EVALUATION OF BENZOYL GLUCURONATE EXCRETION
(SNAPPER) FOR LIVER DYSFUNCTION*
TEST
J, GEORGE SHARNOFF, M.D., MILTON BUDNICK, M.D.,
AND GIZELLA JAKAB, A.B.
From the Departments of Pathology and Medicine, Mount- Vernon Hospital,
Mount Vernon, New York
In 1946 and 1947 Snapper, Greenspan and Saltzman 13-16 proposed a rapid
screening test for liver dysfunction based on the urinary excretion of conjugated
glucuronic acid following the ingestion of a fixed quantity of benzoic acid. Inasmuch as there appeared to be some indication that the test could be useful in
distinguishing surgical obstructive from non-obstructive jaundice17 we undertook
a study of the test. For the purpose of comparison a series of liver tests, of proven
merit and in routine use, were also performed on the patients studied.
The urinary excretion of benzoyl glucuronate following the ingestion of 5
grams of benzoic acid is said to occur only in persons with liver dysfunction. 13 ' 16
The proponents of the test believed that in a person with a normal liver, benzoic
acid is rapidly synthesized with glycine to hippuric acid, whereas a diseased liver
lags in this synthesis, leaving free benzoate to conjugate with glucuronic acid as
benzoyl glucuronate; and that the lag in synthesis of hippuric acid will result in
urinary excretion of benzoyl glucuronate as shown by the increased quantities
found in each succeeding two-hour sample of urine. As indicated in Table 1, in
the presence of minimal liver damage the greatest excretion of benzoyl glucuronate occurs within four hours, and in severe damage at six hours.
This report is concerned with tests in 31 patients with proven liver disease
and in 7 persons used as controls. Of those with liver disease, in 24 the diagnosis
was confirmed at autopsy or by biopsy, while the remaining 7 patients had clinical
acute viral hepatitis, recovered, and were then followed for a sufficiently long
time to confirm their recovery. Of the 31 patients with liver disease, 12 had cirrhosis; 7, viral infectious hepatitis including 2 with homologous serum jaundice;
5, metastatic cancer to liver; 5, extrahepatic biliary obstruction; 1, hepatitis
associated with infectious mononucleosis; and 1 amyloid disease.
METHOD
The Snapper test was performed as originally reported.15 The fasting patients were each given 5 grams of benzoic acid orally. All other medication,
including infusions of glucose, was omitted. Breakfast was also omitted but lunch
was allowed. Urine samples were obtained at two, four and six hours and were
stored in the refrigerator until tested. One one-thousandth part of each two-hour
urine sample was placed in a test tube and diluted to 2 ml. with distilled water.
To each urine sample was added 2 ml. of concentrated hydrochloric acid and 2
ml. of 0.2 per cent freshly prepared aqueous naphthoresorcinol. The test tubes
* Received for publication, October 5, 1950.
234
BENZOYL GLUCURONATB
235
TEST
were placed in a bath of boiling water for ten minutes, and then cooled in running
tap water. Two ml. of iso-amyl alcohol was added to extract a blue color, indicating the presence of benzoylglucuronate. The results were read with the aid
of fluorescent light, and graded as negative (no blue color) or 1, 2 or 3 plus depending on the intensity of the blue color obtained, a 3 plus reading having an
ink-blue appearance. In several instances a greenish blue color was obtained
which interfered with the reading. This was cleared by applying the lead precipitation method of Salt10 as suggested by Snapper and Saltzman.14 Since benzoyl
glucuronate excreted in the urine could be tested by means of the Tollens18
method as modified by Salt,10 this technic was used on the same urine samples
for the purpose of comparison. With the latter method, 5 ml. of urine was placed
in a test tube and then 1 ml. of an alcoholic 1.0 per cent solution of freshly prepared naphthoresorcinol and 5 ml. of concentrated hydrochloric acid were added.
The test tubes were heated in boiling water for one minute, allowed to stand at
room temperature for four minutes and then cooled in tap water. The blue color
TABLE 1
SCHEME U S E D ' I N T H E I N T E R P R E T A T I O N O F R E S U L T S O B T A I N E D WITH S N A P P E R
AND T O L L E N S T E C H N I C S AS R E C O R D E D I N D U P L I C A T E
ABNORMAL PATTERNS
TIME
2 hr.
4 hr.
6 hr.
NORMAL
PATTERNS
-
-
J)egree of Damage to Liver Cells
Minimal
Moderate
++ +++
+++ +++
++
Severe
+
+++ ++
— denotes no blue color.
+ to + + + denotes range from light, blue to ink-blue color.
was then extracted with 2 ml. of ether. The colors obtained were the same as with
the technic recommended by Snapper, Saltzman and Greenspan and were recorded in the same manner.
In Table 1 is outlined a scheme for the interpretation of the results in normal
and abnormal patterns as originally suggested by Snapper and Saltzman.17
The results recorded in the scheme are of determinations done in duplicate. The
same scheme was used for the interpretation of the results obtained with the
Tollens technic.
The other routine liver tests performed consisted of icterus index8 or serum
bilirubin,17 total protein and albumin-globulin fractionation,2 serum alkaline
phosphatase of Bodansky1 and cephalin-cholesterol flocculation.4
DISCUSSION
The results of this study indicate that the urinary excretion of conjugated
glucuronic acid following the ingestion of 5 grams of benzoic acid is of rather low
sensitivity as a test of liver dysfunction. In this respect it has the same weakness
23G
SHARNOFF,
BUDNICK
AND
JAKAB
TABLE 2
COMPARISON O F V A L U E S OF S E V E R A L T E S T S OF L I V E R F U N C T I O N I N 7 C O N T R O L S
AND 30 P A T I E N T S WITH V A R I O U S F O R M S OF L I V E R D I S E A S E
SNAPPER TEST TOLLEKS TEST
TOTAL
PROTEIN
ACE,
SEX
ALBUMIN
ICTERUS
GLOBULIN
INDEX
RATIO
Gm. per
2 hr. 4 hr. 6 hr. 2 hr. 4 hr. 6 hr.
100 Ml.
ALKALINE
PHOSPHATASE
CEPHALIN
FLOCCU"
L ATI ON
Bodansky
Units
24 48
hr. hr.
Cont rols
1. 78,
M
2. 87,
M
3. 62,
M
4. 60,
M
5. 20,
F
6. 73,
F
7. 19,
F
5.2
3.6:1.6
25
6.6
2.6:4.0
5
-
7.0
4.4:2.6
— _
_
5.6
3.4:2.2
5
24- 14-
-
6.8
2.6:4.2
5
6.8
4.6:2.2
5
7.0
4.6:2.4
5
-
14-
-
-
14-
- -
-
14- 14-
-
24-
-
-
14-
14-
-
-
-
4.5 44- 44- P.M.* Acute myocardial infarction
5.2 34- 34- P.M. Acute diffuse
peritonitis
P.M. Acute myocar3.1
dial infarction
8.8 44- 44- P.M. Acute myocardial infarction
3.0 - 14- Infectious hepatitis, 7
mo. previously
5.6
Homologous
serum
jaundice, 18 mo.
2.8
Infectious mononucleosis a n d hepatitis, 4 mo.
Cirrhosis
-
24- P.M. Cirrhosis
-
14- P.M. Cirrhosis
5.8
7.8
2.1:5.7 10-65
2.8 44- 44- P.M. Cirrhosis
9.8
11.
65,
-
6.0
3.0:3.0
3.5 34- 44- B.* Cirrhosis
12.
53,
_
_
-
6.8
3 . 3 : 3 . 5 20-80 14.1 44- 44- B.
29.5
13.
63,
F
14.
57,
M
15.
50,
F
-
24-
24- 14-
6.0
3.3:2.7
5-35
5.6 34- 34- P.M. Cirrhosis
14- 24- 24- 24- 34- 44-
5.8
3.5:2.3
5
3.4
3.7
-
2.7
-
24- 14- -
24- 24-
_ - 14- -
2.8:3.0 5-15
3.4
7.0
5.7
8. 65, 24- 24- 24- 4 + 44- 44M
9. 57, 14- - 2 + 24- 24F
10.
33,
25
Biliary
'
_
_
_ - 14-
-
5
34- B. Cirrhosis
*
24- B. Cirrhosis
cirrhosis
T A B L E 2, Continued
SNAPPER TEST
TOLLENS TEST
TOTAL
PROTEIN
AGE,
SEX
ALBUMIN
ICTERUS
GLOBULIN
INDEX
RATIO
per
2 hr. 4 hr. 6 hr. 2 hr. 4 hr. 6 hr Gm.
100 Ml.
I
Cirrhosis
16.
62,
ALKALINE
PHOSPHATASE
CEPHAUN
FLOCCULATION
Bodansky
Units
24 .48
hr. hr.
(Continued)
5.6
3.1:2.5
5-8
3.3
6.6
-
3+ B. Cirrhosis
17.
53,
-
2+
- _
_
_
6.6
3.4:3.2 10-20 13.3 3 + 4 + B. Cirrhosis
15.9
18.
59,
-
2+
- -
2+
-
6.2
1.5:4.7
70
8.9
19.
45,
M
_
_
_ 4+ 4+ 4+
9.S
4.0:5.8
5-20
4.9 4 + 4 + P.M. Cirrhosis
9.0
_
_ B. Cirrhosis
E x t r a h e p a t i c Biliary Obstruction
7.2
2+2+3+1+1+2+
20.
55,
M
21.
1+ 2+ 2+1+ 1 +
6.6
28,
F
22.
2+ 3+ 3+3+ 4+ 3+ 6.2
77,
M
23.
- 3+ 2+2+ 3+ 1 + 6.2
64,
M
24.
4+ 1+ - 4+ 1+ 1 + 6.3
37,
M
4.7:2.5110-70
8.9
3 + 4 + B.
- 2+
4.0:2.6 50-10 15.2
9.8
2.7:3.5
Carcinoma common bile duct
OP* Common
stone
duct
200 25.2 4+ 4+ P.M. Carcinoma head
of pancreas
3.9:2.3 50-15 IS.8
9.3
2+ 2+ B.
3.9:2.4 30-8
1+ 4+ B. Cholangitis, b i l i -
15.1
10.9
Papilloma a m pulla of Vater
ary obstruction
M e t a s t a t i c Carcinoma of Liver
25.
70,
F
26.
64,
M
27.
65,
F
28.
66,
M
29.
78,
F
2+
-
7.2
2.7:4.5
7.5
3.9:3.6
2+ 1 +
5.5
2.4:3.1
1+
5.S
3.1:2.7
5.3
3.1:2.2
1+ 3 +
-
1+
-
1+
- -
2+
-
1+
-
237
25
7.1
7.7
3+ 4+
P.M.
breast
Carcinoma
8.0
12.0
P.M.Carcinoma
chus
bron-
10
7.4
10.8
P.M. Carcinoma rectum
50
9.4
P.M. Carcinoma sigmoid
4.7
5.4
P.M.
breast
Carcinoma
238
SHARNOFF, BTJDNICK AND JAKAB
T A B L E 2, Concluded
PATIENT
NO.,
AGE,
SNAPPER TEST
TOLLENS TEST
TOTAL
PROTEIN
ALBUMIN'
ICTERUS
GLOBULIN
INDEX
RATIO
ALKALINE
PHOSPHATASE
CEPUALIM
F LOBULATION
DIAGNOSIS
Bodan- 24 48
sky
Units hr. hr.
2hr.4hr.6hr. 2 hr. 4 hr. 6 hr. Gm. per
100 ml.
Acute Viral H e p a t i t i s
30.
— 1+ 2+ - 1+ 2+
33,
F
31.
2+ — — 2+ 1+ 16,
6.3
3.5:2.S
7.5
4.0:3.5
32.
1 + — — 2+ 1+
15,
-
6.6
4.0:2.6
33.
2+ 2+ 2+ 2+ 2+ 2+
18,
6.8
4.0:2.8 40-15 12.4 4+ 4+ Infectious hepatitis
5.6
34.
30,
- 2+ 2+ - 2+ 2+
6.2
3.2:3.0
35.
1 + 3 + 2 + 3+ 3+ 2+
30,
6.4
2.6:3.8 11-3.6 12.5 4+ 4+ Infectious hepatitis
4.6
30
8.3 3+ 4 + Infectious hepatitis
6.3
4.0 2+ 3+ Exacerbation of in4.2
fectious hepatitis
5
30
8.8 4+ 4+ Infectious mononu10.7
cleosis with hepatitis
4.2
-
4+ Infectious hepatitis
36.
1+ 30,
- 2+ 2 +
-
7.1
4.2:2.9 75-15
19.7 4 + 4 + Homologous
jaundice '
37.
2+ 1+
68,
M
- 3+ 3+
-
5.5
3.1:2.4
5.6 4+ 4 + P.M.
Homologous
serum jaundice
20.9
* P.M. indicates diagnosis a t a u t o p s y ; B.,
operation.
100
scrum
diagnosis on biopsy; OP., diagnosis a t
as the hippuric acid test. 6 The same factors may apply here which account for
the limitations of the hippuric acid test, namely, poor absorption of the benzoic
acid by the intestinal tract and possibly poor renal excretion of the conjugated
glucuronic acid.
With the control group (see Table 2) in this series we find first that the Snapper
and Tollens tests gave negative or weakly positive results in all instances, whereas
patients with cardiac disease with minimal parenchymal liver damage caused by
chronic passive congestion gave positive results with the more sensitive (II)
cephalin-cholesterol flocculation test. The latter has also been observed by others. 5, 9 The Snapper and Tollens tests, however, gave poor correlation in 12 patients with cirrhosis (see Table 2), 5 of these patients having false negative results
with each technic. It is in cirrhosis that the hippuric acid test also is least sensi-
BENZOYL GLUCURONATE TEST
239
tive. However, the series of liver tests which were used gave combined results
consistent with cirrhosis in at least 10 of the 12 patients studied. This is based
chiefly on the moderate to marked alteration of the albumin-globulin fractions,
the normal to moderately elevated values for serum alkaline phosphatase (well
below the obstructive jaundice level of 10.0 Bodansky units suggested by Gutman3) and the positive cephalin-cholesterol flocculation tests. In two patients
(Nos. 12 and 17) the serum alkaline phosphatase values were in the obstructive
range; one of these two (patient No. 12) had the highest enzymatic activity and a
biliary type of cirrhosis.
In the 5 patients with extrahepatic biliary obstruction the Snapper and Tollens
tests proved positive in all instances whereas negative results might have offered
the likelihood of distinguishing surgical obstructive from non-obstructive jaundice. It must be emphasized, however, that in only 1 of the 5 patients was an
uncomplicated obstruction by calculus present. Nevertheless the series of routine
liver tests in this group gave far better diagnostic aid, especially the serum alkaline phosphatase values. The latter gave levels in the range found in obstructive
jaundice in 4 of 5 patients and showed no alteration of the albumin-globulin
fractions in a like number of instances. The cephalin-cholesterol flocculation,
however, was positive in all 5 patients. No definite conclusions could be drawn
from the results obtained in the 5 patients with intrahepatic biliary obstruction
as produced by hepatic metastatic carcinoma (see Table 2).
The 8 patients with acute viral hepatitis all gave positive results wit1 the
Snapper and Tollens tests as well as with the cephalin-cholesterol flocculation
test. The Tollens test appeared to be more sensitive than the Snapper Test but
the cephalin-cholesterol flocculation test seemed to be the most sensitive of the
three tests. In only two patients (Nos. 36 and 37), both with homologous serum
jaundice, was the serum alkaline phosphatase value markedly elevated.
Finally, a single patient with presumptive amyloid disease as demonstrated by
a biopsy of the gum, gave a negative Snapper test and a strongly positive Tollens
test. The patient, a 33-year-old Negro woman, had chronic rheumatoid arthritis
with unexplained hepatosplenomegaly. She was not icteric. The blood protein
value was 8.8 to 9.2 Gm. per 100 ml. of blood and the blood globulin value 5.2
to 5.9 Gm. The cephalin-cholesterol flocculation test was strongly positive. The
serum alkaline phosphatase values ranged from 17.1 to 19.5 units. A Congo red
test showed 34 per cent retention of the dye. There was no evidence of other
skeletal disease. The diagnosis of amyloid disease was suspected chiefly on the
basis of an otherwise unexplainable increase in activity of serum alkaline phosphatase. Amyloid disease of the liver has been reported1'2 to produce marked elevation of the serum alkaline phosphatase.
SUMMARY
1. An evaluation was made of the benzoyl glucuronate excretion test (Snapper)
for liver dysfunction in 31 patients with liver disease. In 24 patients diagnosis
was established by biopsy or autopsy. Seven patients were used as controls.
2. The Snapper test was found to have low sensitivity as a test of liver dysl'unc-
240
SHARNOFF, BUDNICK AND JAKAB
tion, especially in cirrhosis, but gave uniformly satisfactory results in viral hepatitis.
3. There seems little likelihood that the Snapper test will prove of value in distinguishing obstructive from non-obstructive jaundice.
4. Employment of a series of liver tests in routine use, including icterus index
or serum bilirubin, serum alkaline phosphatase, total protein and albumin-globulin fractionation and the cephalin-cholesterol flocculation tests gave more consistent diagnostic aid, especially in cirrhosis, viral hepatitis and obstructive
jaundice.
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