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PRECIPITATION OF ABNORMAL SERUMS BY LUGOL'S SOLUTION*
M. SALAZAR MALLEN, M.D., ELENA LOZANO UGALDE, Q.B.P.,t M. REFUGIO
BALCAZAR, Q.B.P., JOSE I. BOLlVAR, Q.B.P., AND SERGIO MEYRAN, Q.B.P.
From the Laboratory of Immunology, Institute N. de Cardiologia, Mexico D. F.
Iodine has been used in different forms as a reagent to detect abnormalities of
serum protein. Botelho 1 used weak Lugol's solution and citric acid to produce
precipitation of protein from diluted cancerous serum. Recently, Huggins, Miller and Jensen 6 described abnormal iodacetate indexes in tuberculous and cancerous serums. We have found that strong Lugol's solution, as recommended by
Weiss,8 for use in Gram's stain, gives precipitation of the serum in patients with
various diseases. The precipitation seems to be related to qualitative changes
in the serum proteins, especially in cases of impaired liver function.
MATERIALS AND
METHODS
Strong Lugol's solution was prepared by mixing 20 Gm. of iodine and 40 Gm.
of potassium iodine in a mortar and dissolving in distilled water to make 300 ml.
Fresh, non-heated serum was used for the reaction. I t has been found that, bilirubin does not affect the test while hemolysis noticeably alters it.
The test was made by mixing one drop of the serum and one drop of Lugol's
solution on a glass slide. The results, recorded within five minutes, are as follows:
1. Negative reaction: the mixture remains transparent; the serum shows only
a change of color, owing to its mixture with Lugol's solution;
2. Strongly positive test: the serum immediately forms a heavy ( + + + + )
amorphous precipitate;
3. Positive test: the serum shoAvs a heavy granular ( + + + ) precipitate;
4. Moderately positive test: the precipitate is lighter ( + + );
5. Weak positive test: turbidity appears and, after a few minutes of rotation
by hand, a fine precipitate ( + ) .
The distinction between positive and negative tests is easy if note is made of
transparency or if positive and negative serums are compared. u'
RESULTS
The test was carried out on the serums of 110 healthy adults of both sexes.
All but three (97 per cent) were completely negative. The three positive tests
were all moderate; one subject had had tonsillitis one week before, the second
had had colitis and the third had been on a low-protein diet for several weeks.
The test was also performed on serums from 119 persons with various diseases.
The majority had hepatic cirrhosis, arteriosclerosis, syphilis and rheumatic fever.
Results of these observations are shown in Table 1. All seven patients with mal* Received for publication, July 25, 1949.
f Q.B.P. is an abbreviation for the title Quimico, Biologo y Parasit61ogo.
39
40
M A L L E N ET
AL.
nutrition gave positive reactions, while 17 of 22 (77 per cent) patients with
hepatic cirrhosis gave positive tests.
SIGNIFICANCE OF THE
REACTION
If the test is done in a test tube, the larger part of the precipitate may be recovered by centrifuging. It is an amorphous, brown substance which is insoluble
in water but readily soluble in weak sodium hydroxide, precipitable with trichloracetic acid, and gives the biuret reaction. Nitrogen determinations by the
Kjeldahl method indicate a high protein content which, added to the protein of
TABLE 1
P R E C I P I T A T I O N BY L U G O L ' S SOLUTION O F S E R U M S FROM P A T I E N T S WITH D I F F E R E N T
CLINICAL CONDITIONS
NO. OF
CASES
NEGATIVE
++++ +++ ++
Rheumatoid a r t h r i t i s ; rheumatic fever
inactive
Brucellosis
Toxic goiter
Gastric cancer
Cholecystitis.
Hepatic cirrhosis
Undernutrition
Diabetes mellitus
Arteriosclerosis
Hepatitis
Congestive heart failure
Obesity
Active rheumatic fever
L a t e n t syphilis
Syphilitic aortitis
Salmonellosis
Gastric ulcer
Totals
7
2
1
2
8
22
7
3
17
7
10
1
11
11
7
1
2
1
0
0
5
5
0
3
16
5
6
1
9
8
4
1
2
119
73
%
/
37
77
100
28
40
18
27
42
21
11
38
the supernatant, equals the total serum protein. This means that the precipitation involves some, but not all, of the proteins of the serum.
In 100 cases the tests with Lugol's solution were run in parallel series with determinations of total protein, albumin and globulin. The comparison of the
absolute quantities of the protein fractions with positive reactions shows that
there is a general tendency for positive serums to have a low albumin and a
high globulin content. Exceptions to this occur, however, as shown in Table 2.
Table 2 SIIOAVS that some relationship also exists betAveen precipitation by
Lugol's solution and presence of an abnormal albumin/globulin ratio. In a feAV
cases, hoAvever, this coincidence Avas not found. The precipitation Avas positive
in 5 per cent of the cases Avith normal albumin/globulin ratios, but some nega-
PRECIPITATION OF SERUM BY LUGOL'S SOLUTION
41
tive serums showed reversed A/G ratios. Considering the exceptions already
noted, a relationship may be established between the albumin/globulin ratio
and the positive reactions. The intensity of the reaction shows definite correlation with the values of the albumin/globulin ratio. In other words, a strong
TABLE 2
A L B U M I N / G L O B U L I N R A T I O S IN R E L A T I O N TO P R E C I P I T A T I O N BY L U G O L ' S SOLUTION
IN
A/G RATIO
2.46
2.19
2.10
2.02
1.89
1.81
1.79
1.78
1.78
1.75
1.73
1.73
1.68
1.67
1.64
1.64
1.60
1.59
1.59
1.56
1.55
1.55
1.52
1.52
1.50
1.48
1.47
1.47
1.41
1.40
1.40
1.40
1.39
TEST WITH
LUCOL'S SOLUTION
_
—
—
-
++
—
—
-
+
—
—
—
—
—
—
—
—
++
++
++++
—
—
100
A/G RATIOS
1.38
1.37
1.36
1.35
1.34
1.31
1.30
1.30
1.30
1.29
1.27
1.26
1.24
1.24
1.18
1.17
1.16
1.15
1.14
1.11
1.10
1.08
1.06
1.05
1.00
1.00
0.99
0.99
0.98
0.98
0.96
0.95
0.94
SERUMS
TEST WITH
LUGOL'S SOLUTION
—
—
+++
—
—
—
—
—
—
++
—
—
—
+
+
+
—
—
+++
+
++
++
++++
—
+++
-
++++
++
++++
+
A/G RATIOS
0.94
0.94
0.94
0.93
0.92
0.92
0.91
0.90
0.S7
0.85
0.85
0.84
0.84
0.S2
0.81
0.80
O.SO
0.80
0.7S
0.78
0.78
0.76
0.75
0.75
0.72
0.72
0.72
0.64
0.64
0.63
0.59
0.54
0.40
0.26
TEST WITH
LUGOL'S SOLUTION
—
—
++
+
+++
+++
+++
+++
++++
++
—
+
++
+ .+ +
+"
—
++++
+++
++
++
+
+++
+++
+++
+++
+++
++++
++
++
+++
++++
++++
++++
+++
precipitation is accompanied, in general, by an abnormal A/G ratio, while the
weak or negative tests are associated with normal or nearly normal ratios
(Table 3).
In 80 cases, it was possible to compare the results of the Lugol test, with the
A/G ratio, thymol turbidity andcephalin-cholesterol flocculation tests (Table 4).
42
MALLEN ET AL.
Serum abnormality was primarily shown by the thymol turbidity, less frequently
by precipitation with Lugol's solution and still less often by the cephalin-cholesterol fiocculation test. In 44 cases (55 per cent) all of the tests were positive.
The thymol test was the only one positive in 11 cases (13.75 per cent), and the
test with Lugol's solution was the only positive test in one case. The cephalincholesterol fiocculation test was never positive by itself.
I t is known that the results of the cephalin-cholesterol fiocculation and the
TABLE 3
D E G R E E OF P R E C I P I T A T I O N OF S E R U M S BY L U G O L ' S SOLUTION IN R E L A T I O N TO A L B U M I N /
GLOBULIN RATIOS
DEGREE OF PRECIPITATION
++++
Number of cases
A/G index (mean)
+++
++
+
-
10
17
13
12
48
0.80 ± 0.27 0.84 ± 0.22 1.06 db 0.34 1.0S ± 0.26 1.47 ± 0.3
TABLE 4
COMPARISON
OF R E S U L T S
OF A L B U M I N / G L O B U L I N
RATIO, PRECIPITATION
BY
LUGOL'S
SOLUTION AND T H Y M O L T U R B I D I T Y AND C E P H A L I N - C I I O L E S T E R O L FLOCCULATION T E S T S
A / G RATIO <
1.25
T E S T WITH
LUGOL'S
SOLUTION
29
11
11
7
7
5
3
3
2
1
1
+ + 1 1 1 + 1 ++
11
+ + 1 1 1 1+ 1+ 1 +
Total 80
Positive: 50 (62.5%)
46(57.5%)
THYMOL TEST
CEPHALINCHOLESTEROL
TEST
+
+
+
1 1 1 ++ 1 1 + 1 1 +
NO. OF CASES
65 (SI. 2%)
42(52.2%)
+
+
+
+
thymol turbidity are not parallel.4 The mechanisms involved in these two reactions are not exactly the same, since the first test does not require the presence
of phospholipids which are necessary for the thymol precipitation. 5 The close
parallelism that we observed between the precipitation with Lugol's solution
and the flocculation of cephalin-cholesterol (both positive in 70 per cent of
cases) may be indicative of the independence of both from phospholipids, since
extraction of abnormal serums with ether does not change either the precipitation or the flocculation.
Other authors 3 ' 4 agree that, in all these reactions, the fundamental change
rests on a qualitative alteration of the albumin fractions in the plasma, resulting
PRECIPITATION OF SERUM BY LUGOL'S SOLUTION
43
in an increased precipitation of the globulins. The following experiments carried
out with abnormal (Lugol-positive) and normal serums seem to support this
assumption.
Separation of the albumin and the globulin fractions was made by precipitation with ammonium sulfate on two Lugol-positive and two normal (Lugol-negative) serums, and the Lugol test was made on different protein fractions. Mixtures were also made of normal (Lugol-negative) and abnormal (Lugol-positive)
TABLE 5
REACTION OP P R O T E I N FRACTIONS FROM N O R M A L AND ABNORMAL S E R U M S WITH
L U G O L ' S SOLUTION
TEST
1
2
3
4
5
0
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
CM. ALBUMIN
6.4S
4.75
3.24
2.73.
1.62
1.18
3.24
1.62
0.81
0.40
0.20
0.10
6.64
6.64
3.32
1.16
0.58
0.29
6.64
3.32
1.66
0.83
N*
N
N
N
N
N
N
N
N
N
N
N
P
P
P
P
P
P
P
P
P
P
VOLUME
CM. GLOBULIN
VOLUME
A / G RATIO
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
2.SO N
2.S6 N
2.SO N
2.S6N
2.SON
2.S6 N
1.64 P
1.64 P
1.64 P
1.64 P
1.64 P
1.64 P
1.40 N
2.SON
2.SO N
2.SO N
2.SO N
2.80 N
0.S2 P
0.S2P
0.S2 P
0.82 P
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
0.05
2.31
1.66
1.15
0.83
0.57
0.46
1.99
0.99
0.49
0.24
0.12
0.06
4.74
2.37
1.18
0.59
0.29
0.19
S.00
4.00
2.00
1.00
TEST WITH
LUGOL'S
SOLUTION
_
—
+
+
+
++
—
+
++
++
+++
+++
+
+
++
++
+++
+++
+
++
+++
+++
* N indicates fraction from normal (Lugol-negative) s e r u m ; P , fraction from pathologic
(Lugol-positive) serum.
globulins and normal and abnormal albumins, so as to have artificial models
with different albumin/globulin ratios to be tested with Lugol's solution.
The results are shown in Table 5. Our data seem to support the hypothesis
that the precipitation of globulins by Lugol's solution is protected to a certain
extent by normal, but not by abnormal albumin. Precipitation of serum by
Lugol's solution could thus be the result of either a decrease of the normal
albumin resulting in a low A/G ratio, or the existence of an abnormal albumin
unable to protect the globulin even in the case of a normal or high A/G ratio..
The behavior of the abnormal, Lugol-positive serums was examined electrophoretically in two instances (Figs. 1 and 2).
44
M A L L E N ET
AL.
Positivity did not seem to be associated with the increase of a given globulin,
but rather, in the two cases studied, with decrease of the albumin component.
FIG. 1. Electrophoretic pattern, case 1 (barbiturate buffer, pH
8.5, 20 MA, 5400 s.). Increase of the "y" globulin.
FIG. 2. Electrophoretic pattern, case 2 (barbiturate buffer, pH
8.5, 20 MA. 5400 s.). Increase of the "a2" globulin.
SUMMARY
1. Strong Lugol's solution precipitates serum proteins of some patients.
The phenomenon was seldom found with serums from healthy persons.
2. The precipitation has a definite relationship with abnormal protein values,
and, more specifically, with changes of the albumin/globulin ratio.
3. Precipitation of serum by Lugol's solution is correlated with positive cephalin-cholesterol and thymol turbidity tests.
PRECIPITATION OF SERUM BY LUGOL'S SOLUTION
/
45
4. Isolated globulins from normal and abnormal serums were precipitated by
Lugol's solution, whereas normal and abnormal albumins were not.
5. Normal albumin protected globulins from being precipitated, while abnormal albumin lacked protecting power.
G. Electrophoretic studies made Avith two Lugol-positive serums did not show
abnormality of a given globulin fraction.
7. The Lugol test is so simple, and positive so often in serums with abnormal
albumin values, that it can be routinely used to recognize or screen such abnormal serums.
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WII.LHEIII, R . : Biochemistry of Malignant T u m o r s . Brooklyn: Reference Press, 1943,
p.S55).
2. DAMBSMEVITSKI, .13.: Suerodiagnosticodel cancer. (Thesis.) F a c u l t a d deCiencias Quimicas, Mexico, 1939.
3. F I B B B O DEL Rfo, L., ARRIETA, A. D . , AND C A N O , F . : Evaluaci6n de la p r e u b a del e n t u r -
biamiento con timol. Rev. mdd. d. Hosp. gen., 8: 7S2, 1947.
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0. MACLAOAN, N . F . , AND B U N N , D . : Flocculation test with clectrophorctically separated
proteins. Biochem. .J., 4 1 : 5S1, 1947.
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