ase in Blood of Patients with Ulcerative Lesions - Clinical Chemistry

Microbiologic Determination of Codehydrogen-
ase in Blood of Patients with Ulcerative Lesions
Saul Caspe and Daniel D. Pitcoff
FOLLOWING THE FINDINGS of the Lwoffs (1) that factor V, identified
as codehydrogenase, is necessary for the growth of the bacilli of the
influenza group, Vilter et al. (2, 3) published an accurate bacteriologic
method for estimating coenzyme in the bloods of normals, pellagrins,
diabetics, and leukemia patients. They noted a reduction of coenzyme
content in the bloods of these diseased patients. As their substrate
for estimating the extent of growth of Hemophilus influenzae or
parainfluenzae, the test organisms used, they employed a proteosepeptone
broth prepared with human whole blood.
In 1947, one of us (S.C.) duplicated some of Vilter’s experiments
using this substrate, but extended their application to include blood
specimens from hospitalized cases of duodenal ulcer on the theoretical
ground that there may be a reduction in the proliferative enzyme
mechanisms in the body accounting, in part at least, for the production
of internal ulcerations and the failure of healing. Coenzyine-1
is the universal center for many cell oxidative reactions and its integral
role as a respiratory and proliferative agent gives added importance
to its estimation in connection with diseases of metabolism.
Since the coenzymes are destroyed by autoclaving, no growth of H.
influenzae was obtained when autoclaved normal blood was tried at
the various dilutions shown in Table I. The Seitz-ffitered proteosepeptone
broth with human blood promoted the growth of this organism
as did the normal blood added to the autoclaved proteosepeptone
human blood broth. The latter represents the control response.
Prom the Department of Laboratories, Jewish Memorial Hospital, New York 40, N. Y.
Received for publication June 26, 1958.
374

Vol. 4, No. 5 DETERMINATION OF CODEHYDROGENASE 375
Table 1. EFFECT OF DILUTIONS OF BLOOD FROM DUODENAL ULCER CASES TJPON Gsowvn or
H. INFLUENZAE m AUTOCLAVED PROTEOSE-PEPTONE BLOOD Bao’rn
Autoclaved normal blood
Seitz-ffltered medium
Normal blood, autoelaved
Patient D.C.
PatientA.D.
PatientS.F.
broth
1
S00
4 4
4 4
4 4
32
22
4
3
4
1
0
1
1000
4 4 4
4 4 2
4 4 4
221
110
Dilutions
1 1
2000 4000
No growth
3 3 2 3 3 1
3 3 1 3 3 0
3 3 2 2 2 1
220 110
±00 000
1
6000
2 2 0
2 2 0
1 1 0
±00
000
1
8000
1 1
1 1
±±
Turbidimetric readings for each of 3 strains of H. mnflueneae 4 = very heavy growth; 3 =
heavy growth; 2 = medium growth; 1 = light growth; ± = Gram negative debris; 0 =
no growth.
Table 1 records the typical results of these early studies using the
proteose peptone broth in tubes-results which were obtained by adding
0.1 ml. broth inoculum of each of three strains of H. iufluenzae to
the various dilutions of blood. The tubes were then incubated at 37#{176}
and inspected for growth by the turbidimetric method. Each dilution
was then streaked on blood agar plates (horse blood), incubated at
37#{176} for 48 hours and read. In every case readings corresponding to
the tube readings were obtained on the plates.
The blood specimens used in this series were given to us as blind
tests of duodenal ulcer cases from the service of Dr. A. F. R. Andresen
of Long Island College Hospital and our results using the Vilter
technic showed a reduction of codehydrogenase in all these specimens,
the degree of reduction corresponded to the severity of the
objective findings. For instance, S.F. showed evidences of a perforating
ulcer extending and penetrating into the pancreas, and his blood
codehydrogenase content was lowest.
Recently we have modified the Vilter technic by using a substrate
recommended to us,1 a modified Leventhal medium (4) using Eugon
broth (Baltimore Biological Laboratories). This substrate is prepared
with defibrinated horse blood prepared as follows: Boil 37 Gm.
of dehydrated brain-heart infusion broth (Difco) in 1 liter of distilled
water, autoclave 20 minutes, at 15 lb. pressure, reboil and allow to
1We are thankful to Miss Grace Leidy in Professor H. E. Alexander’s Department of
Pediatrics, College of Physicians and Surgeons, Columbia University for aiding us with her
experience using this medium and for her contribution in giving us various strains of
H. parainfluenzae used in these studies.
00
00
0
0
0
0
0

376 CASPE & PITCOFF Clinical Chemistry
cool to 95#{176}, and add slowly sufficient defibrinated horse blood to yield
a 10% concentration, filter and pass the filtrate through a Seitz ifiter.
One part of the above medium (Leventhal) is added to three parts
of Eugon broth and the H. parainfluenzae strains are kept viable by
daily transfer into 3 ml of this sterile unautoclaved medium. Thus,
these cultures were kept alive for several months when necessary.
It was deemed wise to use the H. parainfluenzae rather than H.
in! luenzae for the following reasons: past experience indicates both
populations of these organisms respond to the V factor; furthermore
the H. parainfluenzae does not require the X factor but only the V
factor; the danger of serious infection in handling these organisms is
eliminated by using the H. parainfluenzae. Following the Vilter
technic, we originally used three different strains for each test, but
since the deviation in results was slight, we decided to reduce the
number to two strains which were found to be adequate and entirely
satisfactory.
Four normal blood specimens (from subjects 30-45 years old)
were compared upon the growth of H. parainftuenzae in autoclaved
proteose-peptone broth and in autoclaved Leventhal-Eugon broth.
The extent of growth in both types of broth was identical. Our early
studies were then extended to include blood specimens from 5 diabetics,
3 lymphatic leukemia cases, 14 duodenal ulcer, and 8 ulcerative
colitis cases, using the Leventhal-Eugon substrate. These specimens
were diluted with dlistified water and heated at 85#{176} for 10 minutes and
the brown sediments were centrifuged out. The clear supernatants
were used in all tests. All these tests were run on two strains of H.
parainfluenzae. Table 2 records the results of these experiments. We
have noted that with successful treatment and progressive healing of
internal ulcers there is an increase in the codehydrogenase content of
the blood.
The Shank and Hoagland (5) method can be used to measure
turbidity. A standard curve was obtained by using several dilutions
of BaSO4. Photometric measurements were recorded for the various
dilutions of normal blood in broth culture tubes inoculated with H.
parainfluenzae by reading the light transmission against an untreated
tube of broth at 650 m wave length. Reading these measurements
on the standard curve gives the actual units. In table 3, these
unit measurements are compared to the visual turbidimetric measurements.
We are presently engaged in studying the increase of codehydro-

Vol. 4. No. 5 DETERMINATION OF CODEHYDROGENASE 377
Table 2. Gsowru or H. P&aainFi.uERzz iu DILUTIONS OF BLOODS FROM LEUKEMIA,
PoJ.wnt
Vi.5e86 500
DIABETES, AND INTERNAL Ui.cr.a CASES
1
1
1000
Pilutona
V.W. Lymphatic leukemia 1 1 ± 1 0 0 0 0 0 0
C.E. Polycythemia; leukemia 1 1 1 1 ± 0 0 0 0 0
J.H. Leukemia 2 2 2 1 1 1 0 0 0 0
M.K. Diabetes 3 3 1 1 1 ± ± ± 0 0
B.R. Diabetes 1 2 1 1 ± ± 0 0 0 0
A.G. Diabetes 2 3 2 2 1 1 ± ± 0 0
R.W. Diabetes 2 2 2 2 1 1 ± 1 0 0
C.L. Diabetes 3 2 2 2 1 1 1 ± ± 0
4 case? Duodenal ulcer 2 2 2 2 1 1 1 ± 0 0
3 case? Duodenal ulcer 2 2 1 1 1 1 ± ± 0 0
R.S. Duodenal ulcer 2 2 1 1 0 0 0 0 0 0
M.A. Duodenal ulcer 2 2 1 2 1 1 ± ± 0 0
A.C. Duodenal ulcer 3 3 3 3 2 1 ± ± 0 0
W.B. Duodenal ulcer 3 3 2 2 2 2 1 1 ± ±
J.M. Duodenal ulcer 3 3 2 2 2 1 1 1 1 ±
J.K.’ Duodenal ulcer 3 3 2 2 1 2 1 1 ± 0
M.G. Duodenal ulcer 1 2 1 1 1 ± ± ± 0 0
4 case? Ulcerative colitis 2 2 2 2 1 1 ± ± 0 0
H.P. Ulcerative colitis 2 2 1 1 1 ± 0 0 0 0
A.L. Ulcerative colitis 3 3 2 2 2 1 1 ± 0 0
A.R. Ulcerative colitis 2 1 2 1 1 1 ± ± 0 0
A.B. Ulcerative colitis 2 2 1 1 1 1 ± 0 0 0
Normal 4 4 34 22 11 1±
control
Turbidimetric readings for each of 2 strains of H. Parainfluen.sae 4 = very heavy growth;
3 = heavy growth; 2 = medium growth; 1 = light growth; ± = Gram negative debris;
0 = no growth.
Cliuical evidence of healing of ulcer.
bA plural number of cases, each of which gave identical readings, as reported.
Table 3. TURBIDIMETRIC COMPARISON OF THE DILUTION OF NoRMsi. BLOoD UPON THE
GROWTH OF H. PARAINFLUENZAE IN LEVENTRAL-EUGON BROTH
I
500
1
8000
Dilut4or.s
Visual readings 4+ 4+ 3+ 2+ 1+
Shank.Hoagland units 18.5 11.5 6.7 5.0 3.2
I
1000
I
8000
I
4000
1
4000
I
8000
1
8000

378 CASPE & PITCOFF Clinical Chemistry
genase in blood specimens of patients with these metabolic diseases
after treatment with the proliferative enzymes. The results of this
study will be the subject of a later communication.
DISCUSSION
In some cases of ulcerative colitis and duodenal ulcer disease there
is a marked reduction of codehydrogenase in the blood approaching
the reduction of the enzyme seen in lymphatic leukemia. Other cases
of internal ulcer disease show some reduction in blood codehydrogenase
concentration, the significance of which is yet to be determined.
The correlation of these findings with the attempt to classify
ulcer disease, its cause and treatment, is of great importance.
SUMMARY
A microbiologic determination of codehydrogenase by means of H.
parainftuenzae is presented. Data on the blood enzyme concentration
of patients suffering from leukemia, diabetes, and duodenal ulcer
are given.
REFERENCES
1. Lwoff, A., and Lwoff, M., Proc. Roy. Soc. London Series B. 122, 352 (1937).
2. Vilter, B. W., Vilter, 5. P., and Spies, T. D., J. Amer. Med. Asoc. 112, 420 (1939).
3. Vilter, S. P., Koch, M. B., and Spies, T. D., J. Lab. 4 Clin.. Med. 26, 31 (1940).
4. Dubos, B. J., Bacterial and Mycotw Inf ectione of Man, Philadelphia, Lippincott, pp.
478, 491.
5. Shank, H. E., and Hoagland, C. L., J. Biol. Cheni. 162, 133 (1946).