12.0 --- 11.7 --- 11.7 11.7 10.2 10.4
18 11.2 10.0 9.8 10.5 9.7 11.1 10.8 10.5 11.7
19 11.6 9.5 10.0 12.3 13.0 11.1 10.8 11.4 12.5
20 11.4 11.5 10.5 10.8 11.0 -- 10.3 10.0 10.3
21 13.5 12.0 12.3 11.5 10.5 10.8 11.7 11.2 10.4

Mean 11.9 11.3 10.9 11.3 10.8 11.1 10.9 10.5 11.3
+ S. E. .31 .35 .52 .28 .61 .20 .22 .20 .43
P < N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.

 

Table 8. Hematocrit values as affected by repeated bleeding (percent)

 

 

Bleedings (48-hour interval)

 

 

Animal No. l 2 3 4 5 6 7 8 9
16 38 34 -- 33 31.5 33 34 33 37

17 39 36 -- 35 -- 38 37 32 37

18 35 32 -- 33 33 34 36 33 37

19 36 32 -- 38 38 37 37 37 42

20 36 37 -- 34 34.5 -- 34 34 36

21 40 37 -- 37 37 36 37 36 38
Mean 37 35 -- 34 36 36 36 34 38
i_S. E. .82 .97 -- .97 1.41 .95 .61 .79 .87

P < N.S. . -- N.S. N.S. N.S. N.S. N.S. N.S.

 

40
39

Hemato-
crit
Z
32
31
30
13.5
13.0
12.5
Hemo- 12.0
globin
ng/ 1105
100
m1. 11.0
10.5
10.0

 

 

28

Lead treated

 

u_u_. Controls (repeated bleeding)

 

Lead treated
.-.-. Controls (repeated bleeding)

 

 

 

 

1 2 '3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Days

Figure 5. Average hemoglobin and hematocrit change. Lead treated
animals received 100 mg. lead per day.

Table 9.

2

9

Hemoglobin values for each animal at various total dosage levels

(gm./100 m1.)

1

__7

Pretrial Final

 

 

Animal Examina- Examined Cumulative Lead Intake (mg.)
No. tion tion
1 12.3 12.1 9.2**
2 13.8 13.5 -—- 13.6 13.6 10.0**
3 10.7 9.3 --- 10.2 10.2 10.7 8.0**
4 13.0 11.0 12.0 12.2 12.2 12.1 13.2**
5 13.1 --- 13.0 11.0 11.0 11.6 10.1 8.6***
6 12.8 11.4 11.8 11.6 10.8 10.4 9.4**
7 13.8 11.9 ---- 12.2 12.2 11.8 13.2 12.4***
8 13.1 11.8 -—- 10.6**
9 13.6 12.6 12.8 12.0 12.2 12.0 13.4 ll.2***
10 10.0 8.7 8.8 8.6 7.8 9.1 8.8**
11* --- 13.4
12* 12.9 12.2
13* 13.5 14.0
14* 13.0 13.0
15* 11.0 11.4
Mean 12.7 11.4 11.3 11.3 11.3 10.9 10.8 10.7
iLS- E. .26 .51 .74 .48 .62 .38 .88 1.12
P < .01. .025 .01 .01 .001 .005 .001
*Control animals . ‘
**Last sample taken prior to death.
***Last sample taken prior to euthanasia.

30

Table 10. Hematocrit values for each animal at various dosage levels
(percent)

 

Pretrial Final

 

 

Animal Examina- Examina— , Cumulative Lead Intake,(mg.)_
No. tion tion 200 700 900 1100 1400 1600 1800
l 38 38 27**
2 42 41 --- 39 40 30**
3 33 29 --- 29 29 33 22**
4 39 36 37 37 37 39 42**
5 39 --- 37 35 33 35 25 25***
6 41 38 36 36 32 33 26**
7 40 37 --- 35 36 37 40 38***
8 4O 39 --- 33**
9 40 40 38 35 36 38 40 35***
10 30 27 25 24 22 29 22**
11* --- 40
12* 38 36
13* 40 42
14* 39 40
15* 34 34
Mean 38 36 33 34 33 34 31.0 33.0
i_S. E. .75 1.59 2.35 1.52 1.98 1.29 3.46 3.93

P < N.S. .01 .01 .01 .01. .01 .05

 

*Cont rol animals .
**Last sample taken prior to death.

***Last sample taken prior to euthanasia.

31

Table 11. A comparison of hemoglobin (gm./100 ml.) and hematocrit values
(percent)

I—‘—

 

 

 

 

 

Pretrial Final
values values 2 Change
Hematocrit average
(poisoned) 38.2 30.0 -21.2
Hemoglobin average
(poisoned) 12.6 10.1 -20.0
Hematocrit average
(control) 37.7 38.4 + 1.8

Hemoglobin average
(control) 12.6 12.8 + 1.0

 

32

Comparison of 2 dithizone methods for blood lead level determi-

nations (ppm)

Table 12.

 

 

\4
.m
.e
n
t
n
d
a
e
L
e
V
.1.
t
a
1
m
C
_
a
3....
8
mm
FE
a...
in
r
.m
e
[X
PE

Animal

No.

71400. 1600' 1800

900 .1100

700

200

 

tion

Method* tion

 

22

00

22
00

21
00

22
00

22
00

33

Table 12 (cont'd.)

 

 

w
_

h

Pretrial Final

 

 

Animal Examina- Examina- Cumulative Lead Intake (mgi)
No. Method* tion tion 200 700 900 1100 1400 1600 1800
11 I 0.2 0.2
II 0.20 0.19
12 I 0.2 0.2
II 0.19 0.21
13 I --- 0.2
II --- 0.20
14 I 0.2 0.2
II 0.20 0.18
15 I 0.2 0.2
II 0.19 0.20
Mean I 0.2 0.34 0.54 0.41 0.53 0.62 0.89' 1.10
11 0.2 0.32 0.47 0.45 0.50 0.66 0.91 1.08
i_S. E. I 0.0 .026 .048 .032 .04 .07 .13 .09
II .004 .022 .040 .018 .031 .11 .11 .08
P < I .001 .001 .001 .001 .001 .001 .001
II .001 .001 .001 .001 .001 .001 .001

 

*Method I - Trichloroacetic acid method (Hammond 8* aZ., 1956)-

Method II - Ashing method (Hurwitt and Cowgill, 1937).

34
when figured by days prior to death indicated a rise in ppm the closer the

animals approached death (Figure 6).

Trial 4
Following death, composite liver and kidney samples from each poisoned
animal were analyzed to determine tissue lead levels (Table 13). Two
animals had tissue lead levels over 45 ppm, while lead was not detected
in 1 animal. Lead was not detected in the tissues from.the 5 control

animals.

Triali5
In the comparison between bone sections fixed in 10% buffered formalin
and bone sections fixed in formalin saturated with hydrogen.sulfide, sig-
nificant histopathologic differences were not noted._ Positive identifi-

cation of lead could not be made by either method.‘

Trial 6
Kidney sections stained by the Ziehl-Neelsen acid-fast stain did not
reveal intranuclear inclusion bodies. Liver and kidney tissue sections
stained by the method presented by Thompson (1966) using sodium rhodizonate
also failed to identify the presence of lead in any of the poisoned

animals.

Trial 7
No obvious significant differences were noted between the H & E-
stained tissue sections from poisoned animals as compared to control animals
that could be considered as an aid in the diagnosis of suspected lead

poisonings.

35
1.05

1.00

.90
.85
.80
.75
.70
.65
Lead .60

PPm
.55

.35
.30
.25

.20

 

.10

 

18 17 16 15 14 13 12 ll 10 9 8 7 6 5 4 3 2 1 0
Days Prior to Death

Figure 6. Average blood lead levels figured in days prior to death
(lead treated animals).

36

Table 13. Lead levels in liver and kidney composite samples (gravimetric
lead-chromate method)

WV

 

Animal No. Tissue Lead Levels (PPm)
1 41.0 (fetus-44.8)
2 45.5
3 45.1
4 8.0
5 34.2
6 ' 10.0
7 7.7
8 none detected
9 7.3

10 23.8

11 none detected
12 none detected
13 none detected
14 none detected

15 none detected

 

DISCUSSION

This study indicated that accurate laboratory tests are available
to aid in diagnosis of lead poisoning in rabbits. Common variables, such
as age, sex, species, breed, care, chemical dose and route of administra-
tion.were carefully controlled. However, factors unrelated to experimental
design or laboratory procedures were encountered that made interpretation
difficult. Individual animal variation and physical factors, unknown at
the beginning of the trials, caused the greatest alteration in results.

There were 2 unexpected deaths of animals given lead orally, rabbit
No. l and 8. The pregnancy of rabbit No. 1 and the pneumonia (Pasteurella
multocida) of rabbit No. 8 placed added stress on the animals and possibly
were contributory to early death.

Hammond at al. (1956) considered bovine blood containing lead between
0.3 and 0.5 ppm to be suspicious for lead exposure and any blood contain-
ing over 0.5 ppm.to be diagnostic. Even on rabbits No. l and No. 8, using
this criterion, a positive diagnosis of lead toxicosis would have been made.
Examination of the blood smears stained by Wright's stain in rabbits No.
1 and No. 8 for basophilic stippling of erythrocytes would have resulted
in only 1 positive lead diagnosis using standards for the dog suggested
by Zook at al. (1970). He considered the finding of 15 ESE/10,000 R30
to be suggestive of lead poisoning and 40 or more to be pathognomonic. In
this study, if one were to base a diagnosis of lead exposure on the above

criteria, 3 of the 10 animals would have been overlooked.

37

38

Along with an increase in the number of basophilic stippled erythro-
cytes, an increase in the number of polychromatophilic erythrocytes was
noted. This rise was seen in all 10 rabbits with a steady significant rise
in numbers beginning at the 200 mg. lead dosage level. This rise in
number would suggest the possibility of using this procedure as a screen-
ing test for lead exposure. Much lower counts were noted in the rabbits
used in the bleeding effect study.

The present studies emphasize the individual differences of test
animals when exposed to lead. Rabbits No. 5, 7 and 9 were alive at the
end of the experiment, having received 1800 mg. of oral lead. However,
while 1 rabbit had a basophilic stippled erythrocyte count of 7/10,000
RBC, another had over 65 times as many. In general, as the total dosage
of lead was increased, an increase in the number of stippled cells was seen.

The 2 chemical tests for blood lead using dithizone had a very good
correlation. They also compared favorably to the known lead dosage.r At
the 200 mg. dosage level, 9 out of 10 of the rabbits would be considered
suspicious using previously established criteria. At the 700 mg. dosage
level, all rabbits would be considered as having levels diagnostic for
lead exposure. Except in the "added stressed" animals, all rabbits had
blood lead levels in the area of 1.0 ppm just prior to death or at the
end of the experimental period. While no significant difference in final
results was noted between these 2 dithizone tests, a comparison of the
equipment and skill needed to perform both tests revealed that the
trichloroacetic acid method suggested by Hammond was much better than
the ashing method. The necessity of a muffle furnace with accurate
temperature controls to prevent excess lead vaporization and the extreme
skill needed in the back titrating procedure are 2 reasons to preclude

the use of the ashing method. Another advantage of the trichloroacetic

39
acid method was in the shorter total time needed to perform the tests.
The average time for completion of an individual determination was approxi-
mately 2-1/2 hours for method I, while 5 hours were required to run the
ashing method. Neither method should be considered sensitive enough to
be used in situations requiring the quantitative determinations of lead,
such as in a research laboratory. Where this type of need exists, newer,
more sensitive methods, including the atomic absorption spectrophotometer,
should be considered. However, these dithizone methods, when used as
diagnostic aids, can be useful in determining possible lead exposure.,

In this study routine H & E-stained sections as well as special stain-
ing methods (Trials 5. 6 and 7) failed to aid in identifying lead or
animals poisoned by lead. While various reports have indicated the presence
of lead produced inclusion bodies in kidney sections of man and animals.
(Calvery at al., 1938; Fauts and Page, 1942; Morgan at al., 1966; Locke
at aZ., 1969), acid-fast intranuclear inclusion bodies are not always pres-
ent and are not specific (Jubb and Kennedy, 1970). In this experiment and
in this species, inclusion bodies were not observed.. Further work needs

to be done on this aspect of lead toxiCosis to determine‘Whether this test

could be improved to aid in lead poison identification.

SUMMARY

Blood and tissues from lead poisoned rabbits were utilized to compare
various laboratory tests and procedures routinely used as aids in the
diagnosis of lead toxicosis. The 2 most common methods to determine lead
exposure in the living animal include chemical analysis of the blood for
lead concentration and examination of stained blood smears for immature,
abnormal erythrocytes.

Good correlation was noted between blood lead levels and lead dosage.
This correlation was determined by 2 dithizone lead methods. The trichloro-
acetic acid method, as suggested by Hammond et al. (1956) was the more
accurate, simpler and faster test.

Basophilic stippling and polychromatophilic staining of erythrocytes
in Wright's-stained smears increased significantly following ingestion
of lead. Animals receiving lead had a wide range in the number of
stippled cells counted. This would limit its use as a single diagnostic
tool. There was a consistent increase in the number of polychromatophilic
erythrocytes; therefore, this could be considered a more reliable test.

Results of the gravimetric lead-chromate method for tissue lead levels
had little correlation with the known amounts of lead ingested.

Tissue sections stained.with sodium rhodizonateJacid fast stain,
and bone sections treated with hydrogen sulfide failed to aid in identi-

fying lead poisoned animals.

40

REFERENCES

REFERENCES‘

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Alexander, R.: Volumetric determination of lead. Engineering and Mining
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Allcroft, R.: Lead poisoning in cattle and sheep. Vet. Rec., 63, (1951):
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Aub, J. C., Fairhall, L. T., Minot, A. S., and Reznlkoff, P.: Lead
Poisoning. Medicine Monographs, Williams and Wilkins Co., Baltimore,
Md., (1926).

Balbo, W., Gualdi, G., and Marucci, V.: Latest contribution in the study
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Bambach, K., and Burkey, R. L.: Microdetermination of lead by dithizone.
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Benjamin, M. M.: Outline of'Veterinary Clinical Pathology, 2nd-ed. The
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VITA

The author was born on a farm near Hartington, Nebraska, on November
21, 1931. He received his primary and secondary education in the public
schools of Bancroft, Nebraska. After graduation from high school in
1949, he assisted his father on the family farm. In 1953 he was drafted
into the Army and served as heavy equipment operator and motor pool
sergeant.

In 1956 he entered Michigan State University as a preveterinary
medicine student and entered the College of Veterinary Medicine in
September, 1958.

He received his D.V.M. degree in June 1962 and entered a mixed
practice in association with Dr. G. Heyt and Dr. W. Bannink in Sparta,
Michigan.

He joined the Michigan Department of Agriculture in April 1963 as
an area veterinarian and was assigned to Sanilac County. In June 1965
he transferred to the department's control laboratory in East Lansing.

He entered the graduate school in 1966, majoring in veterinary pathology.

The author married Mary Jean Chase in 1953, and they have 3

children: Robert, 16, Nancy, 8, and Kenneth, 3.

46