116

HEPATIC CLEARANCE 0F BILTRUBTN
IN RABBITS

 

Thesis for the Degree of Ph. D.
MICHEGAN STATE UNIVERSITY
MARY JEAN LON
1968

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ABSTRACT
HEPATIC CLEARANCE OF BILIRUBIN IN RABBITS
by Mary Jean Long

The kinetics of bilirubin clearance was studied for
the application as an estimation of erythrocyte life span.
When the mean red blood cell survival time is known, the
activities of the productive and destructive processes can
be calculated from the steady state level of hemoglobin con-
centration in the circulation. These processes are impor-
tant in the diagnosis and treatment of various anemias.

In the first series of experiments clearances of
fed and fasted rabbits were compared. The small difference
noted between the means of these two groups was neither
statistically nor biologically significant.

The second series of experiments was designed to
test the hypothesis that hepatic clearance is independent
of dose. The data were fitted to a straight line by the
method of least squares. The equation for that line is:

y = 38.4 x. From these data the conclusion was drawn that
bilirubin clearance in rabbits is independent of the amount
of the injected bilirubin solution. The mean cell life span

of the erythrocytes of the rabbits was 43 days.

HEPATIC CLEARANCE OF BILIRUBIN IN RABBITS

By

Mary Jean Long

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Pathology

1968

 

DEDICATION

Dedicated to all medical technologists.
This is one small way in which I can express
my sincere thanks to the profession and my
hope that I can pass along a little spark of
incentive or enthusiasm to continue the acquisi-

tion of knowledge and skills.

a 4-'_

 

ACKNOWLEDGMENTS

To Dr. A. E. Lewis, sincere gratitude for counsel
and guidance in this task, for expertise in performance of
the statistical evaluation of the data, for limitless pa-
tience and for the unselfish gift of many hours of his time.

To the Department of Pathology for providing the
original impetus that propelled me on toward this degree,
for providing the graduate fellowships and the part time
assistantship that supported my progress, and for providing
the space and facilities for completing the experimental
work and a comfortable corner for study, "Thank you."

To my guidance committee, Drs. Morrill, Langham,
Lewis and Virginia Mallman, for comments and suggestions
in preparation of this thesis, "Thank you."

To my colleagues, the fellow graduate students, for
advice, companionship, commiseration, and assistance with
the rabbits, my heartiest thanks.

To MSU for six rewarding years of learning and

growing culturally, "Thank you."

Is._

TABLE OF CONTENTS

INTRODUCTION. . . . . . . . . .
LITERATURE REVIEW . . . . . . .

Erythrocyte Life Span .

Hepatic Clearance of Bilirubin. .

The Diazo Reaction. . .

Review of Quantitative Bilirubin Methods

MATERIALS AND METHODS . . . . .

The Rabbit as an Experimental Animal.

BilirUbin C O C O O O O

Hemoglobin and Packed Cell Volume

Chemical Analyses . . .

Experiments with Ultraviolet Absorption
Chemicals Used in Chemical Analyses .

Example of Calculations for One Experime t

EXPERIMENTS . . . . . . . . . .
RESULTS 0 O 0 O O O O O O O O 0
DISCUSSION 0 O O 0 O O O 0 O O O
Hepatic Function Tests.
Experiments . . . . . .
Formulas and Equations.

SUMMARY AND CONCLUSIONS . . . .

LIST OF REFERENCES. . . . . . .

n

I. Bilirubin Clearance and Tolerance Tests. .
II. Bilirubin in Amniotic Fluid. .

III. Erythrocyte Survival

IV. Erythropoiesis and Bile Pigment Formation.
V. Methods for Quantitative Determination of

Bilirubin. . . . . .

VI. Miscellaneous References

GENERAL REFERENCES. . . . . . .

Page

10
12

15

15
16
l6
17
18
l9
19

22
24
30
3O
32
35
39
41
41
42
42
44

45
46

48

.' .rv

 

LIST OF TABLES
Table Page
1. Bilirubin Clearances of Fasting and Nonfasting
Rabbits O O O O O O O O O O O O O O O O O O O O O 25
20 Data for DOSE-Area Curve. 0 o o o o o o o o o o o 26

3. Mean Erythrocyte Life Span in Days. . . . . . . . 28

4. Hepatic Clearance of Bilirubin Independent of
Dose 0 O O 0 O O O O O O O O O 0 O I O O O O O 0 O 29

LIST OF FIGURES

Figure Page

1. Area vs. Dose Curve. . . . . . . . . . . . . . . 27

INTRODUCTION

The purpose of this investigation was to study the
kinetics of bilirubin clearance with particular reference
to its application in the estimation of erythrocyte life
span.

The measurement of mean erythrocyte life span is
an important parameter in the analysis of the mechanisms
involved in the production of various kinds of anemia.
During any steady state the concentration of hemoglobin
(or red blood cells) in the circulation is a resultant aris-
ing from the opposing actions of the rate of erythropoiesis
by the bone marrow and the rate of destruction and removal
of erythrocytes by the reticuloendothelial (R.E.) system.
Thus, anemia may arise from inadequate activity by the bone
marrow or by a shortening of the life span of erythrocytes
arising either from an intrinsic defect in the cells or some
extrinsic hemolytic mechanism. When the mean life span of
erythrocytes is known, the activities of the productive and
destructive processes can be calculated from the steady state
level of hemoglobin concentration in the circulation.

No distinction between old and young red blood cells
can be made by direct examination of blood smears, except
for some basophilia and the generally larger size of reticulo—

cytes. At present, clinically useful estimates of erythrocyte

life span rely on some means of identifying a sample of
erythrocytes. (20-41) These may be classified into two
groups: 1) cohort labeling and 2) random labeling.

Cohort labeling involves the labeling of some build—
ing material of the erythrocyte with an isotope. By feed-
ing the material over a period of two days, the erythrocytes
formed during this interval will be labeled with the isotope.
The life span of the labeled cells is determined by observ-
ing their persistence in the circulating blood. In a nor-
mal subject this may require observations extending as long
as 120 days.

Random labeling involves taking a random specimen
of blood in which, presumably, the representation of each
age group of cells in the specimen will be proportional to
their relative concentrations in the circulating blood.

In random labeling the specimen of blood is incubated with
an isotope and reinjected into the circulation. Changes
in the concentration of the circulating isotope occur more
rapidly than in the cohort method and definite results may
be obtained within a few weeks.

The method of Ashby (20, 21, 78) represents the
first successful technique used for estimation of mean
erythrocyte life span and was used as early as 1919. This
method involves a transfusion of Group 0 cells into a recip-
ient of either Group A or B. At suitable intervals after

the transfusion, blood samples from the recipient are

treated with the appropriate antisera to agglutinate or
hemolyze the recipient's cells, leaving the donor cells in
suspension where they can be counted or otherwise measured.
Essentially this is a form of random labeling, but has lim-
ited practical application because of the difficulty in the
measurements and in the hazards involved in transfusion.
Most of the above methods yield excellent results
but have the severe disadvantage in the requirement of a
minimum of two to three weeks before useful results are ob-
tainable. In order to overcome this objection it has been
proposed that bilirubin clearance be used for the clinical
estimation of mean erythrocyte life span. (29, 30, 35) From
the measurement of bilirubin clearance the rate of bilirubin
formation can be calculated, and since this is chiefly a
reflection of the rate of erythrocyte destruction, the frac—
tion of total circulating hemoglobin destroyed each day can
be calculated. From this fraction the mean erythrocyte life

span is readily estimated.

 

LITERATURE REVIEW

Erythrocyte Life Span

Erythrocyte life span is definite for normal indi-
viduals of each species and is never reported increased
beyond normal for that species, except for the hibernating
marmot and the turtle which have been reported having red
blood cells with life spans increased to eleven months.
All other abnormalities are represented by shortening of
the life span. Measurements of erythrocyte life span have
been found useful in the study of unexplained anemias.

Ashby accomplished the first measurements of eryth-
rocyte life span in 1919. In spite of the objectionable
features of her method it remained the only technique avail—
able until 1942 when Shemin and Rittenberg (37, 46) began
their series of investigations into the biosynthesis of
purines and porphyrins. They administered glycine labeled

15

with the nonradioactive isotope N to human subjects and

to rats and found that the isotopic nitrogen is incorporated
into hemoglobin. Analyses of atom per cent excess of N15
are difficult and require the sophisticated use of a mass
spectrograph thus rendering this technique unsuitable for
ordinary clinical use. In spite of these difficulties this

group under the leadership of I. London (46) employed N15

labeling of hemoglobin to measure the life span of

erythrocytes in various diseases and at the same time meas-
ured the associated rates of conversion to bile pigments.
With these studies it was shown that the mean human eryth-
rocyte life span is 126 I 7 days. Thus the classical esti-
mates of Ashby were confirmed, but an unexpected finding
was that 20-40% of the bilirubin excreted is synthesized
from nonhemoglobin sources and from ineffective erythro-
poiesis in the bone marrow. (42)

Probably the most commonly used technique for the
measurement of mean erythrocyte life span in clinical prac-
tice is the chromium51 labeling technique. When erythrocytes
are incubated in an isotonic solution containing sodium
chromate, the chromate enters the cell and is fixed by the
cytoplasm. Using the radioactive gamma emitter, chromium
Sl (CrSl) random labeling is easily accomplished. An added
advantage in the use of a gamma emitter is the possibility
of detecting loci of sequestration and erythrophagocytosis
by external monitoring. (23) A major disadvantage in the
use of Cr51 is the continued elution of the chromate from
the erythrocytes while they are in the circulation. The
elution rate averages 1% per day, but varies considerably
among individuals. Analyses of the time concentration curves
are complicated by the combined effects of erythrocyte de-
struction, radioactive decay, and variable elution. To
avoid complex mathematical analyses, semi-log plots of the

data obtained during the first two weeks are fitted to a

 

calculated straight line in order to estimate the apparent
biological half-life of the isotope. The result is compared
to empirically determined normal values. The normal value
for the biological half-life of Cr51 under these circum-
stances is approximately 25 days. Because chromium tagging
permits external monitoring, this technique will remain in
use. However, the method that uses diisopropylfluorophos-
phate (DFP) labeled with radioactive phosphorus 32 is the
simplest and most reliable. Although phosphorus 32 is a
fairly strong beta emitter, it is not as readily monitored
with external scintillation counters. DFP is one of a class
of organophosphates which bind firmly to cholinesterase.
Cholinesterase is present on the surface membrane of the
erythrocyte, and incubation of erythrocytes in a solution
containing DFP32 labels the erythrocytes randomly. Some
elution occurs during the first 5-7 days after injection
of the labeled cells, but following this initial brief in-
terval the label persists until the cell is destroyed.
Another way to estimate the life span of the eryth—
rocyte is to calculate hemoglobin destruction rate from the
reticulocyte count by dividing the observed number of retic—
ulocytes by the normal number. (24) This method gives only
an approximation and assumes the presence of adequate eryth-
ropoietic response. In another method, hemoglobin destruc-
tion rate is calculated from the carboxyhemoglobin concen-

tration of the circulating blood. (28, 29, 30, 37, 73) This

 

method cannot be used on smokers or others exposed to car-
bon monoxide in large quantities. One other method uses

sulfhemoglobin measurements.

Hepatic Clearance of Bilirubin

Calculation of erythrocytic survival times from data
of bilirubin clearance tests was originally accomplished
by Lewis and Gershow in 1961. (35) They postulated that
in normal individuals the bilirubin concentration is rela—
tively stable, the amount excreted being equal to the amount
of hemoglobin destroyed and therefore directly related to
the life span of the erythrocyte. The rabbit was chosen
for the experimental work because of the possibility of
toxic reactions to commercially available bilirubin. From
the calculations on the rabbit experiments a mean erythro-
cyte life span of 51.97 days was determined.

Six years later a team of Swedish investigators (29,
30) repeated the experiments of Lewis and Gershow using hu-
man subjects to estimate red blood cell survival by means
of bilirubin clearance. Five normal individuals, 14 patients
with hemolytic anemias and 2 with pernicious anemia were
given intravenous injections of bilirubin. Time versus log
plasma-concentration of bilirubin curves were plotted for
injected doses of 1 mg. per kg. of body weight. Straight
lines were obtained in every experiment. A larger injected
dose, 3 mg. per kg., produced data which when plotted pre-

sented a biphasic curve, with the second part of the curve

 

forming a line parallel to the line obtained with the smaller
dose. They compared red cell survival times by three methods:
the bilirubin clearance method, the radio-isotope random

labeling method (with both Cr51 and DFP32

) and the carboxy-
hemoglobin method. Good agreement was found between the
isotope and bilirubin methods, but the isotope and carboxy-

hemoglobin methods gave poor correlation. They attributed

the wide variation in calculations of red blood cell survival

.- _.d___ _-._T

time using isotopes to the fact that the observation period
was only 26 days. It would be necessary to extend the ob-
servation period for a longer time for reproducible results;
this reemphasizes the fact that for clinical purposes the
isotope methods take too long. Mean values for fractional
turnover rate and plasma clearance of bilirubin were of the
same magnitude in the normals as in the patients with hemo-
lytic disease. This demonstrates that hepatic removal of
bilirubin is not affected by anemia. The total amount of
circulating bilirubin and the bilirubin turnover rate were
higher in the patients with increased hemolysis than in the
controls. Individual variations in the fractional turnover
rate and plasma clearance of bilirubin were wide.

That considerable amounts of bilirubin are broken
down into other excretory substances was demonstrated by
Ostrow, Jandl and Schmid in 1962. (47) Thus the present
method of measuring erythrocyte life span by bilirubin clear—

ance gives values that are too high. In the experiments of

Ostrow SE.2£¢: only 63-80% of Cl4 labeled cells or heme that

was administered to experimental rats was recovered as bili-
rubin—C14. On the other hand, when bilirubin is derived .
from sources other than hemoglobin (42, 46, 48, 50, 52),

this method results in an underestimate of red blood cell
life span. Thus in healthy individuals the bilirubin method
will give values 15-20% too low. These two factors tend g
to equalize one another (48, 49, 52).

An analysis of plasma bilirubin disappearance curves

was performed by Billings, Williams and Richards in 1964 (3). L

 

Thirty-eight subjects were studied: 7 normals, 22 patients
exhibiting various hyperbilirubinemias and 9 close rela-
tives of these patients. Again these data, plotted on semi-
logarithmic paper, produced biphasic curves. The second
portion of each curve had a lesser slope and a lower inter-
cept on the ordinate. They injected a single dose of 2
mg./kg. intravenously and calculated various proportional-
ity constants from the data: "a," the rate at which bili-
rubin leaves the plasma to enter the liver, "b," the rate
at which bilirubin is passed back into the plasma and "m,"
the rate at which unconjugated bilirubin is transferred to
the microsomes for conjugation.

Nixon and Monahan (8) (1967) reported on the use
of the bilirubin tolerance test to diagnose familial non-
hemolytic jaundice, Gilbert's disease. Mathematical anal-

ysis of their bilirubin disappearance curve showed a marked

10

decrease in the rate of uptake of bilirubin by the liver
(Billing's constant "a") and a significant decrease in the
slope of the second phase of the curve.

The procedure for bilirubin clearance tests imitates
closely that of bilirubin tolerance tests; the essential
difference lies in the more sophisticated treatment of the
data in the determination of clearance (1-11). Von Bergman
and Eilbot (1927) (7) introduced the bilirubin tolerance
test for clinical evaluation of liver function. They in-

jected a single standard dose and drew blood samples for

 

analysis (1) immediately before, (2) at 3 minutes and (3) l
at 3 or 4 hours after injection. Results were expressed

in percentage retention and varied from 10-20% in normal

subjects. In 1943 With (7) modified the procedure taking

more frequent blood samples and plotting disappearance of

bilirubin from the plasma. He was unable to detect any dif—

ference in the characteristics of the curves of the patients

with liver disease and those of normal individuals, although

the patients with liver disease tended to show high reten-

tion values.

The Diazo Reaction

 

Paul Ehrlich (65) introduced the use of the diazo
reaction for the demonstration of bile pigment in urine.
He discovered (1883) that bilirubin reacts with diazotized
sulfanilic acid to form a red coupling product. Van den Bergh

and Snapper (1913) (65) were the first to utilize the reaction

11

for the determination of bilirubin in serum. The diazo
reaction is now the basis of practically all methods for

the determination of bile pigment in serum. The general
principle of the reaction is that diazonium salt and an
"accelerator" are added to diluted serum. The diazo reagent
is usually prepared freshly immediately before each deter-
mination by mixing an aqueous solution of sodium nitrite

(B) with a solution of sulfanilic acid in hydrochloric acid
(A). Ehrlich and van den Bergh used alcohol as an acceler-
ator. Any substance that renders the unconjugated bilirubin
soluble so that it can react with the diazonium salt may

act as an accelerator.

Actually two chromatographically separable azo pig-
ments are formed from the unconjugated bilirubin, but they
are regarded as one because they are isomers and have iden-
tical spectra in visible light. In slightly acid solution
this pigment is red with a maximum absorbance at 540 mu.

It also has the prOperties of an indicator, turning blue
in alkaline solution with a maximum absorbance at 600 mu.
The diazo pigment is much more resistant to light and oxi-
dation than the bilirubin alone.

Of the known bile pigments that form red compounds
during the diazo reaction, bilirubin is the only one found
in the blood. Therefore, the formation of a red compound
may be regarded as specific for bilirubin in the blood.

Diazonium salts also react with other organic substances

 

12

in the blood, namely the amines and phenols, to form vari-
ously colored products. These reactions are referred to
as the yellow and brown diazo reactions. Serum proteins
produce a yellow reaction as a result of diazotization of
histidine and tyrosine, and this reaction occurs with all

sera. Uremic sera produce a brown diazo reaction.

Review of Quantitative Bilirubin Methods

The scientific literature abounds with methods for
the quantitative determination of bilirubin (53-71). The
Malloy and Evelyn method employing the diazo reaction is
probably the best known and most often used. Many modifi-
cations of this as well as other diazo methods have been
proposed. Jendrassik and Grof used a buffered caffeine-
sodium benzoate solution as an accelerator of the diazo
reaction and addition of alkali at the conclusion of the
reaction to convert the azo pigment to the blue compound.
Adaptations of the Jendrassik and Grof method have been
published by With (70) who reports, however, that these
methods give erroneous results due to the strong transient
acceleration of the direct reaction by the addition of the
alkali. Nosslin (65) further modified the method of
Jendrassik and Grof by replacing the accelerator solution
with water and terminating the reaction with ascorbic acid
before treatment of alkali. The ascorbic acid combines with
the excess diazo reagent.

In 1960 Meites and Hogg (58) published a method in

13

which serum is diluted with a pH 7.4 phosphate buffer and
read directly in a spectrophotometer at two wavelengths,

455 mu and 575 mu. This method, which is merely an improved
icterus index, has disadvantages: (l) lipochrome substances
may be present in adult blood with increases in absorbencies
at 455 mu, giving false high values; (2) the method tends

to be less accurate at concentrations below 1-2 mgs./lOO ml.

Minimal amounts of hemoglobin in the serum give
falsely low values of bilirubin concentration by the diazo
methods although no adequate explanation exists. McGann
states, (64) "Qualitative as well as quantitative differences
in the effects in individual serum samples make correction
of the van den Bergh type of reaction for the presence of
hemolysis uncertain at the present time."

One solution to the problem of quantitating low con—
centrations of bilirubin in sera is to extract the bilirubin
from the serum specimen with chloroform, gently evaporate
to dryness under a vacuum at a temperature not to exceed
40 C, dissolve the residue in 2.5 ml. of chloroform and read
at 450 mu against a solvent blank (18). The disadvantage
of this method is that when the chloroform is first added
to the serum the proteins precipitate taking an unknown quan-
tity of bilirubin with them, since bilirubin is transported
in the serum in loose combination with protein, largely al-
bumin.

In quantitating bilirubin of amniotic fluid which

14

is important in deciding whether to transfuse erythroblas-

totic infants in utero or not, minute differences in the

 

concentration of the unconjugated bilirubin have to be
measured. (14) Methods have been devised to accomplish

this by scanning the fluid at wavelengths from 300 to 700

mu. The net absorbence at 450 mu is obtained by measuring
the difference in absorbence at the peak at 450 mu and at

the expected slope at 450 mu. The expected slope is obtained
by connecting the lowermost portion of the tracing on each

side of the peak with a straight line (12-19).

 

MATERIALS AND METHODS

The Rabbit as an Experimental Animal

The rabbit was used in this research because (1)
it is readily available, (2) it is of a satisfactory size
and temperament to be handled easily while injecting bili-
rubin and drawing the necessary blood samples, (3) it can
be housed readily, (4) the ear veins are easy sources of
blood samples, (5) it is not affected adversely by bili-
rubin (72).

Fourteen Dutch Belted female rabbits were purchased
and maintained on a standard rabbit pellet diet in individ-
ual cages. Bilirubin solution was injected into each rabbit
on two separate occasions, once fasting and once nonfasting.
A minimum of three weeks between experiments on any one
rabbit was maintained to allow for recuperation of the ear
veins. The rabbits were weighed at the beginning of each
experiment. Vaseline was applied to the ears after each
experiment to assist the healing process, eliminating scab-
bing and formation of excess scar tissue.

The marginal ear vein was used for injections of
the bilirubin solution as well as for obtaining the blood
samples. Shaving the ear hair with a new razor blade facil-

itated withdrawal of the blood samples. A 1 m1. disposable

glass tuberculin syringe with a #25 disposable needle were

15

16
used to obtain the blood specimens.

Bilirubin

Superior grade bilirubin in dry crystalline form
was purchased in 1 gm. amounts. The formula weight was
584.68. The designation superior is applied to organic
chemicals "having a degree of purity controlled at the
highest level consistent with reasonable cost and demon-
strated demand" (76). Fresh bilirubin solution was prepared
at the beginning of each experiment. One hundred milligrams
of the dry chemical were weighed out with a gravimetric
balance. Ten milliliters of 0.1 M Na2C03 were heated to
boiling, cooled to 80 C and the 100 mg. of bilirubin added
to the solution (10). The bilirubin was dissolved by gentle
swirling and allowed to cool to room temperature before in-

jecting into the rabbit. This solution was clear and had

a dark brown color resembling iodine in solution.

Hemoglobin and Packed Cell Volume
Blood was obtained from the marginal ear veins and
transferred into heparinized capillary tubes identical to
those used routinely in a hematology laboratory. For each
sample of blood, four to eight capillary tubes were filled.
The tubes were identified by storing them vertically in a
white plastic rack with slots numbered one through eight.

Hemoglobin was determined by the cyanmethemoglobin method

(74) on the preinjection specimen of each rabbit.

 

17

Microhematocrit values (packed cell volume) were also deter-
mined on the specimens obtained prior to injection of bili-

rubin.

Chemical Analyses

 

The method chosen for performing the quantitative
analysis for bilirubin concentration is an adaptation of
the method of Malloy and Evelyn and is referred to as the
micro total bilirubin (57). This method is that of Malloy
and Evelyn except that all reagents are measured in one-tenth

amounts. One hundred lambda of serum are used with a final

 

solution volume of 1.0 ml. The microcuvette used is 10 x
75 mm. and has a light path of 1 cm.
One experiment was performed using the ultramicro-
method of Knight (60) which utilizes a serum sample of 20
lambda and a final volume of 220 lambda. These determina-
tions were read in a Coleman Jr. spectrophotometer using
an ultramicro cuvette which is rectangular and requires only
100 lambda final volume yet provides a light path of 1.0 cm.
Standards for the bilirubin determinations were pre-
pared by weighing out 20.0 mg. of dry crystalline bilirubin
on a gravimetric balance, dissolving in chloroform in a 100
ml. volumetric flask and filling to the mark with the solvent.
This was labeled "Stock Bilirubin Standard, 20.0 mg./100 ml."
Ten milliliters of the stock standard were diluted to 100 ml.
with absolute methyl alcohol (CH3OH). Dilute standards were

made by adding 2 ml., 3 ml., 4 ml. or 5 m1. of this dilute

18

standard to numbered test tubes and bringing the final vol—
ume to 10 ml. with absolute CH3OH in each tube. Nine-tenths
of a milliliter of each of these dilute standards and 0.1

m1. of fresh diazo AB reagent were added to matched micro
cuvettes (10 x 75 mm.). The rack of cuvettes containing

the reacting standards was placed in the dark for 30 minutes.
The per cent transmittance of each solution was read at 540
mu at the end of this reaction period. The wavelength of

540 mu was predetermined by running maximum absorbence curves
on the spectrophotometer with suitable bilirubin solutions
treated with diazo reagent identically as in the test itself.
Suitable solutions are defined here as solutions that by
trial give readings in the range of 20 to 60% transmittance
over the wavelengths tested. Readings outside this range
represent solutions that are either too light or too dark

for accurate photometry. In other words, the error increases
disproportionately when the transmittance is less than 20%

or more than 60%. Lambda pipettes were used to measure all

the volumes of 100 lambda or less.

Experiments with Ultraviolet Absorption
The absorption spectrum of bilirubin in the UV range
(230-280 mu) was explored to see if any absorption peaks
were present that could be used for simpler micro-analytic
procedures. If any peaks exist they are obliterated or ob-
scured by the protein present and no improvement over exist-

ing methods was possible.

19

Chemicals Used in Chemical Analyses

 

Absolute methanol: the methyl alcohol used in the
chemical analysis conformed to American Chemical Society
specifications for reagent methanol.

Dilute hydrochloric acid: 15 ml. of concentrated
hydrochloric acid, reagent grade, USP specifications were
diluted to one liter with distilled water.

Sodium nitrite: 0.5 gm. pure sodium nitrite was
dissolved in 100 ml. distilled water (Diazo B).

Sulfanilic acid: One gram of dry sulfanilic acid
was placed in a one-liter volumetric flask; 15 ml. of con-
centrated HCl were added and distilled water added to the
mark (Diazo A).

Diazo Reagent: 0.3 ml. sodium nitrite solution
were added to 10.0 ml. of sulfanilic acid solution and

mixed (AB).

Example of Calculations for One Experiment
I. Fitting the data to a straight line by the method of
least squares (75):

The formulas for finding the best "fit" of the ex-
perimental data to a straight line described by the equation
y = A x + B are:

23y = A z x + n B

21xy = A 2 x2 + B z x
in which B is the intercept on the ordinate and A is the

slope of the straight line given by the formula logP=B + At

 

 

x P
Time Conc. in y
min. mg./100 ml. log conc. xy

0 .4

6.5 13.3 1.1239 7.3054
11 14.0 1.1461 12.6071
22 7.8 .8921 19.6262
27 8.1 .9096 24.5592
44.5 4.5 .6542 29.1199
62 3.8 .5763 35.7306
Dose: 4 ml. x 6.63 mg./100 ml. = 26.52 mg.
Q = 26.52 wt.

2.5128

0 = 10.4 mg./kg.
5.3022 = 173 A + 68
128.94 = 7200.5 A + 1738
152.8624 = 4987.59 A + 1738
— 23.92 = 2212.9 A

A = —.0108
5.3022 = -1.8684 + 6B

GB = 7.1706

B = 1.195
log P =
the data)

II. Fractional clearance
%-= .02484 %-= 2.3 x slope

20

42.25
121.00
484.00
729.00

1980.25
3844.00

Hb = 1106 gmo/
100 ml.

Hct = 36%

P' = 004 “190/
100 m1.

log P = y

time = x

injected

= rabbit 2.5128 kg.

1.195 -.Ollt (equation for the line that fits

 

21

A1

A2

 

= 0.0226

III. Area under curve:
mg./100 m1.
Time conc. ._
in min. At. P 2P
0 15.7
6.5 6.5 13.3 29
11.0 4.5 14.0 27.3
22.0 11.0 7.8 21.8
27.0 5.0 8.1 15.9
44.5 17.5 4.5 12.6
62 17.5 3.8 8.3
3.8
A2 = 2U3£ .011 = 152'00
_ mg. min.
A total _ 650.00 100 m1.
IV. Mean cell life:
C (l—Hct) P'
.025 x 0.4 x .64
F = 4114 x 11.6
1
F = 4402

2A Po is determined

from the intercept
188.5 of the ordinate on
122.85 the graph of the
239.8 straight line for
79.5 the equation log P
220.5 = 1.195 - 0.11 t.

145.3

J 996.45

498.225
152.00

650.00

Mean cell life for the erythrocytes of this rabbit is

44.2 days.
V. Clearance:

C:

>K)

[—1

0.4 mg.
7 750 mg. min.7100 m1.

= 1038 mlo/mino

~7Tr

 

 

EXPERIMENTS

At the beginning of each experiment the rabbit was
weighed and a sample of blood obtained for measuring the
base-line bilirubin level, hemoglobin concentration, and
packed cell volume. The bilirubin solution, prepared as 57
described in Materials and Methods, was poured into a ster-
ile 10 ml. syringe, the barrel inserted, air carefully ex-

pelled and the barrel marked at the level of the top of the

 

chamber of the syringe. Using a No. 23 hypodermic needle,

1r“-

the bilirubin solution was injected very slowly into the
marginal ear vein, pausing from time to time to prevent
build-up of pressure within the vein. At the completion
of the injection the needle was slowly and gently withdrawn
with a pledget of cotton applied to the area with pressure
until the danger of hematocyst formation had passed. The
barrel of the syringe was again marked at the end of the
injection, for later measurement of the exact volume of
bilirubin solution injected. A stop watch was started im-
mediately upon removing the needle from the vein. At ap—
proximately 5, 10, 15, 30, 45 and 60 minutes, blood speci-
mens were taken from the marginal vein of the opposite ear
using a No. 25 needle and a disposable 1 m1. tuberculin
syringe. The exact time of withdrawal of each specimen to

the nearest second was noted. Heparinized capillary tubes

22

23

were filled from the syringe and one end sealed with plasti-
cene. At the end of the sampling period the capillary tubes
were centrifuged for 5 minutes in a high-speed microhemato-
crit centrifuge. Packed cell volumes were read on the base-
line specimens and recorded for later calculations of mean
cell life span.

Micro total bilirubin tests were performed on the
blood samples according to the Hogg and Meites (57) adapta-
tion of the Malloy and Evelyn method as detailed above.

Per cent transmittance was read in the Coleman Jr. spectro-
photometer, converted to optical density and multiplied by

a factor predetermined by calculation from the readings of

the standards to yield a concentration in mg. per 100 m1.

Two experiments were done on each rabbit, one with
the rabbit fasting and one nonfasting. For the fasting
experiment the food dish was removed from the cage approx-
imately 18 hours before the start of the experiment. Water

was available to the rabbit at all times.

VT

 

RESULTS

The first series of experiments to determine the
effects of gastrointestinal (and therefore, indirectly,
hepatic) blood flow changes compares the clearances of fed
and fasted rabbits. The results are summarized in Table l.
The small difference noted between the means of these two
groups is neither statistically nor biologically significant.

The second series of experiments is designed to test
the hypothesis that hepatic clearance is independent of dose.
If this hypothesis is false, the deviations of a linear plot
of the areas derived from the time-concentration curves as
a function of dose should show a consistent rather than a
random scatter about the line. The numerical data of this
series of experiments are summarized in Table 2. These data,
fit to a straight line by the method of least squares, are
shown in Figure 1.

During the course of this study, we obtained data
on 14 rabbits for the estimation of erythrocyte life span.
The calculated life spans are summarized in Table 3. The
mean cell life of the red blood cells of the rabbits in these
experiments is 43 days.

Table 4 summarizes the measurements of the hepatic
clearance of bilirubin. Note that the variation, as indi—
cated by the relationships of the standard deviations to

the mean, are comparable in both Tables 3 and 4.

24

25

TABLE l--Bilirubin Clearances of Fasting and Nonfasting

 

 

 

 

Rabbits
Nonfasting Fasting

Experiment Clearance in Experiment Clearance in

number ml./min./kg, number m1./min./kg.
10 2.81 15 2.37
14 3.95 16 2.57
17 1.84 18 1.91
19 2.16 13 5.21
20 1.89 21 1.33
23 3.49 22 3.35
Mean clearance 2.69 Mean clearance 2.79
S.D. 0.88 S.D. 1.36

t = 2004

5% < P < 10%

26

 

 

 

TABLE 2-—Data for Dose-Area Curve
Experiment number Dose in mg./kg, Area
10 16.7 593
13 2.66 52
14 5.87 149
16 8.8 341
17 10.4 564
18 10.0 524
19 6.22 287
20 3.6 191
21 15.1 1135
22 6.47 192
23 35.9 1028
y = 38.4x
Standard error of estimate = 215.27

Y
‘1

27

Area in mg. min./100 m1.

 

 

 

 

1200?
1000“

80:3 1

600'

400

O Fasting
2001- . Nonfas ting
0 Dose in mg ./kg.,
0 e . . . L

Figure 1. Area vs. Dose Curve

28

TABLE 3--Mean Erythrocyte Life Span in Days

 

 

 

Rabbit Mean Cell Life in Days
1 40.44
2 65.87
3 47.53
4 38.91
5 25.71
6 39.37
7 74.23
8 17.15
9 51.81
10 85.17
11 47.68
12 33.22
13 21.46
14 20.92
Mean calculated life span = 43.0 days

S.D. 20.3 days

95% confidence limits of mean I 10.9

29

TABLE 4-—Hepatic Clearance of Bilirubin Independent of Dose

 

 

 

 

No. mg./kg. m1./min./kg,
Experiment Dose Clearance

13 2.66 5.21

20 3.60 1.89

14 5.87 3.95

19 6.22 2.16

22 6.47 3.35

16 8.8 2.57

18 10.0 1.91

17 10.4 1.84

21 15.05 1.33

10 16.7 2.81

15 25.4 2.37

23 35.91 3.49
Mean = 2.74 m1./min./kg.
S.D. = 1.09

95% confidence limits

of the estimated mean i 0.64

DISCUSSION

Hepatic Function Tests

 

The liver plays such an important role in the ani-
mal body that the study of methods for testing its functional
ability has always attracted considerable interest. The
bilirubin tolerance or clearance test is one method for
testing one of the many diverse functions of the liver,
that of excretion of waste products from the blood. This
test is important in the diagnosis of mild liver injury
where plasma bilirubin concentrations are not elevated.
Usually the test is reported as per cent retention of bili-
rubin when it is used as a tolerance test.

In the bilirubin clearance test the bilirubin solu-
tion, which before injection gives a direct van den Bergh
reaction with diazo reagent, is adsorbed by the plasma pro-
teins as soon as it enters the circulation and the van den Bergh
reaction becomes indirect. This can readily be demonstrated
by adding a solution of bilirubin to blood serum in vitro.

Indirect reacting bilirubin is not excreted through
the kidneys. Neither is it stored by the Kupffer cells of
the liver as occurs with other foreign pigments injected
into the blood stream. The bilirubin is not phagocytized
by the R.E. cells; it is totally excreted by the liver.

The rate of excretion of bilirubin injected intravenously

30

31

may therefore be used as a test for liver function, specif-
ically a test of the clearance of bilirubin from the plasma
by the liver.

Clearance is a term used to denote the excretory
capacity of the kidney. Lewis (74) applied the methods and
concepts of renal clearance to hepatic clearance in 1950.

Bilirubin clearance measurements can be used for F“‘
two purposes both clinically and in the study of small ani-
mals. Bilirubin clearance provides: (1) directly, a meas-

ure of one function of the liver, and (2) indirectly, the

 

mean life span can be determined using additional hematologic
data.

Although a quantitative estimate of the function
of the liver is provided by measurements of the clearance
of bromsulfalein (Lewis, 1950), (74) the clearance in this
instance is dependent both on the activity of the hepatic
parenchyma and the blood flow to the liver. The data pre-
sented here demonstrate that bilirubin clearance does not
change with blood flow to the liver, since the clearance
measurements on fed and fasted animals are essentially the
same, differing by about 5% of the mean. Similar studies
with bromsulfalein show a drop of about 40% in clearance
when the animal is fasted. The rabbit is particularly suit-
ed for studies to determine the effect of hepatic flow.
Being a herbivore it has an unusually large gastrointestinal

tract with a correspondingly abundant blood supply.

32

Undoubtedly there are large changes in the blood flow through
this tract and through the portal vein, depending upon the
activity of the bowel. The large effects of fasting demon-
strated by Lewis in his measurements of bromsulfalein clear-

ance substantiate this idea.

Experiments
Aside from the potential clinical utility of the

bilirubin clearance test, demonstration of its applicability

 

in rabbits provides an important investigative tool for

evaluation of the toxic effects of drugs as well as other

'1.

physiologic variables of interest. Hepatotoxicity is gen-
erally easily recognized, but demonstration of possible de-
pressant effects on the activity of bone marrow is generally
far more difficult. Ordinarily, changes in bone marrow
activity are detected by routine hematologic measurements,
such as hemoglobin (Hb), red blood cell (RBC) count, packed
cell volume (PCV), and reticulocyte count. Assuming that
the mean erythrocyte life span in the rabbit is 60 days,
it follows that complete cessation of bone marrow erythro-
poiesis would have to persist for as long as six days before
a drop in Hb concentration of 10% would provide definite
evidence of defective function. Incomplete depression of
erythropoietic rate would, of course, require still longer
time intervals before manifestation.

Since reticulocytes are the youngest cells of the

erythrocytic family normally in the circulation, complete

33

depression of erythropoietic activity would be accompanied
by complete and immediate disappearance of these cells from
the peripheral blood. However, a partial depression amount-
ing, for example, to 50% would merely reduce the reticulo-
cyte count from 1% to 0.5%. If the estimation of the retic-
ulocyte percentage were precise, it could be used as a direct
index of marrow activity. Unfortunately, this measurement
depends on the direct enumeration of supravitally stained
reticulum filled cells, and counts of this kind show the
statistical variation characteristic of the Poisson distri-
bution. This inherent statistical error is so large that

it is not feasible to distinguish between a reticulocyte
count of 1% from one of 0.5%. Recently Maturen (77), work-
ing in this department, showed that reticulocyte counts may
be reliably estimated from the magnesium content of packed
cells. Combined with the measurement of mean red blood cell
life span, using the bilirubin clearance, a fairly complete
picture of the hematologic situation, so far as erythrocytes
are concerned, can be obtained. Thus, for example, if hemo—
lytic anemia were present in a rabbit so that the mean
erythrocyte life span was diminished to about 30 days, pre-
sumably there would be compensatory hyperplasia of the bone
marrow. Assuming reticulocytes represent approximately one
day old cells in the rabbit as they do in humans, the retic-

ulocyte count would reach 2% when the steady state was at-

tained. From this figure alone it would not be possible

34

to determine whether this represented a transient response
of the marrow or an established compensatory hyperplasia.
The bilirubin clearance added to the data would reveal that
the rate of destruction of cells, and therefore the rate
of erythropoiesis was doubled. The clearance value inde-
pendently would reveal any associated damage to the liver.
Before these valuable possibilities of the bilirubin _m—
clearance could be used with confidence the investigations A
summarized in this study were essential. The investigations E

of Engstedt, Johansson, and Nyberg (28, 29, 30) revealed

I' ALL".a-"IJ . J

that the time concentration curve of serum bilirubin after ',§
large injections was biphasic. This biphasic curve would

be consistent with two possible explanations. First, since
it has been established that the excretion of bilirubin in-
volves a series of enzymatic, energy consuming processes
within the hepatic cell (44), high levels of serum bilirubin
might inhibit regulator enzymes, permitting the excretory
mechanism to operate at an accelerated rate. Such is common
in enzymatic processes. If this were the case, clearance
would vary with dose or serum level and could not be used
either as a measure of hepatic function nor as a means of
estimating mean erythrocyte life span. On the other hand,
the biphasic character could just as easily be explained

on the basis of the rates of diffusion from the plasma to
the tissues and back again. This process would be independ-

ent of hepatic activity, and therefore calculation of

35

clearance from the area of the time-concentration curve as
a more or less fixed parameter would be valid. The inde-
pendence of the clearance estimate as related to blood flow
suggests that the limiting process is the concentration of
bilirubin at the hepatic cell membrane. The demonstrated
independence of clearance as a function of dose is consis-
tent with the hypothesis that the biphasic curve of time he
plasma concentration represents the effects of diffusion

of bilirubin to and from the extravascular spaces. F

Formulas and Equations

 

The clearance of a compound is the volume of circu-
lating plasma cleared of that compound in a given interval
of time. Under normal conditions the bilirubin concentra—
tion in the circulating plasma is constant. Bilirubin is
excreted at the same rate that it is formed and conversely,
it is formed at the same rate that it is excreted. If R
is the rate of bilirubin formation in mg./min. and P is the
concentration of bilirubin in the circulating plasma reported
in mg./ml. then C, the clearance of bilirubin from the plasma

in m1./min., is equal to R divided by P.

Hepatic clearance of bilirubin can be represented
by the formula'% where Q is the dose, the amount of injected
bilirubin solution in mg. This value is then divided by

the weight of the rabbit in kg. to eliminate an added

36

variable, the size (or weight) of the rabbit. A is the area
under the curve (in mg. min./ml.) plotted from the data from
the experiments.oC Mathematically A can be represented by

the integral d}7(P-P')dt, in which P is the plasma concen-
tration of bilirubin at a given time, t, and P' is the con-
centration of bilirubin before the injection. Therefore,
clearance is reported in ml./min./kg.

Table 4 summarizes the measurements of the hepatic

. 'r “wen"- any:

clearance of bilirubin. Note that the variation, as indi—

cated by the relationships of the standard deviations to

" 1H.

the mean, are comparable in both Tables 3 and 4. This would
be consistent with our view that the variation in estimates
of the erythrocyte life span may be attributed as much to
the unfavorable conditions as to the precision of the tech-
nical methods. To a large extent these difficulties were
unavoidable, since many of these studies were conducted dur-
ing the summer heat. It was not feasible to allow drinking
water for the animals during the experiments, and even though
the observation periods were generally little more than an
hour, substantial water losses in such small animals can
occur during this interval in warm environments.

It can be concluded from these experiments that
there is no significant difference in the fraction of plasma
volume cleared per minute in the fasting and nonfasting
rabbits. The fraction of volume cleared per minute is ob-

tained from the slope of the line obtained by applying the

37

method of least squares to the time-plasma concentration
data of the experiments. Each experiment has an equation
derived from the experimental data. The aforementioned
equation describes the best straight line "fit" for the
points given by the data. The slope is the fraction that
describes the relationship of the logarithm of the concen-
tration of bilirubin in each timed specimen to the time in ‘TL-
minutes of that specimen. The fraction of plasma volume
cleared per minute is E-and is obtained by multiplying the

V

slope of the line by 2.3 to convert a logarithmic number

 

to a real number. 1~J
Bilirubin clearance data were applied to the calcu-

lation of red blood cell survival time using the equation

(35):

C (l-Hct) P'
F = 4114 V Hb

 

in which F is the fraction of total hemoglobin destroyed
daily, Hct is the packed cell volume expressed as a decimal
g'is the fractional clearance obtained as explained

above, Hb is the concentration of hemoglobin in gm./100 ml.

fraction,

blood. The constant 4114 combines converting minutes to
days and milligrams bilirubin to grams hemoglobin. The con-
centration of bilirubin is expressed in mg./ml. Thirty-five
milligrams of bilirubin are formed from every gram of hemo-
globin destroyed. The mean cell life is the reciprocal of
F. The results of the computations of red blood cell sur-

viAral times for the 14 rabbit experiments are summarized

38

in Table 3. These estimates manifest considerable variation

reflecting the considerable variations in the health of the

rabbits used in this study. Although these data would not

be satisfactory for determining the mean life span of eryth-

rocytes in this species, the design of the experiment in

terms of the hypotheses to be tested is still valid, as are

the conclusions. .__
The mean cell life of the red blood cells of the

rabbits in these experiments is 43 days. That determined

by Lewis (35) in 1961 was 51.97. The literature gives the

value of 50-70 days by the N15 and Fe59 isotope methods (22),

30-64 days by the Ashby (20-21) technique of differential

agglutination, 43 days by sulfhemoglobin method (29), and

45-50 days by radio-iron-tagged red blood cells (Burwell)

(35). The red blood cell survival time of 43 days agrees

with those reported in the literature.

SUMMARY AND CONCLUSIONS

The purpose of these experiments was to study the
kinetics of bilirubin clearance with particular reference
to its application in the estimation of erythrocyte life
span. When the mean red blood cell survival time is known, r-

the activities of the productive and destructive processes

v. in

- 2‘7;

can be calculated from the steady state level of hemoglobin

concentration in the circulation. These processes are im-

num' ‘Wh

portant in the diagnosis and treatment of various anemias.

 

Hepatic clearance of bilirubin in rabbits was meas-
ured 23 times. From the data resulting from mathematical
treatment and evaluation of the experimental data three
conclusions are derived:

1. Hepatic clearance of bilirubin in rabbits varies
widely among animals but no significant difference was found
between fasting and nonfasting rabbits.

2. Bilirubin clearance in rabbits is independent
of the amount of the injected bilirubin solution.

3. Bilirubin clearance may be applied to the cal-
culation of erythrocytic life span. The mean red blood cell
survival time calculated from 14 rabbits' bilirubin clear-
ances is 43 days. This figure agrees with values reported
in the literature for other methods of determining the life

span of rabbit erythrocytes.

39

REFERENCES

ma.

 

 

 

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42

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|l an", A... .’ m:
1

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Watson, C. J.: The Pyrrol Pigments, with Particular
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Yamamoto, T., Skanderberg, J., Zipursky, A., Israels,
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BILIRUBIN METHODS

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Allen, T. E.: Direct Reading Newborn Bilirubin Method
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1955.

Eichman, M. F., Kavitz, E., Porter, N. M.: A Simpli-
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Hart, C., Plant, D.: Evaluation of a Bilirubin Screen-
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Hogg, C. K., and Meites, S.: Modification of Malloy
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Hogg, C. K., Meites, 5.: Direct Spectrophotometry of
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46

Knights, E. M., MacDonald, R. P., Ploompuu, J.: Ultra
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Levinson, S. A., and McFate, R. P. Sixth Edition.
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Macclay, N. F.: An Adaption of the Powell Method for
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Siggaard-Anderson, O., Komarmy, L. E.: A Simple Bili-
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Watson, D., Rogers, J. A.: A Study of Six Representa-
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47

Coburn, R. F., Williams, W. J., Forster, R. E. Effect
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Drabkin, D. L.: The Standardization of Hemoglobin
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N.Y., 1966.

Matheson, Coleman and Bell. Catalogue of Scientific
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Maturen, A. J. Jr.: Biochemical Evaluation of Reticu-
locytes in the Rabbit. M.S. Thesis, Dept. of Pathology,
Michigan State University, 1968, 54 pages.

Wintrobe, M. M.: Clinical Hematology, sixth edition,
1967, Lea and Febiger, Philadelphia.

GENERAL REFERENCES

Bishop, C., Surgenor, D. M.: The Red Blood Cell. A Com-
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Brauer, R. W.: Congress on Liver Function. Amer. Insti-
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l 9 5 8 o Pf an.“

Copenhaver, W. M., Johnson, D. D.: Bailey's Textbook of
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Fallstrom, S. P., Bjore, J.: Endogenous Formation of CO
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1968.

 

”-
hag-

Fog, J., Bakken, A. F.: Conjugated and Unconjugated Bile
Determinations in Icteric Sera by Direct Spectro-
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Hoffman, W. S.: The Biochemistry of Clinical Medicine,
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Disease. Hoebner-Harper, N.Y., 1957.

Martinek, R. G. Improved Micro Method for Determination
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1966.

Meites, S., Traubert, J. W.: Use of Bilirubin Standards.
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London, I. M.: The Metabolism of the Erythrocyte. The
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Page, L. B., Culver, P. J. A Syllabus of Laboratory Exam-
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48

49

Rand, R. N., diPasqua, A.:

A New Diazo Method for the

Determination of Bilirubin. Clin. Chem. 8:1962,

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A Uniform Bilirubin Standard. Recommendations of the

College of Amer. Pathology Stds. Committee.
J. Clin. Path. 39:90-91, 1963.

Am.

 

VITA

Born: March 28, 1914, in Duluth, Minnesota, as Mary Jean
O'Brien

High School Completion: East Grand Rapids, Michigan, 1931

8.5., Michigan State University, 1935, Chemistry FT?-

Married, 1936, to Lt. Frederick C. Long, U.S.A.F.

Children: Lora Elizabeth Rikans, 3.3. and M.S., M.S.U. in I

Foods and Nutrition, presently in biological research

 

at Dow Chemical Company in Midland E;J
Christopher Eugene Long, B.S., M.S.U., presently

completing a Ph.D. in solid state physics at Florida
State University

Laboratory Technician, Parke, Davis and Company, 1935-1937,
Detroit, Michigan

Registered M.T. (ASCP), 1954

Laboratory Technician, Blodgett Memorial Hospital, 1951-54

Medical Technologist, Blodgett Memorial Hospital, Grand
Rapids, Michigan, 1954-59

M.S., Michigan State University, 1961, Biochemistry

Supervisor Clinical Chemistry, Blodgett Memorial Hospital,
Grand Rapids, Michigan, 1960-62

Instructor, Department of Pathology, Michigan State Univer-

sity, 1962-66

Member of The Lansing Society of Medical Technologists,
The Michigan Society of Medical Technologists,
The American Society of Medical Technologists,
Associate Member of Sigma Xi, Honorary Member of
Alpha Delta Theta, Member of Sigma Delta Epsilon.
Registered with The National Registry of Clinical Chemistry,
1968
Awards: Warner-Chilcott Scholarship, 1959
Dow Chemical Company Fellowship, 1960
Hilkowitz Award for original research, 1961
Scientific Products Foundation Award, First in
Biochemistry, 1961
American Society of Medical Technologists, Third
award, 1966
M.S.U. Graduate Office Tuition Scholarship, 1966-67

M.S.U. Graduate Office Fellowships, 1967, 1968

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