REFRACTOMETRIC DETERMINATION OF TOTAL SOLIDS

AND WATER OF SERUM AND URINE

BY MILTON E. RUBINI* AND A. V. WOLF

(From the Department of Cardiorespiratory Diseases, Walter Reed Army
Institute of Research, Walter Reed Army Medical Center,
Washington, D. C.)

(Received for publication, July 19, 1956)

Refractometric analysis of total solids in serum and urine is simple, ac-

curate, and rapid. Adolph (1) demonstrated the high coefficient of corre-
lation between refractive index and total solids of dog plasma and subse-
quently employed refractometry as an index of the total solids of human
serum (2). Blohm (3), using a dipping refractometer, showed for human
urine that the reading given by that fluid was proportional to its dissolved
total solids, and he determined a refractometric coei&ient of proportion-
ality. The application of refractometry is extended in the present paper
with the determination of several new coefficients for total solids of serum
and urine.
The refractive index of serum has been used as a measure of total pro-
tein concentration (4, 5), but it has been supposed that high accuracy in
this analysis can be obtained only when the albumin-globulin ratio is
known (6) and when the lipide content is low (7). However, at least
when refractometry is used as a measure of total solids, the albumin-
globulin ratio is not vitiating, as we shall show, nor is there significant
influence of the usual lipide content of serum on the determination.
Refractometry is particularly advantageous for urine, since otherwise
its total solids can be analyzed satisfactorily1 only by relatively elaborate
procedures such as lyophilization. Estimates of urinary solids by means
of specific gravity coefficients are considerably less accurate (3, 8).

Methods
A Bausch and Lomb dipping refractometer, with or without an auxiliary
prism, was used with a sodium vapor lamp (D line). In accord with the
simpler precedent, we employ the instrumental increment of the refractom-
eter which is the difference between the scale reading for the solution
being tested and the scale reading for water, both being taken at 17.5” f
0.2”. In a few cases, water and solution were examined at room tempera-
ture, the instrumental increment being essentially constant so long as
* Major, Medical Corps.
1 “Satisfactorily” does not imply “accurately.” As with sea water, it may be
impossible exactly to determine total solids of urine by evaporative techniques.
869

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870 TOTAL SOLIDS IN SERUM AND URINE

both fluids are at the same temperature (2, 9). With the auxiliary prism
only 1 drop of sample is needed, and less than 5 minutes are required, for
each determination of a series.
Total solids of serum (man, dog, rat) were determined gravimetrically
after being dried to constant weight in air at about 98”. Serum protein
was determined by a biuret method (10). Total solids of urine (man,
dog, cat) were determined by a lyophilic “cryochem” process (8, 11).
Specific gravity of urine, D$, was obtained at 20” with a Westphal balance
and empirically converted to D ii: by multiplying the measured value by
1.0016.
Serum and urine may be stored for over 2 months at -18” and thawed
without a significant change in the refractive index; similarly, they may be
stored at ordinary refrigerator temperatures for over 2 weeks. Sediment
of cold urine was redissolved by suitable warming before refractometry.
The coordinates of the graphs were chosen to yield the more useful form
of equations rather than to denote independent and dependent variables.
All coefficients of correlation and standard errors of estimate, given in con-
nection with lines conditionally fixed at the origin, strictly relate to lines
of best fit determined by the actual data.2

Results
Serum-Fig. 1 illustrates the high correlation between the instrumental
increment of the refractometer and total solids of serum. The equations
(As), = 0.2242 R and (A,), = 0.2049 R, derived statistically, give the
lines of best fit which are conditionally fixed at the origin. The constants
of proportionality are obtained from ZXY/ 2x2, a formula kindly called
to our attention by Dr. Ardie Lubin.
Lines of best fit determined from the actual data, i.e. not conditionally
fured at the origin, are scarcely more accurate in the range of observed
values and tend to be less accurate as they are extrapolated toward their
intercepts. However, such lines were represented as follows:
Man, (As)mp= 0.2283 (R - 0.76) and (As). = 0.1884 (R + 3.71)
Dog, (A& = 0.2311 (R - 1.02) and (As). = 0.1949 (R + 2.34)
Rat, (As), = 0.2247 (R - 0.69) and (As). = 0.1837 (R + 3.97)
All points, (A& = 0.2318 (R - 1.35) and (AS). = 0.1939 (R + 2.38)
*The following symbols are used: A, serum or plasma concentrations; D, den-
sity of urine; D$, specific gravity of urine, density measured at 20” and referred to
the density of water at 15”; K, slope of lines, A/R or U/R; N, number of observations;
n, refractive index; R, instrumental increment of the refractometer (scale reading
of solution minus the scale reading of water at the same temperature) ; r, coefficient of
linear correlation; 8, script or subscript denoting total solids; S,, standard error of
estimate; s, subscript denoting gm. per 100 gm. of solution; U, urine concentration;
0, subscript denoting gm. per 100 ml. of solution; w, subscript denoting gm. per 100
gm. of water; X, an abscissa value; and Y, an ordinate value.
 M. E. RUBINI AND A. V. WOLF 871

FIQ. 1. Relations and correlations between total solids of serum and instrumen-
tal increment of refractometer. Points for the (As), curve (gm. per 100 gm. of se-
rum) are omitted to avoid confusion with those of the (A& curve (gm. per 100 gm.
of serum water).

TABLE I
Relations and Correlations between Total Solids of Serum, As, and
Instrumental Increment of Refractometer, R
S, is the standard error of estimate of Ae in gm. per cent.

iv T (Ashv, R (AL+, R
L=fshdR (Ash/R

Lu = 0.113
0.9918 r = 0.9837
Man 118 0.2242 0.2045
s, = 0.118
kg = 0.177
0.9857
Dog 68 0.2252 0.2062
iv = 0.9818
0.166
r = 0.9986
Rat 25 0.2205 0.2035
s, = 0.077 Lu = 0.9783
0.089
Total 211 0.2242 0.2049 r = 0.9856
k, = 0.145
0.9905 s, = 0.149
Do& 144 0.2227 0.2059
Lg = 0.125
0.9649 i, = 0.9668
0.110
-
* For Iines conditionally fixed at the origin (see the text).
f Calculated from original data supplied by Dr. E. F. Adolph, personal communi-
cation.

The conversion of SWto S, is exactly given by S, = 100 SW/(100 + S,).

Table I suggests that this conversion may be slightly more accurate for
estimating (As)* because the coefficient of correlation appears to be slightly
higher between R and (A&.
The relationship of As to R is not appreciably changed by varying pH,
872 TOTAL SOLIDS IN SERUM AND URINE

buffer content, or protein, water, and salt content as follows: (1) pH,
from 5.82 to 10.22 by adding HCl or NaOH or by removing CO, under
reduced pressure; (2) buffer, by the addition of NaH2POd or NaHC03

, 2. /, /I 0, I, I.
I~~-- ‘~~ --I 0”
I: E
18 - * MAN
Y - DOG “r*
84 5
E x CAT x
= 14 -
%
d, IO-
g
d 6-
,
u)

32 c
I I I too
0; IO 20 30 40 50 60 70 80
R
FIG. 2. Relations and correlations between total solids and water of urine and
instrumental increment of refractometer. (77~1~0)~= 100 - (Us)..

I”” .“““‘a ‘I
20- * MAN
% 0 DOG
cr
. CAT
= 16-
0”

-2 l2-
s -

FIG. 3. Relations and correlations between total solids of urine and urinary speci-
fic gravity. The former was calculated as the product of the (Us). and D:$ values.

to serum (phosphate was increased 2 to 8 m.eq. per liter; bicarbonate 8

to 32 m.eq. per liter.) ; (3) protein, by addition of albumin or globulin to
serum or serum-saline mixtures. (A,& of the modified sera ranged from
2.36 to 17.82 gm. per 100 gm. of water. Albumin-globulin ratios varied
from 0.13 to 14 in solutions containing 1.77 to 13.9 gm. of protein per 100
ml. of solution. Water was either added to serum or partly removed by
 M. E. RUBINI AND A. V. WOLF 873

drying in air at 60”. Sodium chloride was added either as dry salt or as
0.9 per cent solution.
Urine-Six pairs of relations, correlations, and standard errors of esti-
mate are given below. The first of each pair (M) derives from 190 samples
of human urine; the second (T) derives from a total of 233 samples (the
samples were from twenty-one dogs, twenty-two cats, and the above 190
human subjects). All equations represent lines of best fit conditionally
fixed at the origin.

1M (Us), = 0.2447
R; r = 0.9981; &, = 0.103 gm. y0
1T (US), = 0.2406
R; r = 0.9992; S, = 0.141 gm. %
2M (Us), = 0.2578
R;r = 0.9974; AS',= 0.131 gm. ‘%
2T (Us), = 0.2708
R; T = 0.9989; 8, = 0.198 gm. y0
3M (Us), = 0.2503
R; r = 0.9980; 8, = 0.108 gm. %
3T (U,$, = 0.2507
R; r = 0.9995; S, = 0.123 gm. To
4M D% = 0.0011644 R + 1.0000; r = 0.9723; S, = 0.00165
4T D::: = 0.0009949 R + 1.0000; r = 0.9848; S, = 0.00214
5M (U,& = 212.6 (D% - 1.0000); r = 0.9701; S, = 0.419 gm. ‘%
5T (US), = 245.7 (D:iz - 1.0000); r = 0.9850; S, = 0.644 gm. %
6M (U,), = 639.52 (n - 1.33320); r = 0.9993; S, = 0.062 gm. y0
6T (Us), = 637.10 (n - 1.33320) ; r = 0.9998; AS’, = 0.070 gm. %

Fig. 2 illustrates equations 1M and 1T; Fig. 3, equations 5M and 5T.

Equations which relate refractive index with other variables may be
obtained by combining equations 6M or 6T appropriately with other
equations. Although the high coefficients of linear correlation make
practical the use of the above equations, the true relations are probably
slightly curvilinear; no attempt has been made to determine these. It
should be noted that equations 6M and 6T are the best of all for accurately
estimating Y from X.

DISCUSSlON

We have not investigated the contentions of earlier workers as to the

usefulness of refractometry for the determination of serum protein. How-
ever, our data do not support the view (6, 9) that the determination of
total protein should be adversely affected by variations in the albumin-
globulin ratio. We find that sera modified by the addition of protein and
saline to encompass a loo-fold range in the albumin-globulin ratio (see
“Serum” under “Results”) and pathological human sera selected for a
wide range of total protein and albumin-globulin ratio have essentially the
same refractometric coefficient as normal sera. Further, various protein
fractions in saline (Fig. 4) do not differ appreciably from serum in the
specific instrumental increment of the refractometer ( = l/K).
3 Empirically and approximately, Dir = 0.9966 D$.
874 TOTAL SOLIDS IN SERUM AND URINE

Therefore, to the degree that non-protein solid is constant in plasma,

and because it contributes only a small fraction of the refractivity, empiri-
cal relations between R or n and total protein of serum should prove
sufficiently accurate for many purposes. As a measure of total solid, and
thence of water* of serum, refractometry is at least as good as gravimetry,
considering the accuracy, speed, and size of the sample required.
Refractometry is equally useful as a measure of urinary solids and water,
and offers an additional advantage in that gravimetry of urine depends upon
a more elaborate method of drying than does serum.

I I I I
0 IO 20 30 40 50 60 70
R

FIG. 4; Relation between total solids of solutions of proteins of human serum dis-
solved in physiological saline and instrumental increment of refractometer. The line
drawn is that of Fig. 1, derived from observations of 211 human, dog, and rat sera:
(A& = 0.2242 R. The following proteinswere used: (a) human albumin, 25 per cent
solution, prepared from Red Cross plasma by Armour Laboratories, Kankakee, Illi-
nois, lot No. FP-21; (b) hyperimmune r-globulin, approximately 10 per cent solution,
Wyeth, Inc., Philadelphia, Pennsylvania; (c) purified protein fractions, dry, pre-
pared from Red Cross plasma by E. R. Squibb and Sons, New York, lot Nos. 97-RR
(Fraction II), 782 through 788 (Fraction IV-7), and 1438-J (Fraction V).

Blohm’s refractometric coefficient (3, 12) for the estimation of urinary

solids in man is 2.43.6 Our estimate of such a coefficient, derived from
equation 3M, is 2.50. Blohm’s specific gravity coefficient (“Haeser type”)
relating the second, third, and fourth decimal places of the increment,
D::: -1, and urinary solids is 0.218; our estimate, derived from equation
5M, is 0.213. Blohm’s coefficient and our coefficient are not strictly com-
parable in a mathematical sense because Blohm. simply used the arithmetic
mean of the individual ratios of total solid to instrumental increment of

4 (AH~o)~ = 100 - (&)s.

6 That is, 2.43 R = gm. per liter of urine. This coefficient is an average based on 63
protein-free sugar-free human urines dried at room temperature, in a vacuum, over
concentrated sulfuric acid.
 M. E. RUBINI AND A, V. WOLF 875

the refractometer or specific gravity increment, while our coefficients were

derived from a least squares regression line. However, by applying our
method to Blohm’s original data, the yield was 2.44 and 0.217, respec-
tively ; by applying Blohm’s methods to our data for man, the refracto-
metric and specific gravity coefficients are, respectively, 2.49 and 0.203.
We thus confirm closely Blohm’s numerical value for the refractometric
coefficient as well as his view that refractometry is superior to specific
gravity as a measure of total urinary solids. In many instances in which
total solid or specific gravity is desired, refractometry will provide an
accurate and practical measure of these in place of direct determination.
Possible interferences with refractometric analysis have not been ex-
haustively evaluated, nor have they been much in evidence in either
Blohm’s series or our series. The critical boundary may be less sharp
than that usual in lipemia. However, in only one of our cases, a patient
with nephrosis, whose lactescent serum contained 7 to 11 per cent total
lipides, was it impossible to make a reading. Other grossly turbid, slightly
hemoglobinemic, or jaundiced sera could be read.
Acetone added to urine increases the R but reduces the (US), value.
Each volume per cent of added acetone raises the apparent total solids
(i.e., KR) about 0.3 gm. per cent. It is interesting to note that a solution
of NH3 has a positive R value which varies with the concentration of the
dissolved gas, but its 8 is zero and its D$ is less than 1. Possibly “free”
ammonia in urine under unusual conditions would lessen the accuracy of a
refractometric determination of total solids.
The validity of refractometric coefficients for total solids has not been
tested with highly abnormal urines, as in certain pathological cases. More-
over, in the latter days of the prolonged fast of Benedict’s (13) subject, it
was found that the specific gravity coefficient for total solids was “con-
siderably larger than that accepted for normal people . . . probably ex-
plained by the fact that there were products of defective fat katabolism
in the urine.” The refractometric coefficient might conceivably be modi-
fied appreciably also, under these circumstances, although it generally
tends to vary less than that for specific gravity (compare Figs. 2 and 3).
However, where glucose, urea, or sodium chloride was added to dilute and
concentrated urines in amounts up to 10, 10, and 3 per cent, respectively,
the refractometric coefficient remained essentially the same.
Application of refractometry is sometimes hampered by relative costli-
ness of instruments and the inconveniences which attend temperature
regulation. Relatively inexpensive, temperature-compensated hand re-
fractometers (14), adaptable to various ranges of refractive index and cali-
brated in suitable units, may be expected to stimulate the employment of
this method in new areas.
876 TOTAL SOLIDS IN SERUM AND URINE

SUMMARY

Analysis of total solids and water content of serum and urine by refractom-
etry is shown to be accurate, precise, and rapid. Refractometric coefli-
cients for total solid of serum (man, dog, rat) and urine (man, dog, cat)
and specific gravity coeficients for urine are presented.
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