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A COMPARISON OF THE GRADIENT TUBE

AND THE MICROuKJELDAHL
NITROGEN METHODS FOR THE
DETERMINATION OF SERUM PROTEIN
CONCENTRATION

Thasis far Iha Dagmar of M. S.
MICHIGAN STATE COLLEGE
.éane? Hope “Travar

1949

THESIS

This is to certify that the

thesis entitled

"A Comparison of the Gradient Tube and the
Micro-Kjeldahl Nitrogen Methods for
the Determination of Serum Protein

Concentration"

presented by

Janet Hope Traver

has been accepted towards fulfillment
of the requirements for

_.-.M;Sg_____degree inIQQd§_&1d Nutrition

Major profefir w —-

Date_ May 25, 19A? __.._-

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L

A COEPARISON OF THE
GRADIENT TUBE
AND THE
MICRO-HEIDAIE NITROGEN
MTHODS FOR THE
DETMBVATION OF SERUM. PROTEIN
C 0130331?ka ION

By

Janet Hope graver

A THESIS

submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SC IENCE
Department of Foods and Nutrition
School of Home Economics

1949

ACEW WLEDGLmITT
The writer wishes to express her grateful appreciation
to Dr. Margaret Ohlson, Dr. Dena Cederquist, Dr. Carl
Hoppert, Dr. KaithIMQ,Call, Professor Charles Ball, and
Miss Wilma Brewer for their help and suggestions Which

made this study possible.

217998

7T1 7'27" 7‘ '1 fllTI‘I ~""I"r"
i;L‘.L..‘J OJ: v-3 genie -J

Page

E:TRODTJCTIOIIQ.QQQQQOOOcocoon-o0.000000000000000000000 1
REVIEW OF THE LIT:RATURE............................. 6

Kethods for Determining Protein C ncentration..... 5
Accuracy of Specific Gravity'rethcds for Deter-
minirg Protein Concentration................... 12
Factors Ynich Alter Specific Gravity.............. 17
Use of Serum and Plasma for Protein Determinations l9
EEEQEEEElflmnfimit.n.u.u.u.u.u.u.u.u..21
Collection of Samples............................. 21
Specific Gravity Hethod........................... 23
Total Nitrogen hethod............................. 26
Non-protein Nitrogen method....................... 29

FESEIIITSOOOOOOOOOOIOOOOOOOOOIOOOOOIOOOOOOOOOOOOOOOQQQO

Accuracy of the Hethods for Total and Eon-protein
Nitrogen....................................... 32
Correlation Between Specific Gravity and Protein
Nitrogen Concertration.....,....t........f..... 36
Effects of Salt and Sugar on the Specific Gravity
of an Albumin Solution......................... 40
DISCUSSION or mars... 42
Use of the Gradient Tube to Determine Protein.
Concentration.................................. 42
SUZEARY AID CCICLFS FS.............................. 4?
LITERATURE CIT:D..................................... 49

HFEmIJECQOOOCIOIOOOOOIOOOOOOOOOO0......00....COAAQOO 61

TABLES

Number Title Page
I Total Eitrogen Recoveries for Three

Serum Samples with'Varying Digestion

Times. _ 33
II Effect of Sampling and Dilution on the

Determination of Total Kitrogen. 34
III Specific Gravity, Total and Non-protein

Nitrogen, and Protein Concentrations

for Twenty Samples of Rat_Sera. 37
IV Effects of Sodium Chloride and Glucose

on the Specific Gravity of an Albumin

Solution. _ _ ‘ 41
V Formulae Used to Relate Specific Gravity

and Protein Concentration. 45

 

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1-77“: - *n .-'m

.LU'I alum

Number Title
I Gradient Tube Cylinder Readings for
Solutions of Known Specific Gravities.
II Percent Transmissions for Solutions of
Known Nitrogen Concentrations.
III Relation Between Specific Gravity

and Protein Eitrogen Concentration.

31

38

INTRODUCTION

INTRODUCTION

Plasma and serum protein determinations have
been used with other clinical tests to assess nutritional
status (Ybumans et al., 1943). Normal human plasma and
serum.values reportedly range between 6.0 and 8.0 grams
protein percent (Epstein, 1912; Rowe, 1916; Linder, "
Lundsgaard, and‘Van Slyke, 1924; Bruckman, D'EsOpo, and
Peters, 1930; Peters and Eisenman, 1933; Cameron and
White, 1942; Kagan, 1942; Bieler, Ecker,_and Spies, 1947).
The significance of values near the extremes of the
normal range is a matter of speculation. .

The concept of a state of dynamic equilibrium
between plasma proteins and food and body proteins was
formulated by Madden and Whipple (1940). ncperimentai
data to support this concept were published by
Schoenheimer and Rittenberg (1940). These workers fed
amino acids containing isotopic nitrogen to animals and
found that the greatest concentration of the isotOpe
was present in the serum.proteins. ._.

Since proteins are closely associated with many
vital functions of the body, it might be expected that a
constant concentration of blood protein would exist.
Experimenters have demonstrated, however, that blood
proteins may vary in concentration.

Some experimental work has shown that plasma

or serum protein concentrations have decreased when

-2-
animals were fasted or maintained on diets low in
protein (Frisch, Mendel, and Peters, 1929; metcoff,
Favour, and Stare, 1945; Field and Dam, 1946). On the
other hand, Leethem (1945 and 1947) demonstrated that
animals on high protein diets (78 percent casein or
lactalbumin) did not show significant increases in plasma
protein concentration.

The quality as well as quantity of the dietary
protein also may be a factor in altering blood protein
concentration. Some proteins appear to be more efficient
than others for the regeneration of plasma proteins (Kerr,
Hurwitz, and Whipple, 19183‘Madden and Whipple, 1940).
This is probably due to varying amino acid contents of these
proteins, for Madden and his cowworkers (1943) have shown
that the ten amino acids essential for the growth of the
rat are needed for the production of serum proteins in the
dos. ,

The relation between blood and dietary proteins
also has been demonstrated in.humans. , Peters, wakeman,
and Eisenman (1927) observed that the plasma protein
concentrations of 19 cases of severe malnutrition
ranged from 3.44 to 7.17 grams percent. .A decrease of
0.73 grams protein percent plasma was found by Keys et a1.
(1946) when they examined blood from 54 men Who had been
existing for six.months on 49 grams of protein per day.

'Most of this protein came from.vegetable sources. The

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recommended daily allowance for an adult men is 70 grams
of protein (Food and Intrition Board, 1943). Halters,
Rossiter, and Lehmann (1947) exaninei blood from 12
Indian vnrr prisoners VihO had been eatir fs diets low in
protein. 1e control group as nine normal Indian
subjects. 'ean concentti011s of 5.;4 and 6.89 grate
protein percent serum respectively for the prisoners_and
the controls were noted. A relationship was observed
between protein intake and sernn protein concentration
by Arnell, Goldman, and Bertucoi (1945) in a study of
11 re”1snt women with nutritional edema.

Other studies} lave revealed normal protein
concentrations in populations ovis ting on low protein
diets. Stuart (1945) dete mined serum.albumin
concentrations on Branch children who, during the war,
had received approximately 63 grams of protein a day
but only 19 grams from and a1 sources. He observed no
edema or hypoproteinemia arorv t“e children studied.
‘Youmans et a1. (1943) studied a middle Tennessee rtfai

pepujatiO“ of 776 white and colored peeple. .A dietary

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Gated rear intakes of 64 grams of prote n

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pc day for the 1d1ites and 48 grans of protein per day for
the colored. The mean serum.protein concentrations were
6.95 and 6.96 grams protein percent respectively. There
appeared to be no cor relation betteen ire dietary i1;t 1res

of calories or protein and the serum.protein concentration.

 

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The protein intakes in these studies although less
than the recommended dietary allowance for this
nutrient, evidently were not low enough to produce
changes in the blood protein concentrations. ’

Arnell, Goldman, and_Bertucci (1945) stated
hat " close correlation between the protein content of
the diet and alterations in the composition of the blood
cannot be expected"; however, a pepulation study of
protein intakes and blood protein concentrations might .
reveal some relationship betweendietary and blood protein
values. In other words, would a minimal protein intake
be correlated with a blood protein value in the lower
portion of the "normal" range? Similarly, would a very
high protein intake be correlated with a blood protein
value in the upper portion of the "normal" range? If such
a relationship existed, a plasma or serum protein deter- .
mination would be of value in assessing nutritional status.
The need for a simple but accurate method for

the determination of serum or plasma protein concentration
for survey studies is obvious. Although several methods
have been suggested, their accuracy for the determination

of serum.or plasma protein may be questioned.

In the study to be reported, the use of one of
the short methods, the Linderstrom-Lang gradient tube, as
described by Lowry and Hunter (1945) has been investi-

gated. The accuracy of serum protein concentration

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calculated from specific gravity, as determined by this
method, was found by comparison with values obtained from
midro-Kfleldahl determinations. The relative effects of
salt and sugar on the specific gravity of an albumin

solution were also determined.

PEI IEW OF TEE LITEPATUPJS

Fethods for Determining Protein Concentration

The methods commonly used to determine protein
cohcentration may be divided into two groups, physical
and chemical. Firstly, it is assumed that non-protein
substances have a negligible effect on the physical
prOperty being tested, and, secondly, that there is no
appreciable uncorrected variation resulting from changes
in the non-protein fraction. Kirk (1947) stated that
"it is important to note that the physical prOperty
itself can generally be measured quite accurately and
reproducibly, but the relation of that prOperty to
protein concentration may be uncertain due to the
failure of one of the basic assumptions".

One of the physical prOperties which has been
related to protein concentration is specific gravity.
This property may be measured by the use of a gradient
tube (Ponder, 1942; Lowry and Hunter, 1945; Hoch and
Earrack, 1945) which consists of a cylinder containing
kerosene and bromobenzene or chlorobenzene so mixed
that the density of the mixture varies linearly
throughout a portion of the column. Thus, a substance
of unknown specific gravity, e.g. serum, drapped into
the column will come to equilibrium at a point Where
the mixture and the serum have the same specific gravity.
The specific gravity of the serum, however, may be

altered by solution of the bromobenzene or chlorobenzene

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in the serum. This alteration in specific gravity
necessitates the standardization of the time between
delivery of the serum into the mixture and reading
of the cylinder. Solutions of capper or potassium
sulfate of known density are utilized to relate
cylinder readings to specific gravity.

Barbour and.Hamilton (1926) calculated
specific gravity from the time required for a 10 cubic
millimeter drOp of a substance to fall 50 centimeters
through a mixture of xylene and bromobenzene of a
density slightly lower than that of the substance
being tested. This method has two disadvantages
not present in the gradient tube method; the size of
the drop and the timing must be very accurate.

Specific gravity also may be determined by
allowing drops of a substance to fall into a graded
series of copper sulfate solutions of known densities
and noting the rise or fall of the drOps (Phillips
et al., 1944; Adams and Ballou, 1946; meyer et a1.,
1947). Simeone and Sarris (1941), using a similar
method, noted the movement of glass beads, calibrated
for density, in the solution being tested. Mbrtensen
(1942) devised a.method using glass beads, one heavier
and one lighter than the sample. The beads were
placed in a pipette with the sample and the pipette
inverted. The position at which the beads collided

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-8-
was used as an indication of specific gravity.

The refractive index of a protein solution
is another physical prOperty which has been used to
determine concentration. This method has been used
by Neuhausen and Rioch (1925), Siebenmann (1937), and
Sunderman (1944), but according to Kirk (1947) this
method for determining protein concentration is not
as sensitive as the specific gravity methods.

Bateman (1947) utilized another physical
prOperty of proteins, that of surface tension and the
related spreadingon surfaces, to determine concen-
tration. He spread the protein over the surface of
a concentrated salt solution and measured the area
of the film When it was compressed to a standard
pressure. The Ejeldahl determination of nitrogen
was used to relate area to protein concentration.

Other methods based on physical prOperties
also have been used. Infrared absorption spectroscppy
was used by Buswell and Gore (1942)_and electrOphoresis
by COOper (1945). At present these methods are used
primarily in research (Kirk, 1947).

Protein concentration may be determined by
the weight of precipitated proteins after washing and
drying. Gravimetric determinations have been done by
Barnett, Jones, and Cohn (1952) and by Boyd (1939).

The difficulty in procuring quantitative separations
of protein is a major problem.with this method of

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analysis (Kirk, 1947).

Looney and welsh (1939) precipitated total
protein with sulfosalicylic acid in the presence of
gum ghatti and determined the concentration turbidi-
metrically. The volume of precipitated protein after
centrifugation has been used to determine protein
concentration by Rytand (1939) and by Lewis (1946).
Kirk (1947), however, reported that it is difficult to
standardize the conditions of precipitation and
centrifugation sufficiently to make this procedure
quantitative.

Among the chemical methods used to determine
protein concentration is the Kjeldahl with its several
variations. After a sample is digested with acid in
the presence of a catalyst, the ammonia formed upon
the addition of alkali may be distilled into boric acid,

' and the ammonium.salt formed titrated with standard acid
(Ma and Zuazaga, 1942). The ammonia also may be

distilled into standard acid and the excess acid

titrated with standard base (Robinson, Price, and Hogden,
1937). 'Van Slyke (1927), using a gasometric method,
measured the amount of nitrogen produced by the addition

of hypobromite after digestion. Earcali and Reiman (1946)
eliminated distillation by titrating the ammonium salts
formed upon-digestion with sodium.hydroxide in the presence

of formaldehyde. However, calcium, barium, cOpper, iron,

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and phosphates interfere, so the latter method of
analysis would probahly not be applicable to blood.
Levey (1948) suggested the use of the Conway diffusinn
cell for serum protein determine ations. In this method
the ammonia from the digested sample is allowed to_
diffuse into boric acid and the ammonium.salt formed
titrated with standard acid. Direct Hesslerization
of the digested sample and colorimetric determination
of the compound formed is another variation of the
KJeldahl method commonly used (Hubbard, 19313 Schales,
Ebert, and Stead, 1942).

It is possible to estimate protein concen-
tration by determining the amounts of certain amino
acids which are present. The Folin phenol reaction
for tyrosine (Greenberg, 1929) and the Sakaguchi
reaction for free guanidine and, therefore, arginine.
(Albanese, Irby, and Saur, 1946) are but two examples.

The biuret reaction, a test for the presence
of peptide linkages, has been used to determine protein
concentration (Robinson and Hogden, 1940). Stiff (1949)
devised a new modification of this method by determining
the amount of COpper held in solution with protein.

Another method for estimating protein concen-
tration was used by Fishberg and Dolin (1930), The
proteins were titrated directly with acid; the amount

of acid required to take the protein solution from one

-11-
pH to another was found to be a function of the protein

concentration.

Of the many methods available for the deter-
mination of protein concentration, Kirk (1947) stated
that the KJeldahl nitrogen method, the biuret reaction,
and specific gravity have shown themselves to be
particularly useful despite the many limitations
inherent in each method.

Accuracy of Specific Gravity‘hethods for Determining

Protein Concentration

Kagan (1938) stated that " since the protein
content of serum is between 75 and 80 percent of the dry
weight, a close correlation with specific gravity is to
be expected". Formulae have been calculated to relate
specific gravity to protein concentration by experi-
mentally determining both specific gravity and protein
concentration on serum.and plasma samples. Nevertheless,
some workers disagree as to the validity of this
procedure.

Some investigators have compared the protein
concentration values secured from the gradient tube and
the Kdeldahl methods. Lowry and Hunter (1945) analyzed
serum.samples from 240 patients with a variety of
diseases but with no liver damage. They reported a
mean deviation between the values from the two methods
of 0.24 gram percent for a rather wide range of protein
values, 3.5 to 13.5 grams protein percent. Twenty-five
normal serum.samples were analyzed by Koch and Marraok
(1945) who reported good correlation; however, they
recorded differences up to 1.9 grams protein percent.
Ponder (1942) stated that the gradient tube method
gives values correct to tO.1_gram protein percent and

concluded that this method may be used for clinical

purposes.

I)

~13-

The results obtained from the OOpper sulfate
and the Edeldahl methods also have been compared. A
mean difference of +0.15 gram protein percent (cOpper
sulfate value minus Kjeldahl value) was reported by
Atchley and co-workers (1945). heyer et a1. (1947)
using 47 samples of dog plasma found a correlation
of 0.9061 between the results obtained by using these
methods._ These workers also reported that 68 percent
of the samples agreed within 0.2 grams protein percent.
On the other hand, Adams and Ballou (1946) discovered
that only 35 percent of the 128 human serum.samples they
analyzed agreed within 0.2 gram.protein percent and
reported a correlation of 0.74. Cook, Keay, and
Mp,Intosh (1945) found good correlation between the
results of the two methods but large individual
differences and, therefore, concluded that specific
gravity gives only an approximation of plasma protein
concentration.

'Values for protein concentration obtained from
the falling drOp method of determining specific gravity
have been compared with values obtained from the
Kaeldahl method. A.highly significant correlation of
0.9522 for dog plasma was reported by heyer and co-
workers (1947). They found that 77 percent of the
samples gave results that agreed within 0.2 gram protein
percent and concluded that the falling drop method may be

-14-
used clinically, Kagan (1938) testing the falling
drop method with both serum and plasma reported mean
deviations from the macro-KJeldahl of:t0.16 gram
protein percent for serum andiO.23 gram protein
percent for plasma. On the other hand, a very low
correlation of 0.18 was revealed by Looney (1942)
when he compared values obtained from the falling drOp
method and theturbidimetric method. Although Looney
believed the latter method to be an accurate standard,
the use of this method for a standard is not univer-
sally accepted (Kirk, 1947).

Protein concentration calculated from specific
gravity measured directly by the weight of a measured
volume of plasma was found to agree fairly closely with
the Kieldahl gasometric values by Moore and vsn.Slyke
(1930); the maximum deviation was 0.6 gram.protein percent.
weech, Snelling, and Goettsch (1933) uSing canine plasma
verified the close relationship between specific gravity
and protein concentration and the formula calculated by
(there and van Slyke (1930) to relate specific gravity and
protein concentration. weech, Snelling, and Goettsch
(1933) also demonstrated the applicability of this
formula at specific_gravities as low as 1.0121 which
represented 1.84 grams protein percent as determined
('by the Kdeldahl method. Extremely'high correlations,
0.993734 for dog serum and 0.991532 for dog plasma,

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were reported by weech, Reeves, and Goettsch (1936).
They used a pycnometer to determine specific gravity
and the Kjeldahl method to determine protein content.
On the other hand, Zozaya (1935) found a correlation
of 0.28 between the values obtained from these methods.
He thought this poor agreement was due to the use of
serum, but this assumption was shown to be false by
other workers (Weech, Reeves, and Goettsch, 1936)

The glass bead methods also have been tested
for their accuracy in determining protein concentration.
Simeone and Sarris (1941) analyzed 118 samples of human
serum.finding an average deviation of 0.25 gram.protein
percent between the values from their method and the
micro-Kjeldahl method. Hortensen (1942) compared his
method. with the Kjeldahl and found fair correlation.

The disagreement between workers as to the
validity of the specific gravity methods to determine
serum and plasma protein concentration may be due to
variations in serum and plasma constituents otherthan
protein which also affect specific gravity. These

constituents are discussed in the next section.

. In view of the conflicting evidence in the
above reports, the use of specific gravity methods to
determine protein concentration cannot be assumed to

give satisfactory results. For this reason, this

13

 

study was designed to contribute more information
concerning the correlation between the results'obtained

from the gradient tube and the micro-Kaeldahl methods.

Factors Which Alter Specific Gravity

Blood constituents other than proteins which
may vary in concentration have been shown to effect the
specific gravity of serum.and plasma. _ simeone and
Sarris (1941) demonstrated that glucose produces a
linear error, but the glucose concentrations used were
as high as 1050 milligrams percent serum. A.glucose
concentration at this level would represent an abnormal
condition. for normal post-absorptive concentrations
range from 90 to 120 milligrams percent blood (Meyers,_
1924). The work of Simeone and Sarris (1941) suggests
that normal concentrations of glucose do not produce
a large error in protein concentration as calculated from
density. They found a difference of only 0.0? gram
protein percent when calculated from specific gravity at
a glucose level of 200 milligrams percent When contrasted
to the calculated protein concentration at a glucose-
level of 100 milligrams percent. A.blood glucose level
of 200 milligrams percent might be possible in the
normal individual after the ingestion of carbohydrate.

.It has been recognized that salts have an
effectzon the specific gravity of serum.and plasma V .
(Barbour and Hamilton, 1924; Looney, 1942). However,
Kagan (1938) found no relation between the level of salt
and the deviation between the protein values obtained

from the falling drop and the K3eldahl methods.

1-18- . ...._

Lowry and Hunter (1945) stated that " in order
to influence the apparent serum protein concentration
[calculated from specific gravity] by as much as 0.1
gram percent, it would be necessary to double the normal
concentrations of either serum lipids, the blood glucose,
or the non-protein nitrogen ".

Zozaya (1935) believed that the specific
gravity of serum depends on the relation between the
bound and the free water. He stated that bound water
is dependent on the kind and percent of the protein
fraction, euglobulin having a greater density per unit
concentration than albumin. _‘Nugent and Towle (1934),
however, stated that " beef serum albumin and serum
globulin exert effects upcn the specific gravities of
their synthetic solutions which.are identical within
the limits of experimental error of the methods usually
employed for the accurate determination of specific

gravity values ".

Use of Serum and Plasma for Protein Determinations

Both serum and plasma have been used for the
determination of protein; however, it has been shown ‘
that the addition of oxalate or heparin to blood alters
the protein concentration. 3A rather conclusive study
was done by Peters, Eisenman, and Bulger (1925). They
determined serum protein concentrations on defibrinated
blood before and after the addition ofan_anticoagulant.
When 0.2 gram neutral potassium.oxalate percent was added
to the defibrinated blood, a decrease of 0.3 to 0.4 gram
protein percent was noted, although plasma could be
expected to contain about 0.3 gram protein percent more
than serum.(Kagan, 1942). _ __ _

p, Serum, oxalated plasma, and heparinized plasma

were analyzed by‘Kagan (1942). ' He observed that there
was nO' consistent difference between the protein concen-
tration of oxalated plasma and_serum and that heparinized
plasma gave higher results than oxalated plasma. This ‘
effect of oxalate also was noted by Gettler and Baker (1916),
by Lehman and Scott (1935), and by Cameron and White (1942).

As was previously noted, Kagan (1938) found a
lower mean deviation between the protein values obtained
from the falling drop and the micro-K3eldah1 methods for
serum.than for plasma. He (Hagan, 1942) stated that
" the values obtained for oxalated plasma are not as

reliable as those for serum, probably because the amount

 

6..

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-20-
of water withdrawn from the red blood cells varies with
the amount of oxalate used as well as with the varying

effect that oxalate has on the cells of different persons".

The actual protein concentration of the serum
of living organisms is probably better represented
by experimentally determined serum protein values than
by experimentally determined plasma protein values.

For this reason, serum was used throughout this study.

EGKQE’TEITAL PROCEDT "TS

Collection of Samples

The blood samples used for testing the gradient
tube method for the determination of serum protein
concentration were obtained from albino rats (250 to 500
grams) which had been used for other experiments. The
animals at the time of sacrificing had been maintained

for at least one month on the following ration:

 

 

 

 

‘ ' Percent
Casein 13.7
Yeast ' 8.4
Salt mixture - 4.2
Cod liver oil 1.1
Corn oil ’ ' 4.2
Cerelose and/or

corn starch 68.4

 

_ The metabolic activity of the rats was kept
as uniform as possible by removing both the food and
water the evening before the animals were killed. The
period of fasting ranged from 16 to 18 hours. _Halatol_
was injected intraperitoneally, the heart exposed, and a
blood sample taken with a 10 milliliter syringe and a
number 25 needle. Four to nine milliliters of blood
could be secured in this manner. Hemolysis was
minimized by removing the needle before the blood was
transferred to a 15 milliliter centrifuge tube.

. The sample was allowed to clot and the serum
transferred to a corked centrifuge tube.. The serum
was centrifuged for 15 minutes at 3300 revolutions per
minute (diameter of head, 20 inches), decanted, and

recentrifuged. The serum samples were tightly corked

14

l)

 

-22-

whenever possible throughout the analyses to prevent
evaporation and absorption of ammonia. Aliquots were
taken for the gradient tube, the‘Kjeldahl total
nitrogen, and the non-protein nitrogen determinations

within one hour after centrifugation.

Specific Gravity'Method

A gradient tube was established as described
by Lowry and Hunter (1945). ‘ Mixtures of kerosene and
brcmobenzene (see Appendix) were used; the lighter
mixture was layered over the heavier in a 500 milliliter
graduated cylinder. The graduated cylinder was placed
in a larger cylinder containing the jacket solution and
paraffin used for a seal (see diagram in rppendix). A
spiralled COpper wire was used for mixing. About five
minutes of mixing were required to form the gradient.

Potassium sulfate solutions of known specific
gravities were prepared. The specific gravities of the
solutions covered the range of the specific gravities
usually found in serum. These solutions were used to
relate the cylinder readings of the gradient tube to
specific gravities.

Both serum and standards were allowed to reach
room temperature before use. Pipettes calibrated to
0.01 milliliter were employed for delivery into the
gradient. The pipette was rinsed two times with each
standard before use; a clean pipette was used for the
serum sample. The cylinder readings of the center of
the drops were recorded four:tone-half minutes after
delivery. Fine sea sand was used to settle the drOps,

and the gradient was covered while not in use to prevent

evaporation.

-24-

A.graph was constructed (Figure I.) by plotting
the cylinder readings of the standard solutions against
their specific gravities. The specific gravity of the

serum was determined from such a graph.

In order to determine the relative effects of
salt and sugar on the specific gravity of serum, an
albumin solution of a concentration_approximatinghuman
serum protein concentration was prepared. Glucose and
sodium chloride were added to portions of this albumin
solution. >

Normal serum sodium values range from 390 to
330 milligrams percent (HaWk, Deer, and Summerson, 1947,
p. 596) and serum.chloride from.547 to 576 milligrams
percent as calculated from 98 to 106 milliequivalents
per liter (HaWk, Oser, and Summerson, 1947: P. 574).
Sodium chloride in amounts representing 650, 670, 690,
and 710 milligrams percent were added to portions of
the albumin solution.

Normal post-absorptive blood glucose values
range from 70 to 120 milligrams percent depending on
the method of analysis used (Hawk, Oser, and Sunnerson,
1947, pp. 520 to 529). Glucose in amounts representing
85, 120, and 250 milligrams percent were added to portions
of the albumin solution. The specific gravities of all
the solutions were determined by the gradient tube and the
nitrogen concentration by the micro-Ejeldahl method.

(J

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Specific Gravity

Figure I. Gradient Tube Cylinder Readings

for Solutions of Known Specific Gravities.

1.0141

Total Nitrogen Method

Special van Slyke-Ostwald blood pipettes were
used to transfer at least two 0.5 milliliter samples pf
serum to 10 milliliter volumetric flasks. The samples
were diluted to volume with 0.9 percent sodium chloride
(Robinson, Price, and Hogden, 1957). At least two
two milliliter aliquots of each dilution were transferred
to 30 milliliter Ejeldahl flasks. _ Three milliliters of
selenium digestion mixture and three glass beads were
added to each (Robinson, Price, and Hogden, 1937). . The
aliquots were digested until colorless (one to one and
one-half hours), the necks of the flasks washed with
ammonia-free distilled water, and the heating continued
for one-half hour. A.diagram of the digestion rack
may be found in the Appendix. . '

. After the flasks had cooled, 10 milliliters
of ammoniaffree distilled water and a piece of red _
litmus paper were added. Before the cooled flask was
connected to the distillation apparatus (see diagram
in Appendix), the apparatus was steamed out for at least
five minutes. Five milliliters of two percent boric
acid and three drops (approximately 0.005 milliliter) of
the indicator (us and Zuazaga, 1942) were placed in a'50
milliliter Erlenmeyer flask Which in turn was floated in
a beaker of water. The tip of the delivery tube was
placed Just above the surface of the liquid before eight

-27-

milliliters of concentrated sodium hydroxide were added
to the Kjeldahl flask from the reservoir. Immediately
the delivery tip was lowered into the boric acid-indicator
mixture, and the Kjeldahl flask was heated with a micro
burner. After the sample had been distilled for 10‘
minutes(Robinson, Price, and Hogden, 1957), the collecting
vessel was lowered until the delivery tip was again Just
above the liquid. The delivery tubewas washed in the
following manner: the micro burner was removed, the clamp
on the trap opened, and-ammunia-free distilled water was
allowed to flow from the reservoir into the Kjeldahl
flask; thesample was reheated for one minute and water
from the reservoir was allowed to flow from the reservoir
into the collecting flask. The tip of the delivery tube
was rinsed and the Erlenmeyer flask removed for titration.
The total volume of the distillate wee kept at approxi-
mately lfi'milliliters_CMa and Zuazaga, 1942)._ The
distillate was titrated with 0.01 N hydrochloric acid to
the purple end peint of the indicator (pH 4.52); the
amount of acii required was read to 0.1 milliliter and
estimated to the nearest_0.01 milliliter. Aiblank using
two milliliters of 0.9 percent sodium chloride was
analyzed for each five digestions. _

, “The protein concentration was calculated
from the protein nitrogen concentration; pretein==

factor (total nitrogen - non-protein nitrogen).

I)

-28-
Although 6.25 is the factor commonly used to relate

the concentrations of protein and protein nitrogen,

Cook (1946) found that 6.6 is more applicable to blood
proteins. Both factors were used to calculate protein
concentration in this study.

Five milliliters of the ammonium sulfate
standard solution containing 0.125 milligram of nitrogen
per milliliter were used to determine the time required
for distillation and also were added to serum aliquots
to determine recovery of nitrogen. Digestion times for
one-half, one, two, and four hours after the solutions
were colorless were tested to determine the digestion

time required for maximum recovery of nitrogen.

‘\

Non-protein Nitrogen Method

lAt least two 0.5 milliliter samples of serum
were pipetted into 15 milliliter centrifuge tubes and
4.5 milliliters of 10 percent trichloroacetic acid
added (Robinson, Price, and Hogden, 1937). _The
corked tubes were allowed to stand with occasional
shaking for at least two hours. - The samples then
were centrifuged for 15 minutes at 2000 revolutions
per minute (diameter of head, 20 inches) and the
supernatant decanted. _ Two one milliliter aliquots
of the supernatant were transferred to 18 x 25
centimeter pyrex test tubes Which had been calibrated
to seven milliliters. One milliliter of diluted
capper sulfate digestion mixture (hawk, Deer, and
Summerson, 1947, p. 495) was added, and the aliquots
were heated over a micro burner until a few drops
remained. Two dr0ps of 50 percent hydrogen peroxide
were added and the tubes reheated. The precess was
repeated using one drOp of hydrogen peroxide. The
digests were diluted to seven milliliters with
ammonia-free distilled water. Blanks using one
milliliter of 10 percent triohloroacetic acid were
also digested.

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hmmonium sulfate standard Digestion Ammonia-free
(0.02 mg. N/ml.) mixture distilled water
m1. m1. m1.
0 1 6.0
1.0 l 5.0
1.5 1 4.5
2.0 1 400
2.5 1 3.5

 

 

 

 

 

After all the tubes had been thoroughly chilled
with running water, three milliliters of Nessler's
reagent were added to each, and in five minutes the
percent transmissions were determined with a Cenco-_
Sheard-Sanford photolometer using a green filter (wave
length, 525 millimicrons). The undigested blank was
set at 100 before the percent transmissions of the _
standard solutions were determined.. Similarly, the
digested blank was set at 100 before the percent
transmissions of the samples_were determined. ‘_ _

H .A curve was drawn 9n semi-logarithmic paper
(Figue II.) by plctting the percent transmissions of the
standard solutions against their nitrogen content. One
milliliter of the ammonium.sulfate standard solution_
containing 0.02 milligram nitrogen per milliliter was
added to supernatant aliquots to test the recovery of

nitrogen.

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Figure II. Percent Transmissions for

Solutions of Known Nitrogen Concentrations.

Accuracy of the methods for Total and Hon-protein Nitrogen

Recoveries for the total nitrogen were deter-
mined on three serum samples. Table I summarizes the
results. Osborn_and.Krasnits (1934) also noted a
maximum.recovery and then a decrease with longer
digestion times when selenium was used as a catalyst.
Although Chibnall, Rees. and Williams (1943) suggested
an eight hour heating period after clearing for macro-
Kjeldahl complete digestion,_Ccok, Keay, and Hg Intosh
(1945) found no differences among two, four, eight, and
sixteen hours total digestion for the micro-Kaeldahl.
These workers secured recoveries between 98 and 102
percent. _ .

The differences in the titration values
for aliquots from the same diluted serum.sample and
from different dilutions of the same serum sample
from.the 20 rats are summarized in Table II. The
differences also are expressed in terms of protein ‘
concentration (grams nitrogen times 6.6) and in terms
of percent of the mean protein concentration.

The titration values used to calculate
protein concentration by Milan (1946) were within
0.2 milliliter of 0.2 N acid for 0.2 milliliter of
plasma. The quantity being titrated in this study
represented 0.1 milliliter serum. All titration values

were averaged.to calculate protein concentration.

43

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Table I

Total Nitrogen Recoveries for Three Serum Samples

with Varying Digestion Times

 

 

 

 

 

 

 

 

 

Time heated Serum Sample Recovery Mean
after sample plus recovery
colorless 0.625 mg. N

hours mge N % 1118. N fl % %
f 0.792 1.407 98.5
0.796 1.415 99.1
0.801 1.416 98.3
0.800 1.409 97.7
0.786 1.430 100.0
0.793 1.416 99.6

99.4
1 0.834 1.451 98.8
0.824 1.422 95.7
0.804 1.421 98.7
0.815 1.418 96.4

97.4
2 0.644 1.258 98.2
0.668 1.269 96.1
0.675 1.264 94.2

96.2
4 0.644 1.251 97.0
0.658 1.255 95.4
0.663 1.258 95.2

 

 

 

 

 

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-35-

The blank determinations averaged 0.4 milli-
liter (range 0.20 to 0.55 milliliter) 0.01 N acid or
5.8 percent of the mean titration which is somewhat
higher than the three percent reported by Cook. Keay,
and mg Intosh (1945). Perhaps this was due to the
fact that the distillations and titrations were done
in a room in which other determinations were being

done.

The recoveries for non-protein nitrogen
ranged from 95 to 105 percent which was considered
sufficiently accurate due to the relatively low
concentration of non-protein nitrogen when corpared

with the total nitrogen.

Correlation Between Specific Gravity and

Protein Nitrogen Concentration

The results of the analyses of the 20 samples
of rat serum may be found in Table III. The means and
the standard deviations of the values have been reported.
Sex differences could not be compared. because there
were nearly twice as many male as female rats. A corref
lation of 0.9938 was found between the specific gravities
and the protein nitrogen concentrations.

A scatter diagram representing the relationship
between the specific gravities and the protein nitrogen
concentrations in grams percent serum is shown in Figure
III. The formulae for the lines which best fit the
data were calculated and rearranged* so that protein_
nitrogen and protein concentration could be calculated
from specific gravity. The following formulae were
calculated:

(1) Protein nitrogen (P) in grams percent serum:
_ 2252.1 (specific gravity - 1.0075).
(2) Protein (P) in grams percent serum (protein N x6.25):

P=325.7 (specific gravity - 1.0075).

*Protein=A+B (specific gravity) (or)

Protein =' 13 (specific gravity+ A543). ' ' "

B:=the sum of (the deviations of the specific gravities
from their meanthhe deviations of the concentrations
of protein from their mean)€%the sum of the squares of
the deviations of the specific gravities from their
mean.

A=the mean of the specific gravities - (B x the mean of
the concentrations of protein).

Table III
Specific Gravity. Total and Non-protein Nitrogen. and
Protein Concentrations for TWenty Samples of Rat Sera

 

 

 

 

 

Specific Total Nonsprotein Protein Protein

gravity nitrogen nitrogen (protein (protein

minus nitrogen nitrogen
non-protein XG.25) x6.6)

nitrogen

grams %' grams % grams % grams %
1.0197 0.636 0.022 3.98 4.20
1.0218 0.752 0.041 4.70 4.97
1.0233 0.791 0.029 4.94 5.22
1.0236 0.834 0.030 5321 5.50
1.0241 0.832 0.048 5.20 5.49
1.0248 0.880 0.016 5.50 5.81
1.0248 0.914 0.041 5.72 6.03
1.0253 0.922 0.041 5.76 6.08
1.0254 0.925 0.045 5.78 6.11
1.0255 0.935 0.039 5.84 6.17
1.0256 0.953 0.039 5.96 6.29
1.0259 0.958 0.046 5.98 6.32
1.0260 0.988 0.044 6.18 6.52
1.0266 1.013 0.042 6.33 6.68
1.0270 1.022 0.038 6.39 6.74
1.0274 1.024 0.029 6.40 6.76
1.0283 1.081 0.024 6.76 7.14
1.0283 1.107 0.029 6.92 7.30
1.0287 1.111 0.035 6.95 7.33
1.0306 1.190 0.046 7.44 7.85

IMean
1.0256 0.943 0.036 5.90 6.23
S. D.

0.0024 0.013 0.009 0.81 0.86

 

 

 

 

 

 

1.0310
1.0300 /
1.0290 ./

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1.0270

 

 

 

 

 

 

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1.0250 /.
1.0210 /7

1.0230 /

1.0220 / ,

1.0m /

1.0200 / /
1.0190

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

 

 

Specific Gravity

 

 

 

 

 

 

 

 

 

 

 

 

 

Protein Nitrogen Grams Percent

Figure 111. Relation Between Specific Gravity

and Protein Nitrogen Concentration.*

*Line calculated from formula (1) .

~39-
(3) Protein (P) in grams percent serum (protein Nx6.6):
P:=343 (specific gravity - 1.0075).

The theoretical specific gravity for serum
containing no protein was calculated to be 1.0075. The
other numbers in the above formulae are factors calculated
to convert specific gravity to the desired expression.

The standard error of estimate for formula (1)
was found to be 0.0145 gram nitrogen percent; for

formulae (2) and (3) was 0.09 gram protein percent.

Effect of Salt and Sugar on the Specific Gravity

of an Albumin Solution

Table IV summarizes the effects of the addition
of various amounts of sodium.chloride and glucose on the
specific gravity of an albumin solution. Within the
range used. each_increase of 20 milligrams sodium chloride
percent resulted in the specific gravity being increased
by 0.0004 and the calculated protein concentration by
0.15 gram percent.

No differences could be detected between the
specific gravities of the albumin solutions containing
85 and 120 milligrams glucose percent, but a value of
250 milligrams percent increased the calculated protein
concentration by 0.14 gram percent when compared with _
the protein values calculated at glucose concentrations

characteristic of normal fasting blood.

Table IV
Effects of Sodium Chloride and Glucose on the
Specific Gravity of an Albumin Solution

 

 

 

 

Specific Protein Protein
gravity 2:343 (Sp. Gr.~1.00'75) eldahl
protein
nitrogen
x6.6)
milligrams %' grams %' grams %
Sodium.ohloride
added
0 1.0292 7.44 5.15
650 1.0558 9.71 5.14
670 1.0362 9.84 5.13
690 1.0567 10.02 5.15
710 1.0571 10.51 5.11
Glucose added
0 1.0292 7.44 5.15
85 1.0294 7.51 5.10
120 1.0294 7.51 5.14
250 1.0298 7.65 5.17

 

 

 

 

 

F RESULTS

SCUSSIOV C

'T‘T
H‘d-

USe of the Gradient Tube to Determine Protein Concertration

Although a high correlation of 0.9958 was found
to exist between specific gravity and protein concen-
tration under the experimental conditions described,
several points should be mentioned concerning the use of
the gradient tube for the determination of serum protein
concentration.

The effect of variations of glucose within the
normal post-absorptive range on the specific gravity of
an albumin solution was not detectable. However, a
higher blood glucose value of 250 milligrams percent,
which would be possible after the ingestion of carbo-
hydrates, would probably affect the accuracy of specific
gravity methods for the determination of serum protein
concentration. Although in Lanv nutrition surveys_
carbolydrate ingestion is not controlled, an abstinence
period of several hours probably would be desirable
if the specific gravity of serum is to be determined.

The addition of salt, varied within the normal
range, had a marked effect on the specific gravity of
an albumin solution. In this study salt concentration
may not have been an important factor in the accuracy
of the gradient tube to determine_scrum protein concen-
tration, for it is possible that experimental animals
under controlled conditions such as diet and period of

fasting and water abstinence would.have a more uniform

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-43-
serum salt concentration than a group of humans. It
is interesting to note that the high correlations between
specific gravity and protein concentration reported by
Weech, Reeves, and Goettsch (1956), 0.995734 and 0.991532,
and by Meyeret a1. (1947), 0.9522,_were observed with
canine plasma and serum While the specific gravity and
protein concentration of human plasma and serum.usually
showed a lower correlation (Adams_and'8allou, 1946); ,.It
is possible that these‘differences were due to a larger
variation in the salt concentrations of the human
samples.
» . _ Several investigators have reported the effects
of large doses of salt and of salt abstinence on blood
sodium chloride content. Two humans were given 50 grams.
of sodium chloride a day for two days by Torbert and Cheney
(1956) who noted increases in plasma chloride (expressed
as sodium.chloride) from 555 to 582 milligrams percent
(94.9 to 99.6 millieuuivalents chloride per liter) and from
554to 502 rilligrams percent (94.8 to 103.0 milliequivalents
chloride per_liter).i Bourdillon (1937) fed a human 210
millieouivalents (12.5 grams) of sodium chloride and
observed an increase in serum sodium from.l42.7to 147.1 ‘
milliequivalents per liter and in serum.chloride from102.8
to 108.4 milliequivalents per liter. Based on the data _
obtained in the present study, there would be a difference

of approximately 0.22 gram protein percent between the

JJ

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-44-

protein values calculated from specific gravities at

the lower and the higher sodium and chloride concen-

trations reported by Bourdillon (1957). An error of

this magnitude, however, w0fi7d probably be clinically

important only at borderline values. ‘d _
Cutler, Power, and Wilder (1943) analyzed the

blood from 28 patients (with no Addison's disease) who

had been on a salt free diet for two days.” Plasma sodium

values between 500 and 522 milligrams percent (150.5 to

140.0 milliequivalents per liter) and plasma chloride

values between 533 and 571 milligrams_percent_(95.9 to

104.6 millilquivalents per liter) were observed.

‘. Although these eXperiments demonstrated__
changes in blood sodium chloride concentrations, the_
values reported were within or near the normal ranges
of serum sodium.values, 129.4 to 154.0 milliequivalents
per liter (a composite range from the literature),_
reported by Snyder and Katzenelbogen (1942-) and the .
serum chloride values, 95.2 to 115.9 milliequivalents
per liter, reported by Eisenman (1929). If blood salt
and glucose concentrations were determined in a.
nutrition survey, the effects of these substances in
extreme concentrations on the determination of protein
values from specific gravity could be considered.

.A comparison of.the formulae used to calculate

protein concentration from specific gravity determined

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~46-
bY various methods may be found in Table V. It was
assumed that when no factor was mentioned, 6.25 was used
to convert grams protein nitrogen to grams protein. r"he
formula calculated from the data in this study using the
conversion factor, 6.25, was included for comparison. It
should be noted that formulae differ not only with the
method used to determine specific gravity and the inves-_
tigator but also with the substance being analyzed. . Ehe
protein.value calculated_frcm the formula from this study
is with one exception the lowest of those listed. The
lower value might be due to a species difference in
blood salt concentration, for Anderson et al. (1930)
found rat blood chloride concentration slightly higher
than human. It is obvious that the formulae calculated
in this study could not be applied to human studies

without further investigation.

If the conditions for Studies with human
subjects could be sufficiently controlled, the gradient
tube might prove to be an accurate as well as convenient
method for the determination of serum protein concen-

tration in nutrition surveys.

8 ILL "11’ A173 COLTS US IOl-TS

S L1lnfitf‘AYD COIN SIDES

The correlation between specific gravity and
protein nitrogen (total nitrogen minus non-protein
nitrogen) was determined on 20 samples of rat serum.
Specific gravity was determined by the linderstrom-
Langg adient tube method, total nitrogen by the
micro-Kjeldahl nitrogen method, and non-protein nitrogen
by: esslerization. The effects of physiological
concentrations of blood glucose and so: u sodium chloride
on the specific gravity of an albumin solution also
were deterrinei.

A correlation of 0.9958 was found to exist
between soecific gravity and protein nitrogen concen-
tration under the experinertal conditions described.
Formulae based On th .e daia v.ere calculated to convert
spe cific gravity to protein and protein nitrogen
concentrations. The standard error of s~tivste an:
0.09 grew protein percent.

Yo differences co'ld be detected bstneen the
specific grevities of the albumin solutions conta inirg
concentrations of glucose characteristic of nornal
post-absorptive blood. However, a r"T'ucose concen-
tration of 250 milligrm opercent increased the
Cal cu: ate1 protein concentration by 0.14 gr.r percent

when coupe red with the protein value at normal post-

abs 0 rpt ve glucose concentrations.

-48-

Similarly, within the range used, each increase
of 20 milligrams sodium chloride percent increased the
calculated protein concentration by 0.15 gram.percent.
Such an error probably would not be important clinically
except at borderline values. In this study salt
concentration may not have been an important factor in
the accuracy of the gradient tube method for the deter—
mination of serum protein concentration, for it is
possible that the blood of experimental animals under
controlled conditions would have a more uniform salt
concentration than a group of humans.

It was concluded that if the conditions for
studies with human subjects could be sufficiently
controlled, the gradient tube might prove to be an
accurate as well as convenient method for the deter-
mination of serum.protein concentration in nutrition

surveys.

L ITmTTFE . C ITID

ilrirnmnr c I'll-ID

Adams,IM. A. and A. N. Ballou 1946 A,Comparison
Between the values for Plasma or Serum Protein
as Obtained by the Specific Gravity and'Eicro-
Kjeldahl‘Methods. J. Lab. Clin. Med., 31:507-
515.

Albanese,.A. A.,'V. Irby and B. Saur 1946 The
Colorimetric Estimation of Proteins in various
Body Fluids. J. Biol. Chem., 166:231-237.

Anderson, A. K., H. E. Honeywell, A. C. santy and
S. Pedersen 1930 The Composition of Normal Rat
Blood. J. Biol. Chem., 86:157-165.

Arnell, R. E., D. W. Goldman and F. J. Bertucci 1945
Protein Deficiencies in Pregnancy. J. Am. med.
Assoc., 127:1101-1107.

Atchley, J., R. Bacon, G. Curran and K; David. 1945
A Clinica1.Eva1uation of the COpper Sulfate method
for Measuring Specific Gravity of Whole Blood and
Plasma. J. Lab. Clin.IMed., 30:830-858.

Barbour, H. G. and W. F. Hamilton 1924 Blood Specific
Gravity: Its Significance and a Newimethod for Its
Determination. Am. J. Physiol., 69:654-661.

Barbour, H. G. and W. F. Hamilton, 1926 The Falling
Dray Method for Determining Specific Gravity.

J. Biol. Chem., 69:625-640.

I)

r.
4
x
‘2
. .
9
u
r.

1’?

IJ

/)

1"

fl

[J

-50...

Barnett, C. W., R. B. Jones and B. B. Cohn 1952 The
Imaintenance of a Normal Plasma Protein Concentration
in Spite of Repeated Protein Losses by Bleeding.

J. Exp. Med" 55:683-695.

Bateman, J. B. 1947 Determination of Protein in Serum
and Cerebrospinal Fluids by Formation of Unimolecular
Films. J. Cell. Comp. Physiol., 29:85-89.

Bieler, M. N., E. E. Ebker and T. D. Spies 1947 Serum
Proteins in Hypoproteinemia Due to Nutritional
Deficiencies. J. Lab. Clin. med., 32:130-138._

Bourdillén, J. 1937 Distribution in Body Fluids and
Excretions of Ingested Ammonium Chloride, Potassium
Chloride, and sodium.Chloride. Am. J. Physiol., 1203
411-419.

Boyd, E.Ifi. 1939 Separations of Lipids in Gravimetric_
Acetone Method for Plasma Total Protein. Proc. Soc.
Exp. Biol. Med., 42:263-264.

Bruckman, F. S., L. M. D'Escpo and J. P. Peters 1930
The Plasma Proteins in Relation to Blood Hydration.
IV. Malnutrition and the Serum Proteins. J. Clin.
Invest., 83577-590. -

Buswell, A. M. and R. C. Gore 1942 Quantitative
Spectros00pic Analysis of Proteins. J. Phys. Chem.,
46:575-581. ,

Cameron, A. T. and F. D. White 1942 The Diagnostic Value

of Plasma Protein. Canadian Med. J., 46:255-261.

4‘)

f.)

I)

-51-
Chi-b11811, A. Co, M. W. 3338 and E. F. Nlljii‘m‘ 1943

The Total Nitrogen Content of Egg Albumin and Other
Proteins. Biochem. J., 37:354-359. .. .

Cook, R. P. 1946 Tne Determination of Nitrogen and of
Protein in Pooled Samples of Human Plasma. Biochem.
J., 40:41-45.

Cook, R. P., D. H. Keay and D. G. Mg Intosh 1945 Hathods
for Determining Plasma Protein. Brit. Had. J., 2:
456.457. . _ .. . . .

COOper, G. R.‘ 1945 Electrophoretic Analysis of Mixtures
of Protein. J. Biol. Chem., 1583727-728.

Cutler, H. H., L. H. Power and R.IM. Wilder 1943 Concen-
trations of Chloride, Sodium, and Potassium in Urine
and Blood: Their Diagnostic Significance in Adrenal
Insufficiency. J. . Led. Assoc., 123:28- 30.

Eisenman, A. J. 1929 A.Iote on the Van Slyke Method for
the Determination of Chloride in Blood and Tissue.

J. Biol. C1 em., 82:4ll~4l4.

Epstein, A. A. 1912 A Contribution to the Study of the
Chemistry of Blood Serum. J..Exp._Med.. 16:719-731.
Field, J._B. and H. Dam. 1946 Influence of Diet on Plasma
Fibrinogen in the Chick. J. Nutrition, 31:509-523.

Fishberg, E. H. and B. T. Dolin 1930 Determination of
Serum Proteins. Proc. Soc. EXP. Biol. Hed., 28:

205-206.

’L‘

F)

I)

0'1

f)

-52-

Food and Nutrition Board 1948 Recommended Dietary
Allowances, Revised. National Research Council.
Reprint and Ciro. Series No. 129.

Frisch, R. A., L. B. Mendel and J. P. Peters 1929
The froduction of Edema and Serum Protein Deficiency
in White Rats by Low Protein Diets. J. Biol. Chem.,
84:167-177.

Gettler, H. O. and W. Baker 1916 Chemical and Physical
Analysis of Blood in Thirty Normal Cases. J. Biol.
Chem., 25:211-222.

Greenberg, D.IM. 1929 The Colorimetric Determination
of the Serum Proteins. J. Biol. Chem., 82:545-550.

Hank, P. B., B. L. Deer and W. H. Summerson 1947
Practical Physiological Chemistry. The Blakiston
Company. Philadelphia, Pa., 123h’ed.

Hill, R. M. and‘V. Trevorrow 1941 Plasma Albumin,
Globulin, and Fibrinogen in Healthy Individuals from
Birth to Adult Life. J. Lab. Clin. Hed., 26:1858-
1849.

Hech, H. and J. Harrack 1945 Estimation of Serum Protein
by the LinderstroméLang Gradient. Brit. Med. J., 2:
876-873. .

Hubbard, R. S. 1931 The Determination of Blood Proteins
by a Direct Micro-KJeldahl Hethod. J. Lab. Clin. lied"
16:500-503.

I?

13

/J

I)

I)

-53-

Kagan, B. H. 1938 A.Simp1e Method for the Estimation
of Total Protein Content of Plasma and Serum. J.

Clin. Invest., 17:373-376.

Kagan, B. H. 1942 Studies on the Clinical Significance
of Serum.Protein. I. The Protein Content of Normal
HUman‘Venous and Capillary Serum and Factors Affecting
the.Accuracy of Its Determination. J. Lab. Clin. Hed.,
27:1457-1463.

Kerr, W. J., S. H. Hurwitz and G. H. Whipple‘ 1918
Regeneration of Blood Serum Proteins. II. Influence
of Diet Upon Curve of Protein Regeneration Following
Plasma Depletion. Am. J. Physiol., 47:370-378.

Keys, A., H. L. Taylor, 0. Mickelsen and A. Henschel 1946
Famine Edema and the Mechanism.of Its Formation.
Science, 103:669-670.

Kirk, P. L. 1947 The Chemical Determination of Proteins.
In Anson, H. L. and J. T. Edsall Advances in Protein_
Chemistry. Academic Press Inc., Pub1., New york, N. Y.,
‘Vol. III.

Leathem, J. H. 1945 The Plasma Protein Concentrations
and Organ weights of Rats on‘a High Protein Diet.
Endocrinology, 37:157-164.

Leathem, J. H. 1947 Plasma Protein Concentrations and
Organ weights of Rate as Related to a High Protein Diet.
Proc. Soc. Exp. Biol. med., 64:90-92.

I)

I.)

I)

1‘)

f)

-54—

Lehman, W. and F. H. Scott 1935 Note on the Total
Protein Content of Plasma and Serum. J. Biol.

Chem., 111:43-44.

Levey, S. 1948 .A Simple Method of Determining Non-
protein Nitrogen, Total Protein, and Albumin in Blood
Serum.Samples by USing Conway Cells. Am. J. Clin.
Path., 18:435-438.

Lewis, J. H. 1946 A.Centrifuge method of Determining
Blood Proteins. Arch. Path., 42:350-354.

Linder, G. 0., C. Lundsgaard and D. D. Van Slyke 1924
The Concentration of Plasma Proteins in Nephritis.

J. Exp. Had., 39:887-920.

Looney, J. H. 1942 The Relation Between Specific
Gravity of Blood Serum and Its Protein Concentration.
J. Lab. Clin. Med" 27:1463-1469.

Looney, J. M. and A. I. Walsh 1939 The Determination of
Globulin and Albumin in Blood Serum.by the Photo-
electric Colorimeter. J. Biol. Chem., 130:635-639.

Lorry, O. H. and T. H. Hunter 1945 The Determination
of Serum Protein Concentration with the Gradient Tube.
J. Biol. Chem., 159:465-474.

‘Ha, T. S. and G. Zuazaga 1942 jHicro-Kjeldahl Deter- .
mination of Nitrogen. A New Indicator and e Improved
Rapid Hethod. Ind. Eng. Chem" Ana1.vEd., 14:280-282.

Madden, S. C., J. R. Carter, 1. 'A. Kettus, I... L. Miller,
and G.‘H. Whipple 1943 Ten Amino Acids Essential for

I)

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a
t .5
\
'9 n
"D Q
\
QR A
a .
f‘ O
Q 5
.- O
Y“ ‘
N t
1
‘\ A
' o
in g

I)

4

r)

-55-

Plasma Protein Production Affective Orally or Intra-
venously” J. Exp. Med" 77:277-295. _ _

Madden, S. C. and G. H. Whipple 1940 Plasma Proteinsxs
Their Source, Production, and Utilization. Physiol.
Rev., 20:194-217.

Marceli, K. and W. Reiman 1946 KJeldahl Determination of
Nitrogen. Elimination of Distillation. Ind. Eng. Chem.,
Anal..Ed., 18:709-710.

Markham, R. 1942 A Steam Distillation. Apparatus Suitable
for Micro-K3 eldahl Analysis. Biochem. J., 36:790-791.

Metcoff, J., C. B. Favour and F. J. Stare 1945 Plasma
Protein and Hemoglobin in the Protein Deficient Rat.

A Three Dimensional Study. J. Clin. Invest., 24:82-91.

Meyer, F. I... W. E. Abbott, M. Allison and C. M; Key 1947
A Comparison of Plasma Protein Concentration, Hemo-
globin, and Hematocrit Values Determined by Chemical
Methods and Calculated from Specific Gravity. Arch.
Biochem., 12:359-366.

Meyers, V. C. 1924 Chemical Changes in the Blood and
Their Clinical Significance. Physiol. Re‘V’., 4:274-
328.

Milan, D. F. 1946 Plasma Protein Levels in Normal
Individuals. J. Lab. Clin. Med., 31:285-290.

Moore, N. S. and D. D. Van Slyke 1930 u The Relationships
Between PlasmaSpecific Gravity, PlasmaProtein Content,
and Edema in Nephritis. J. Clin. Invest., 8:337-355.

1‘3

9

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‘30
armr-
t
1

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e t " ' Q
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i
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ng J S‘ 0‘
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°’ _. 4 Q P 1.
..
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e- W
N "
v a \ 0 ‘
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A 6 Q Q O \
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n
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fl " I e ‘
,\
n

-56..

Mortensen, R. A. 1942 A Rapid Methodhfor the Deter-
mination of Serum Protein. J. Lab. Clin. Med" 27:
693-700.

Neuhausen, B. S. and D. II. Rioch 1923 The Refracto-
metric Determination of Serum Protein. J. Biol.

Chem., 55:353-356.

Nugent, R. L. and L. W. Torlc 1934 The Specific Gravity

of Synthetic Solutions of Serum Albumin and Serum
_ Globulin. J. Biol. Chem., 104:395-398.

Osborn, R. A. and A. Krasnitz 1934 A Study of the .
KJeldahl Method. III. Further Comparisons of Selenium
with Mercury and with Copper Catalysts. J. Assoc.
Official Agri. Chem.,l7z339-342.

Peters, J. P. and A. J. Eisenman 1933 The Serum Proteins
in Diseases Not Primarily Affecting the Cardiovascular
System or Kidneys. ‘Am. J. Med. Sc., 186:808-833.

Peters, J. P., A. J. Eisenman and H. A. Bulger 1.925 ~
The Plasma Proteins in Relation touBlood Hydration. '
I. In Normal Individuals and in Miscellaneous Conditions.
J. Clin. Invest., 1:435-450. ‘ _ .

Peters, J. P. and D. D. Van Slyke 1932‘ Quantitative .
Clinical Chemistry. Vol. II. Methods. The Williams
and Wilkins 00., Baltimore, Md. _

Peters, J. P., A._M. Wakeman and A. J. Eisenman 1927 The
Plasma Proteins in. Relation to Blood Hydration. III.
The Plasma Proteins in Malnutrition. J. Clin. Invest.,
3:491-495.

I)

I)

I)

I?

f)

Phi-111135. R. A., D. D. Van Slyke, V. P._ Dole, K. Emerson,
Jru P. 13- Hamilton and R. M. Archibald . 1944 Copper
Sulfate Method for Measuring Specific Gravities of
Whole Blood and Plasma with Line Charts for Calcu-
lating Plasma Protein, Hemoglobin, and Hematocrit
from Plasma and Whole Blood Gravities. Bull. Hygiene,
19:140.

Ponder, E. 1942 A Simple Method for Determining Plasma
Density and Plasma Protein Concentration. J. Lab.
Clin. Med" 28:232-234. ‘

Robinson, H. W. and C. G. Hogden 1940 The Biuret. Reaction
in the Determination of Serum Proteins. J. Biol.
Chem., 135:707-725. ,

Robinson, H. W., J. W. Price and C. G. Hogden 1937 ~ The
Estimation of Albumin and Globulin in Blood Serum. . I. _A
Study of the Errors Involved in the Filtration Procedure.
J. Biol. Chem., 120:481-498. '

Rowe, A. H. 1916 The Albumin and Globulin Content of Runan
Blood Serum in Health, Syphilis, Pneumonia, and Certain
Other Infections, with the Bearing of Globulin on the
.Wassermann Reaction. Amh. Int. Med., 18:455-473.

Putand, D. A. 1939 A Simple Rapid Method for the Deter-
mination of; Total Protein and Albumin Concentrations in
Blood Plasma, Serum, or Other Body Fluids. J. Lab.
Clin. Med., 24:439-443.

IA

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-58..

Schales, 0., R. V. Ebert and E. A. Stead, Jr. 1942
Capillary Tube Edeldahl MBthod for Determining Protein
Content of Five to Twenty Milligrams of Tissue Fluid.
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Schoenheimer, R. and D. Rittenberg 1940 The Study of
Intermediary Metabolism of Animals with the Aid of
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Schousboe, J. 1939 use of Krogh's Precision Syringe
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129:571-375. ’ ‘ r

Siebenmann, C. 1937 Refractometrichethods for Deter-
mining Total Protein. Biochem. J., 31:205-211.

Simeone, F. A. and S. P. Sarris 1941 A.Simple‘Method ‘
for the Determination of Serum Protein. J. Lab. Clin.
iMed., 26:1046-1052.

Sobel, A. E., A. M. Mayer and S. P. Gottfried 1944 A
Convenient Titrimetric Ultramicromethod for the
Estimation of Urea and.Kjeldahl Nitrogen. J. Biol.
Chem., 156:355-363. _ ‘

Snyder, R. and S. Katzenelbogen 1942 W The Distribution
of Sodium, Potassium, Calcium, Magnesium, Inorganic
Phosphorous and Chloride Between the Blood Serum and

Cells of Normal Individuals. J. Biol. Chem., 143:

223‘226.

1

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f)

I!

-59-
Stiff, H. A. 1949 A.New Colorimetric method for the

Determination of Serum.Protein. J. Biol. Chem.,
177:179-185.

Stuart, H. C. 1945 Review of the Evidence as to the
Nutritional State of Children in France. Am. J.
Pub. Health, 35:299-307.

Sunderman. 3'. w. .1944 A Rapid Method for Estimating
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Concentration from.the Refractive Index of Serum.

J. Biol. Chem.,153:139-142.

Torbert, H. C. and G. Cheney 1936 Reduction in
Colloidal Osmotic Pressure of Blood Serum After
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‘Van Slyke, D. D. 1927 Casemetric‘Mlcro-Kjeldahl Deter-
mination of Nitrogen. J. Biol. Chem., 71:235-248.

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Blood'Volume Changes in Protein Deficiency. Lancet,
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Relationship Between Specific Gravity and Protein
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J. Biol. Chem., 1133167-174. _ _

weeoh, A. A., C. E. Snelling and E. Goettsch 1933 The
Relation Between Plasma Protein Content, Plasma Specific
Gravity, and Edema in Dogs Maintained on a Protein
Inadequate Diet and in Dogs Rendered Edematous by

I)

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-60-

Plasmaphoresis. J. Clin. Invest., 12:193-216.
Youmans,.J. 13.. E. W. Patton, W. R. Sutton, R. Kern
and R. Steinkamp 1943 Surveys of the Nutrition of
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33:955-964.
Zozaga, J. 1935 A.Physicochemical study of Blood Sara.
J. Biol. Chem., 110:599-617.

APP EHD IX

Gradient Tube*

 

‘ Scale‘ '
one inch equals
epproximat ely
five inches.
Kl?
A» 500 mdlliliter graduated cylinder.
3- Ungraduated cylinder.
C- Gradient.
D- Jacket solution.
E— Paraffin seal.
FE Cover.

*Lowry and Hunter (1945).

Digestion Rack*

 

 

 

 

/

Scale
one inch equals
approximately
five inches.

Key

Micro burner (flame one inch high).
Asbestos board.

Heavy wire.

Ring stand; and clamp.

ti“???

{Ma and Zuazaga (1942).

Distillation Apparatus*

 

 

 

C. - -G
- -—- -J
Scale - _
-—-~H one inch We :2.-_.:.,
approxima ely i=_;-,{_:i-::. - K
311: inches. 235-5}

Key

Hot plate. '

300 milliliter Erlenmeyer flask containing ammonia-free
distilled water and boiling chips.

Funnel. '

Watch glass. - ,

Trap and eduelizer of steam pressure. '
Rurvoir containing concentrated sodium hydroxide.

30 milliliter KJeldahl flask containing digested sample.
Micro burner.

I- Reservoir containing ammonia-free distilled water which
has been adjusted to the purple color of the indicator.
- 5O milliliter Erlenmeyer flask containing boric acid and

indicator.
K- 250 milliliter beaker containing water.

T?

 

$??T??

q

Rubber tubing was boiled in dilute ammonium hydroxide,
w‘ashed with distilled water, and boiled in two percent
boric acid (Sobel, Mayer and Goettfried, 1944?.
*Peters and Van Slyke (19323: Hill and Trevorrow 190.1):
Markham (1942).

Solutions

Albumin solution (C.P. blood albumin containing 15% salt):
20 milliliters of albumin solution (approximately 6.5
grams per liter) were diluted to 25 milliliters.

162.5 milligrams of sodium.chloride were dissolved
in approximately four milliliters of water, 20
milliliters of the albumin solution added, and
the mixture diluted to 25 milliliters.

This was repeated with 167.5, 172.5, and 177.5 milli-
grams of sodium chloride and with.21.2, 30.0,
and 62.5 milligrams of glucose.

Ammonia-free distilled water:

10 milliliters of concentrated sulfuric acid and a
fewlorystels of potassium.permanganste were
added to 1500 milliliters of distilled water.
The mixture was redistilled and the distillate
collected after the first 50 to 100 milliliters
were discarded. The distillate used was.
tested with Nessler' s reagent for the presence
of ammonia.

The pH of a portion of the water was adjusted to the
Vpurple color of the indicator.

*Ammonium.sulfate standards flC.P.-neutrality A.C.S. tested):
‘ The ammonium.sulfate was dried to constant weight at

105 degrees centigrade (Sobel,‘Meyer, and

*Solutions were made with ammonia-free distilled water.

Gottfried, 1944). 0.5896 gram was diluted
to one liter (0.125 milligram nitrogen per
milliliter). 80 milliliters of thisIsolution
were diluted to 500 milliliters once a week
. (0.02 milligram nitrogen per milliliter).
*Boric acid - 2 percent: .

10 grams of boric acid were diluted to 500 milli-
liters with boiled water.

*Digestion mixtures: I . ..

One milliliter of selenium.oxychloride was diluted
to 250 milliliters with concentrated sulfuric
acid and added to 250 milliliters of saturated
potassium sulfate. .. III _ .

300 milliliters of 85 percent phosphoricIacid, 5O
milliliters of five percent copper sulfateI
solution, and 100 milliliters of concentrated
sulfuric acid were combined. The solution

I was diluted one to ten for use.

Gradient Jacket solution: I

Two grams of copper sulfate and 0.6 milliliter of

concentrated sulfuric acid were diluted to
I one liter.
Gradient mixtures: . - I I _ _

150 millilitersIof’kerosene*fIwere combined with 100

milliliters of bromobenzene*** and adjusted to

*Solutions were made with’ammonia-free distilled water.

HEimer and Amend, odorless.
*‘HEimer and Amend, special for falling drOp method.

a specific gravity of 1.07.

180 milliliters of kerosene were combined with 70
milliliters of bromobenzene and adjusted to a
specific gravity of 0.99.

Hydrogen peroxide - 30 percent.
Hydrochloric acid standard solutions:

0.1 N acid was made by carefully adjusting the
acidity of an acid solutioanhich was approxi-
mately 0.1 N. The acid was titrated with
sodium hydroxidewhioh had been standardized
against potassium.acid phthalate (A.C.S.
standard). . I

0.01 N acideas made by diluting the 0.1 N acid.
This was done once a week.

Indicator:_

0.1 gram of bromrcresol green was diluted to 100
milliliters with95 percent ethanol. 0.1
gram.methyl red was diluted to 100 milliliters
with 95 percent ethanol. Five parts of the
brom-cresol green solution were combined with
one part of the methyl red solution for use.

Nessler's reagent: I

100 grams of mercuric iodide and 70 grams of .
potassium iodide were added to 400 milliliters
of distilled water. 100 grams of sodium
hydroxide were dissolved in 500 milliliters

of distilled water and added to the above
solution. The combined solutions were made
up to one liter and diluted one to one for
use (Hawk, Oser, and Summerson, 1947. P. 1230 -
method of_Bock and Benedict).
Potassiumsulfate standards:

Potassium sulfate (A.C.S. standard) was dried to
constant wsight at 110 degrees oentigrade and
the following amounts were each diluted to 500

 

 

 

 

 

milliliters:
Potassifim sulfate Specific gravity
of solutions

grams

8.8200 1.0141
11.5400 1.0184
17.0200 1.0270
19.7900 1.0313
22.5500 1.0356

 

The stock solutions were kept in a refrigerator
in paraffined sealed bottles. Smaller bottles
of the solutions were mbfrigerated and
maintained for use with the gradient tube.
They were renewed every two weeks.
*Sodium.chloride - 0.9 percent.
*Sodium.hydroxide (C.P. pellets):
100 grams of sodium hydroxide were added to 100
milliliters of distilled water and allowed

*Solutions were made with ammonia-free distilled water.

to stand a week. The solution was filtered
and stored in paraffined bottles.

*Trichloroacetic acid - 10 percent.

*Solutions were made with ammonia-free distilled water.

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MICHIGAN STATE UNIVERSITY LIBRARIES

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