THE CLINICAL APPLICATION OF THE HIPPURIC ACID
AND THE PROTHROMBIN TESTS*
ARMAND J. QUICK
Department of Pharmacology, Marquette University School of Medicine, Milwaukee
A survey of recent advances in medicine and surgery yields
numerable examples of problems solved by laboratory methods
which had completely defied all approaches by purely clinical
means. No better illustration can be cited than the recognized
dangers confronting the jaundiced patient who required surgical
treatment. All the surgeon's clinical experience availed him
little in determining the extent to which the liver had been injured; nevertheless this information was highly desirable for
determining the type of anesthesia, the possible postoperative
course, and numerous other factors. Furthermore while it was
clearly recognized that every jaundiced patient was potentially in
jeopardy of serious and occasional fatal hemorrhage, it was
neither possible to predict clinically which subjects would bleed,
nor to take steps to prevent this catastrophic complication. Two
simple tests developed during the past six years have been accepted rather widely by American surgeons as definite aids,
particularly in the surgery of the biliary tract in meeting the two
problems just presented. The first of these tests is the hippuric
acid synthesis as a means for determining liver function; the
second, the quantitative method for estimating the prothrombin
concentration of the blood.
THE HIPPURIC ACID TEST FOR LIVER FUNCTION 1 -
2
Principle. When benzoic acid is taken into the body, it is
combined with glycine and the resulting compound, hippuric
acid, is excreted in the urine. The reaction is:
Benzoic acid + Glycine = Hippuric acid
* Received for publication, July 13, 1939.
Read before the Eighteenth Annual Meeting of the American Society of
Clinical Pathologists, May 12, 1939, St. Louis.
222
HIPPUBIC ACID AND PROTHBOMBIN TESTS
223
This physiological process requires first, glycine which the
organism must synthesize, since this compound is not stored in
the body; and secondly, a mechanism to combine the glycine with
benzoic acid. In man, both processes appear to reside principally
in the liver. Consequently, in hepatic damage the output of
hippuric acid is reduced, and clinically there is evidence that the
decrease is fairly proportional to the hepatic impairment. The
actual test consists in administering a definite quantity of sodium
benzoate and then determining the amount of hippuric acid
excreted during a fixed period. In the original test, the sodium
benzoate is given by mouth and the hippuric acid which is
excreted during the ensuing four hours is determined. Recently,
an intravenous modification was developed3 in which a smaller
amount of sodium benzoate is given and the quantity of hippuric
acid determined which is produced in one hour.
The oral test. The patient is given 6 grams sodium benzoate dissolved
in 30 cc. of water flavored with oil of peppermint, which serves as a satisfactory
vehicle. A half glass of water should be drunk immediately. More water
may be taken but, in order to keep the urine down to a volume convenient
for handling, excessive drinking of water should be discouraged. It is desirable
that the test be done in the morning one hour after the patient has had a light
breakfast consisting of toast or cereals, and coffee, tea or milk. Immediately
before the test, the bladder should be emptied, and thereafter, a complete
specimen collected every hour for four hours. The patient must be instructed
to take no drugs for two days prior to the test.
Each specimen is carefully measured, and solid ammonium sulfate added
in the proportion of 5 grams for every 10 cc. of urine. (Weichselbaum and
Probstein4 suggested adding sodium chloride to the urine to decrease the solubility of hippuric acid. Their modification has distinctly increased the accuracy
of the test and has further led to the investigation of other salts. It was found
that ammonium sulfate is somewhat superior to sodium chloride.) When
the salt has dissolved, the urine is either filtered or centrifuged, and then acidified with hydrochloric acid. Usually 1 cc. of the concentrated acid is sufficient,
but it is necessary that the urine be distinctly acid to Congo red or thymol
blue. It is desirable to add a slight excess of the acid. After vigorous stirring,
the specimen is allowed to stand 30 minutes before filtering off the crystalline
hippuric acid. It is convenient to use a 4.5 cm. filter paper on a 2.5 cm. filter
plate. The hippuric acid is dried and weighed without removing it from the
filter paper. Another filter paper of the same size can be employed as counterweight.
The intravenous test. It is best that the test be done in the morning an hour
224
AEMAND J. QUICK
after the patient has had a light breakfast. A solution containing 1.77 gram
of sodium benzoate (equivalent to 1.5 gram of benzoic acid) in 20 cc. of distilled
water is given intravenously. This solution is prepared with the usual precautions taken in making any preparation intended for intravenous use.* The
injection should require 5 minutes or more. If the solution is given faster, the
patient is apt to experience a pain in the upper arm. The patients voids before
the test, and completely empties his bladder exactly one hour after the injection.
In order to increase the volume of the urine, it is advisable to have the patient
drink a glass of water a short time before the injection. The hippuric acid is
determined in the same manner as in the oral test.
If the hourly output in either of the tests exceeds 150 cc, it is advisable to
concentrate the urine on a water bath to about 50 cc. before adding the ammonium sulfate and precipitating the hippuric acid. Before concentrating
the urine, it should be made slightly acid with acetic acid. Hippuric acid is
easily hydrolyzed in alkaline solution, therefore the urine should not be allowed
to undergo ammonical fermentation. Toluene is a good preservative.
Calculation. The amount of precipitated hippuric acid can be determined
either by titration or by weighing directly. To the amount obtained, one
must add the quantity which remained in solution. It has been found that
100 cc. of urine containing 50 grams of ammonium sulfate will dissolve approximately 0.1 gram of hippuric acid. To express the hippuric acid in terms
of benzoic acid the weight of the former is multiplied by 0.68.
Interpretation. The average healthy adult will excrete from 3 to 3.5 grams
of benzoic acid as hippuric acid under the conditions of the oral test. For
convenience 3 grams is chosen as the average normal for calculating the output
in terms of per cent of normal. On this basis an excretion over 90 per cent
is considered non-pathological. It is likely that an excessive excretion, i.e.,
over 120 per cent is not normal, but at present it is not possible to explain such
results. The output of hippuric acid in the intravenous test has been found
to range from 1.0 to 1.4 gram (equivalent to 0.7 to 0.95 gram of benzoic acid).
Since 1 gram of hippuric acid can be accepted as the minimal normal output,
it is unnecessary to convert the weight of the hippuric acid to benzoic acid for
calculating the per cent of normal as it is done in the oral test.
Although the intravenous method has not been used extensively, sufficient
data have been obtained to show that it is equally as satisfactory as the oral
test. The intravenous procedure was introduced to meet certain demands
not met by the oral method. Its main advantages are: first, it can be given
to a patient who would vomit were he given sodium benzoate orally; secondly,
the collection of urine is simpler since only a one hour specimen is required;
and thirdly, it eliminates the danger of a false low hippuric acid output due
* Ampules of sodium benzoate suitable for the intravenous test have been
prepared by Hynson, Westcott, and Dunning, Baltimore, and by George A.
Breon Inc. Kansas City, Mo.
HIPPURIC ACID AND PROTHROMBIN TESTS
225
to gastric stasis, or obstruction in which the sodium benzoate might fail to be
absorbed properly. The oral test, however, has been found entirely satisfactory
in all but an occasional patient and it offers the one great advantage that it
does not require intravenous injections.
No attempt will be made to review the literature that has
accumulated since this test was introduced in 1933. The test
rests on two sound physiological principles. In the first place, it
determines important functions, namely the synthesis of glycine
and the conjugation of benzoic acid with glycine. This latter
reaction probably involves the same mechanism that the body
employs for the formation of glycocholic acid (cholic acid +
glycine = glycocholic acid). In the second place, the test
actually determines the maximum functional capacity of the
liver, since the organ is given more benzoic acid than it can
convert into hippuric acid in the period of time allowed. Under
these conditions, one measures not only the output of work for
ordinary physiological demands, but also the reserve.
Aims of the test. 1. To test the functional efficiency of the liver.
By means of the hippuric acid test, it is possible to obtain a fairly
quantitative measure of the functional state of this important
organ. To be sure the statements are still made that the reserve
of the liver is so great that defective function can only be demonstrated when it is too late, and that because of the multiplicity
of functions possessed by the liver, the measurement of a single
function cannot give reliable information concerning the functional status of the organ as a whole. Clinicians, and surgeons
in particular, are beginning to realize that these theories are
best reserved for erudite physiological discussions, and ignored
in clinical surgery. It is now recognized, as Snell6 has emphasized, that when a patient with biliary tract disease has an hippuric acid output of 50 per cent or less of normal, he is a poor
operative risk. By postponing the operation and waiting until
the condition of the liver has improved as indicated by the hippuric acid test, it is possible as Boyce has stressed to reduce the
mortality. In many other ways, a knowledge of the liver's
functional condition is of great value in surgery as well as in
medicine.
2. To aid in the differential diagnosis. The hippuric acid test
226
ARMAND J . QUICK
alone will not furnish a differential diagnosis since it only gives
a quantitative measure of the function of the liver. Nevertheless
if the information furnished by the test is used intelligently it
may help greatly in arriving at a diagnosis. Thus, for example,
if a patient suddenly develops a marked jaundice, a low hippuric
acid output would definitely point to a toxic hepatitis, whereas
a normal output would suggest biliary obstruction. In icterus
of long standing, the test is much less satisfactory for diagnosis,
since any obstruction is apt to cause secondary hepatic injury.
TABLE 1
T H E H I P P U R I C ACID T E S T AS AN A I D I N THE P R O G N O S I S O P H E P A T I C D I S E A S E S
E X C R E T I O N OF BENZOIC ACID AS HIPPURIC
ACID
'A
m
H
O
-<
lhour 2 hours 3 hours 4 hours
PEE
CENT
NORMAL
grams
grams
grams
grams
grams
F
27
0.27
0.65
0.57
0.99
0.84
0.98
0.42
0.59
2.10
3.21
70
107
M
70
0.26
0.57
0.71
0.74
2.28
76
0.15
0.25
0.41
0.29
1.10
37
0.10
0.13
0.25
0.27
0.33
0.29
0.42
0.36
1.10
1.05
37
35
F
59
REMARKS
Total
Catarrhal jaundice
One month later
Malignancy of the liver and
gall bladder
One month later
Atrophic cirrhosis with ascites
One month later after intensive treatment
3. To help in prognosis and in evaluating therapeutic measures.
By means of repeated hippuric acid tests it is possible to follow
fairly accurately the course of the various hepatic diseases as
illustrated in table 1. With this test Boyce6, has demonstrated
the beneficial action that glucose has on the liver, thus confirming
the numerous clinical observations. The effectiveness of the
treatment which I 2 have outlined for liver damage has likewise
been demonstrated by the hippuric acid test.
4- To furnish new information concerning the liver. A test such
as the hippuric acid offers a new approach to many clinical problems. Recently it has been found by Bartels 7 , Boyce8 and others
that the hippuric acid output is greatly reduced in severe hyper-
HIPPURIC ACID AND PROTHROMBIN TESTS
227
thyroidism. The question naturally arises whether these results
indicate serious organic damage of the liver, or whether they
merely signify that a liver drastically depleted of glycogen has
temporarily an impaired detoxifying power, which can however
be restored as soon as the liver is again able to store glycogen.
The hippuric acid test may perhaps also lead to a reconsideration of atrophic cirrhosis. At times a patient with a marked
cirrhosis of the liver has been a rather surprisingly good output
of hippuric acid, while often however the synthesis is markedly
reduced. It is interesting to speculate whether a cirrhotic liver
per se, especially when the process is quiescent, actually is functionally impaired, or whether perhaps the reduced hepatic efficiency is due mainly to damage resulting secondarily from mechanical obstructions, and impaired circulation.
In pregnancy the hippuric acid test seems to give rather weird
results9. There is a tendency for the parturient woman to have
a low hippuric acid output before delivery, and a normal function
soon after the baby is born. At present it is not possible to
explain these results.
It should be pointed out, however, that a low hippuric acid
output does not necessarily and invariably denote damaged
hepatic function. It seems justifiable to state that if the hippuric
acid output is normal, it is quite probable that serious liver
pathology does not exist. On the other hand, a reduced synthesis cannot blindly be accepted as absolute evidence of liver
impairment. It is well known and probably a little over emphasized that renal diseases interfere with the hippuric acid test.
Likewise, it is conceivable that other factors may influence the
synthesis of glycine or the conjugation of benzoic acid, and it is
well to bear these possibilities in mind when interpreting the
results obtained with this liver function test. From the results
that have already accumulated, it is clear that only in rare incidences will an abnormally low hippuric acid output mean anything else except an impaired liver function. Nevertheless, the
more the hippuric acid test is employed with criticalness, intelligence, and alertness, the more will it contribute to the solution
of hepatic problems.
228
ARMAND J. QUICK
QUANTITATIVE DETERMINATION OF PROTHROMBIN10' «
Principle. The coagulation time of blood or plasma can be
employed as a measure of the prothrombin concentration, if the
other factors in the clotting process are made constant. The
coagulation mechanism is concisely presented in the form of
two equations:
Prothrombin + thromboplastin + calcium = thrombin.
Fibrinogen + thrombin = fibrin.
Since the clotting time is proportional to the concentration of
thrombin, one can assume that it is also proportional to the concentration of prothrombin provided thromboplastin, calcium and
fibrinogen are made constant, for under those conditions the
amount of thrombin formed is dependent upon the concentration
of prothrombin present in the blood. On this basis, the author's
quantitative test for prothrombin was developed. The method
consists essentially in adding to oxalated plasma, an excess of
thromboplastin and then recalcifying with a fixed quantity of
calcium chloride. The coagulation time is a direct measure of
the concentration of prothrombin and can be evaluated by
referring to a chart which was made by plotting the clotting time
for known concentrations of prothrombin.
The test. By venipuncture 4.5 cc. of blood are drawn and mixed immediately with 0.5 cc. of 0.1 M sodium oxalate. Plasma is obtained by centrifugation.
To 0.1 cc. of plasma are added 0.1 cc. of thromboplastin and 0.1 cc. of 0.025
M calcium chloride. The time from the addition of the calcium chloride to
the formation of the clot is accurately recorded with a stop watch. With an
active preparation of thromboplastin, the coagulation time should be 111 to
12i seconds for normal plasma. By means of the accompanying chart, the
prothrombin concentration can be readily evaluated.
The test requires three solutions:
1. 0.10 M of sodium oxalate. Dissolve 1.34 gram of anhydrous sodium
oxalate, c.p. in 100 cc. of distilled water.
2. 0.025 M of calcium chloride. Dissolve 1.11 gram of anhydrous calcium
chloride, c.p. in 400 cc. of distilled water.
3. Thromboplastin. About 0.3 gram of dehydrated rabbit brain are thoroughly mixed with 5 cc. of a freshly prepared physiological sodium chloride
solution and incubated at 50 C. for 10 to 15 minutes. The milky supernatant
HIPPURIC ACID AND PROTHROMBIN TESTS
229
liquid is used which is obtained after the coarse particles have been removed
either by very slow centrifugation or by spontaneous sedimentation.
The rabbit brain is prepared by completely removing all blood vessels, i.e.,
stripping off the pia, and then mascerating the brain in a mortar under acetone.
By replacing the acetone several times, a non-adhesive granular powder is
obtained, which is dried on a suction filter. The material is then placed in
small ampules which are evacuated for 3 minutes by means of an oil vacuum
pump, and then sealed. Such a preparation retains its full activity apparently
indefinitely.
70r-t
1
1
1
1
1
1
1
1
1
1
20
30
40
50
60
70
Concentration of Prothrombin in Plasma (Human!
80
90
100
60—1
50
V
<n
\
§40
^
,c
\
4>
\
if 30
V
too
\
c
\
o 20
- ^ —
10
%
10
CHART 1. THE QUANTITATIVE DETERMINATION OF PROTHROMBIN IN
HUMAN PLASMA
The relationship of the clotting time of recalcified plasma (with excess
thromboplastin) to the concentration of prothrombin.
Clinical value of the prothrombin test. With the discovery
that a food accessory substance known as vitamin K is essential
for the synthesis of prothrombin, came the recognition that a
deficiency of this new vitamin can bring about a hemorrhagic
diathesis characterized by a diminished prothrombin concentration of the blood. Theoretically a fall in prothrombin may occur
if the intake of vitamin K is insufficient, if its absorption is
230
ABMAND J. QUICK
defective, or if its utilization for the production of prothrombin
is impaired.
An inadequate intake of vitamin K probably never causes a
prothrombin deficiency, since there is good evidence that the
intestinal bacteria synthesize significant quantities of this substance. In newborns, however, a unique situation is encountered.
Apparently little vitamin K or prothrombin is stored in the fetus,
therefore the available prothrombin in the newborn baby quickly
Ffer
Cent
100
•o
m 80 •
ji)
is
•|
•
•
60
e
•S 40
.§
"G
Io2 0
.
o
1
2
3
4
5
6
7
8
Age in Days
CHART 2. T H E PEOTHEOMBIN LEVEL IN NEWBOENS
It should be noted that although the prothrombin concentration is nearly
normal at birth, it often drops precipitously during the first days of life. A
prompt spontaneous restoration usually occurs.
becomes exhausted, and often drops to a strikingly low level.
The condition appears to be purely a vitamin K deficiency and
can be prevented by feeding the baby a concentrate of this
vitamin. Normally, the prothrombin remains low only for one
or two days, as shown in chart 2, and it appears probable that
as soon as a bacterial flora is established in the baby's intestinal
tract, sufficient vitamin K is synthesized to meet the normal
physiological requirements. In the hemorrhagic disease of the
newborn, which appears to be due to a delayed restoration of
the proper prothrombin level, vitamin K acts as ajspecific cure12.
HIPPTJRIC ACID AND PROTHROMBIN TESTS
231
In adults the common cause of a decrease in the prothrombin
of the blood is faulty absorption of vitamin K. Since the latter
is a fat soluble substance, the presence of bile in the intestines is
essential for efficient absorption. Logically, one would expect
to find prothrombinopenia to occur in patients with prolonged
complete biliary obstruction, or with a biliary fistulae that brings
about a loss of bile externally. Clinically these expectations have
been convincingly established. When the prothrombin decrease
is due solely to inadequate absorption of vitamin K, the response
obtained by giving a concentrate of this vitamin together with
bile salts is often spectacular. This is illustrated by the patient
presented in chart 3.
It is now widely accepted that the liver pays an important
r61e in the synthesis of prothrombin, and it is believed that the
organ has an essential part in converting vitamin K into the
prothrombin component. The bleeding tendency observed in
acute yellow atrophy of the liver undoubtedly is characterized
by a low prothrombin, although clinical data are not yet available. In many jaundiced patients the decreased prothrombin is
very likely due to hepatic damage. Such cases will respond
poorly to vitamin K and bile salts unless the function of the
liver is improved.
It is a common finding that the effectiveness of vitamin K to
elevate the prothrombin level becomes markedly reduced after
operation. The patient in chart 3 illustrates this. I t seems
logical to assume that the anesthesia, the trauma and possibly
other factors cause a temporary damage of the liver which results
in an impaired synthesis of prothrombin.
Without attempting to discuss the therapeutic aspect, a word
concerning the employment of vitamin K must be said. In the
treatment of the hemorrhagic disease of the newborn, the vitamin
seems to be fully effective without simultaneous administration
of bile salts. On the other hand in biliary obstruction, vitamin K
usually only becomes highly effective if bile salts are also given.
If, however, in addition to biliary obstruction, hepatic damage
exists, vitamin K even with adequate amounts of bile salts may
not bring about any significant elevation of the prothrombin.
In addition to the administration of vitamin K, an attempt should
232
AKMAND J. QUICK
therefore be made to improve the function of the liver by means
of glucose, calcium, salts, and gelatine. Because the prothrombin
response to vitamin K is not constant, it is desirable to do a
quantitative determination of this clotting factor before operation
I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 2021 22 2324 252627
Days
CHABT 3. THE RESPONSE OF THE PROTHROMBIN LEVEL TO VITAMIN K THERAPY
The patient a man of 56 years of age was operated for a malignancy of the
biliary tract which caused a complete obstruction. A cholecystogastrostomy
was done. The recovery which started on the fourteenth day was probably
due to a restoration of hepatic function and to the reappearance of bile salts
in the intestines. The figures in parentheses after K refer to the number of
capsules of Klotogen (a vitamin K concentrate) which were administered.
Bile salts were also given.
even though the patient has been treated with bile salts and a
concentrate of vitamin K..
STJMMAEY
1. The technic is described for performing the oral and the
intravenous modification of the hippuric acid test for liver
function.
HIPPTJBIC ACID AND PROTHROMBIN TESTS
233
2. The value of this test is pointed out in determining quantitatively hepatic function, in aiding differential diagnosis, in evaluating therapy, in following prognosis, and in obtaining new information concerning the physiology of the liver.
3. The method for determining quantitatively the prothrombin
of the blood is described.
4. The relationship of vitamin K to prothrombin is discussed,
and comments made on the therapeutic use of vitamin K.
REFERENCES
(1) QUICK, A. J.: The synthesis of hippuric acid: a new test of liver function.
Am. J. Med. Sci., 186: 630, 1933.
(2) QUICK, A. J.: Clinical value of the test for hippuric acid in cases of disease
of the liver. Arch. Int. Med., 67: 544, 1936.
(3) QUICK, A. J., OTTENSTEIN, H . N., AND WELTCHEK, H . : The synthesis of
hippuric acid following the intravenous injection of sodium benzoate.
Proc. Soc. Exp. Biol, and Med., 38: 77, 1938.
(4) WEICHSELBAUM, T. E., AND PROBSTEIN, J. G.: The determination of
hippuric acid in urine. J. Lab. and Clin. Med., 24: 636, 1939.
(5) SNELL, A. M., AND PLUNKETT, J. E.: The hippuric acid test for hepatic
function: its relation to other tests in general use. Am. J. Dig. Dis.
and Nutrit., 2: 716, 1935.
(6) BOYCE, F . F.: Hepatic and biliary tract disease. Ann. Surg., 109: 351,
1939.
(7) BARTELS, E. C , AND PERKIN, H. J.: Liver function in hyperthyroidism
as determined by the hippuric acid test. New England J. Med.,
216: 1051, 1937.
(8) BOYCE, F. F., AND MCFETRIDGE, E. M.: Studies of hepatic function by
the QUICK hippuric acid test. I I . Thyroid disease. Arch. Surg.,
37: 427, 1938.
(9) HIRSHEIMER, A., AND MILLER, M . B.: The hippuric acid excretion test
in pregnancy. Am. J. Obst. and Gyn., 37: 363, 1939.
(10) QUICK, A. J., STANLEY-BROWN, M., AND BANCROFT, F . W.: A study of
the coagulation defect in hemophilia and jaundice. Am. J. Med.
Sci., 190: 501, 1935.
(11) QUICK, A. J.: The nature of the bleeding in jaundice. J. Am. Med.
Assn., 110: 1658, 1938.
(12) WADDELL, W.W., AND GUERRY,'DUP. : Effect of vitamin K on the clotting
time of the prothrombin and the blood with special reference to
unnatural bleeding of the newly born. J. Am. Med. Assn., 112:
2259, 1939.