T U G A M E R I C A N J O U R N A L OF CLINICAL
PATHCLOGV
Vol. 39, N o . 1, p p . 42-15
J i m m u y , 1963
C o p y r i g h t © 1063 b y T h e Williair.s & Wilkins Co.
Hrinted in
U.S.A.
TOTAL PROTEIN/PROTEIN NITROGEN RATIO OF HUMAN SERUM
A FACTOR CONSISTENT WITH TOTAL SOLIDS
E M M A DEI, C A R M E N C H I A R A V I G I J O , A. V. WOLF, P H . D . , AND P H O E B E G. P R E N T I S S , M.S.
Department
of Physiology,
University
of Illinois
College of Medicine,
Chicago,
Illinois
the factor of Sunderman and associates11
and recommends that 6.54 (15.3 per cent
nitrogen) be adopted in lieu of 6.25 (16.0
per cent nitrogen).
When total serum protein is determined
by means of conversion of nitrogen content,
using the conventional factor 6.25, the concentration obtained is inconsistent with that
of the independently determined nonprotein solids and total solids. Sunderman and
associates11 demonstrated that much of the
discrepancy arises from the conversion factor, noting that if its value is taken at 6.54,
the apparent error is practically eliminated.
Wolf and co-workers14 allude to refractometric analyses of total solids of serum that
agree with this opinion. Actually, factors
from 6.21 to 7.14 appear in the literature. 2 ' ''• "
In this paper we describe the determination of a total protein/protein nitrogen ratio
by means of a new gravimetric and a standard Kjeldahl analysis. Such a factor, when
it is used to determine total serum protein
from protein nitrogen analysis, yields a value
in appropriate relation with total solids in
the serum. Heretofore the use of the incorrect factor, 6.25, was of little adverse consequence. With the advent of newer refractometric technics for determination of
total solids of body fluids, 3 ' 4 ' 8 ' »• 13' 14 it
becomes important to revise the factor appropriately. This paper provides independent affirmation of the essential accuracy of
METHODS
Blood samples were drawn from 20 men
and 10 women who were apparently normal
and were aged 17 to 50 years, and serum
was obtained from the samples. Total solids
and water of this serum, and of its substituent deproteinized fluid, were determined
by drying in air at 45 to 50 C. for 21 hr.
This treatment was shown to leave no water
residue in terms of assay with Karl Fischer
reagent.7 It yields somewhat higher values
for total solids than conventional drying
at 100 C. or the vacuum-drying of Armstrong and co-workers'2 at 50 to 80 C. Serum
was "deproteinized" by the following
methods.
1. Mixing 100 parts with 1 part of 10
per cent acetic acid and heating in a syringe
at 95 to 98 C.14 The resulting coagulum was
squeezed through a 21-gage syringe needle
to break it up, and the extruded mass, after
centrif ligation, yielded a supernatant,
largely deproteinized fluid that still contained glycoprotein and possibly small
amounts of other uncoagulated protein in
solution.
2. Pressure (ultra) filtration through
Polypore protein enrichment membranes of
pore size 0.004 /x.
Total solids of all fluids were checked by
TS meter, and the refraction (104 X refractive index increment over water, at the same
temperature) of fluids was also measured
by dipping refractometer with a sodium
light.
Kjeldahl analysis6 provided nitrogen content of all fluids. Total lipids, measured by
chloroform-methanol extraction10 of the
dried residue of "deproteinized" serum, were
Received, June 22, 19(52; accepted for publication, October 4.
Miss Cliiaraviglio is a Fellow of the National
Council of Scientific and Technical investigation,
Argentina. Her present address is Institute of
Physiology, University of Cordoba, Cordoba,
Argentina. Dr. Wolf is Professor of Physiology
and Head of the Department, University of
Illinois College of Medicine, Chicago, Illinois.
Mrs. Prentiss is Principal Laboratory Technologist
in the Department of Physiology.
This work was supported in part by P H S Research G r a n t H-45I7 from the National H e a r t
Institute and by Research G r a n t 9569 of the National Science Foundation.
42
Jan. 1963
TOTAL P R O T E I N ' / P R O T E I N N I T R O G E N
approximately 0.0002 Gm. per Gm. and
0.0005 Gm. per Gm. for filtrate and supernatant fluids, respectively. Residual protein
(largely glycoprotein) in the "deproteinized"
serum was analyzed by measuring the difference in nitrogen of "deproteinized" serum
before and after precipitation of residual
protein by 5 per cent phosphotungstic acid
in N HC1 (0.2 ml. per ml. of sample).12
CALCULATIONS*
value
A',, _ G,,
J)
D
Nt
D
S
N.
w.
w.
w.
]
\_w. w,
J
D
N.
(3)
S/W, and its reciprocal in (3) are obtained
by direct gravimetry.
In order to obtain N./W, for (3) we proceed as follows. Using "deproteinized"
serum, measure its water, IF,/, and its total
solids, G,i, gravimetrically. G,t includes an
amount of uncoagulated protein, M,i, that is
greater when derived from the acid-heat
procedure than from filtration. It also includes lipid (nonprotein), L,,, which differs
from that of the precursor serum by an
amount, AL,/. Analysis revealed L,, of filtrate
to be essentially negligible (0.0002 Gm. per
Gm.) and that of supernatant fluid to average 0.0005 Gm. per Gm. Assuming average
values for the lipid of the precursor serum,1
we have adopted probable values of ALd as
0.0062 Gm. per Gm., in the case of filtrate,
and 0.0052 Gm. per Gm., for supernatant
fluid. When these are taken with our analytic values of Mtl, we obtain the virtual
* Tlie following symbols are used: S = Gm. of
serum sample; D = C m . of "deproteinized" serum
sample; P = Gm. of total protein in a sample; A' =
Gm. of nonprotein solid; IK = Gm. of water; M =
Gin. of uncoagulated protein, largely glycoprotein; L = Gin. of total lipid; G = Gm. of total
solid; and l\ = Gm. of protein nitrogen. The lower
case subscripts, s and d, denote references to either
S or D fluid samples.
(5)
(«)
(1)
(2)
(•I)
where D/l-F,, is measured by direct gravimetry. The conversion of units in (5) permits us to use the fact that
and to substitute (6) in (3). Thus we obtain
a gravimetric analysis of total protein from
P,
SS
Multiplication of both sides of (2) by W./S
yields
s
J)
D _ A'„t
\Vt
II',,
N,i
P.
AL,, _ 1\1,,
From (4) we can obtain
Since
P. + N. + W, = S
43
JtATIO
\_W.
II',,
J S
(7)
From Kjeldahl analysis of weighed quantities of serum, we find Ks and K,/S. The
conversion factor is then
(P,/S)/(K,/S)
=
P./K.
= total protein/protein nitrogen
(S)
RESULTS
From Table 1 and Equation (1), after
multiplying each term by 100/S, we determine average percentages as follows: 100
P./S = 7.24, 100 N./S = 1.56, 100 G./S =
8.80, and 100 W./S = 91.20, in which the
sum of total solids and water is 100 per cent.
The average, nonlipid, nonprotein solid is
given by 100 N./8 - 100 L./S = 1.56 0.62 = 0.94. The number of cases, Ar, totals
53.
With serum total solids at 8.80 per cent,
and with such serum having a specific gravity of 1.027, the average protein concentration in our series was approximately
7.44 Gm. per 100 ml. (5.58 to 8.69), and
the average total solids was 9.04 Gm. per
100 ml. (7.21 to 10.26).
We applied our method to the determination of the protein/nitrogen factor of Armour Protein Standard Solution of crystalline (lipid-free) bovine albumin, stated to
f In the case of the s u p e r n a t a n t fluid whose contained solids have been diluted by addition of
acetic acid, I\!J/\V,I is corrected by multiplying by
1.02.
C H I A R A V I G L I O ET
Vol. 39
AL.
TABLE 1
SUMMARY OF D A T A USED IN CALCULATION OF P R O T E I N / N I T R O G E N FACTORS FOR H U M A N SERUM
(53 C A S E S )
Procedure
5/11'.
D/Wi
Gd/D
ild/D
K./S
P./S
P,/K,
,v
Filtration
1.0961 ± 0.0075 1.0109 0.0108 0.0002 0.0721 ± 0.0055 0.0110 0.55 ± 0.33 24
Acid-heat coagulation 1.0967 ± 0.0095 1.0145 0.0143 0.0030 0.0720 ± 0.0063 0.0111 0.57 ± 0.20 29
0.0724
Weighted overall av0.56
erage
TABLE 2
R E F R A C T I V I T I E S OF H U M A N SERUM AND S U B S T I T U E N T S B A S E D ON F I L T R A T E S O N L Y — A V E R A G E S OF 21
C A S E S , W I T H STANDARD D E V I A T I O N S
's
rd
100 Gs/S
100 Gd/D
158.1 ± 10.1
IS.3 ± 1.0
8.77 ± 0.57
1.08 ± 0.10
contain "approximately 16.0 per cent" nitrogen. From 10 analyses, 5 nitrates yielded
15.6 per cent, 5 supernatants yielded 15.8
per cent, and the overall average was
15.7 =fc 0.2 per cent, or a factor of 6.37. Our
Kjeldahl analyses of protein nitrogen agreed,
within 0.3 per cent, with the stated protein
nitrogen of the Armour Standard.
Table 2 lists refractometric analyses of
serum and ultrafiltrate. The refraction, r,
is defined13 as r = (A?i)104, where An is the
difference between refractive index of a
sample and that of water at the same temperature (.1.7.5 C ) . The refractivity, R, is
defined here as the refraction per Gm. per
100 Gm. Thus Ra = r,/(100 G./S) and
Rd = r„/(100 Gd/D).
Rd
R.
18.03 ± 0.46
16.84 ± 0.S7
98 C.9 (to "constant weight," usually 24 hr.),
and discrepancies with the data reported
here depend largely upon this fact.
Our average factor for human serum protein, 6.56, is in reasonable agreement with
the factor of Sunderman and associates,11
6.54. It is consistent with the constitutive
requirement that protein solids and nonprotein solids equal total solids of serum as
determined by independent methods. Inasmuch as there is no significant difference
between the 2 factors, and because that of
Sunderman and associates11 was determined
directly, we recommend specifically the
adoption of the latter, 6.54, as a practical
working laboratory constant.
SUMMA KY
DISCUSSION
Inasmuch as we have no independent
measure of refractivity for serum lipids, we
do not estimate the refractivity of serum
protein. Actually, results of this latter determination2 and that of Rs depend upon the
method of drying protein and serum. Preliminary tests reveal that the apparent
serum solids obtained from 24-hr. drying
at 45 to 50 C. compared with 104 C. are
approximately 6 to 7 per cent greater; the
refractivity of the solids obtained at 104 C.
will be correspondingly greater. The refractivity of serum solids upon which scales
of the TS meter are based is derived from
gravimetric analyses at approximately
A new method of gravimetric analysis for
total protein of human serum that permits
determination of the total protein/protein
nitrogen ratio has been devised. Our results
affirm the validity of the ratio, 6.54, which
has been recommended in lieu of the conventional factor, 6.25. The factor 6.54 is
consistent with the constitutive requirement
that protein solids and nonprotein solids
equal total solids of serum as determined
by independent methods.
SUMMA RIO I N
TNTERLINGUA
Esseva elaborate un nove methodo pro le
analyse gravimetric del proteina total in sero
human, permittente determinar le propor-
Jan. 1968
TOTAL r i t O T E l N / l ' R O T E I N
tion de proteina total a nitrogeno proteinic.
Nostre resultatos supporta un proportion de
6.54 a 1. Isto es recommendate in loco del
factor conventional 6.25. Le factor 6.54 satisface le requirimento constitutive que le
summa del solidos proteinic e del solidos nonproteinic sia equal al solidos total del sero
secundo determinationes per methodos independente.
NITROGEN
MEUN,
HATIO
M . , AND T A Y L O R , H . L . : P r e p a r a -
tion and properties of serum and plasma
proteins, I V . A system for t h e separation
into fractions of t h e protein and lipoprotein
components of biological fluids and tissues.
J . Am. Chem. S o c , 68: 459^175, 194G.
0. H A W K , P . B . , O S E R , B . L., AND SUMMERSON,
W. H . : Practical Physiological Chemistry,
E d . 13. New York: Blakiston, 1954, p p .
547-548.
7. M I T C H E L L , J . , J R . , AND S M I T H , D . M . : A q u a m e -
t r y . N e w York: Interscience, 1949, pp. S2-SG.
8. R E M P , D . C ,
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