Folic Acid Activity in Leukemia and Cancer1

P. B. RAMA
RAO,B. LAGERLOF,
J. EINHORN,
ANDP. G.
(From the Departments of Internal Medicine, Pathology, Radiumhemmet, and King Gustav V Research Institute, Karolinska
Sjukhuset, Stockholm 60, Sweden)

SUMMARY
The serum folic acid activity (SFAA) was measured using Lactobacillus casei as
the test organism. The SFAA was lower for patients with malignant tumors than
for the control group. The lowest values were for malignant lymphoma and leu
kemia. No relation was found between the SFAA and either the spread of the tu
mors, the therapy administered, the age of the patient or the degree of wasting. Al
though the SFAA was significantly lowered in leukemia, the folic acid activity (FAA)
was higher in white cells of such patients than in those of healthy controls.
The FAA was also determined in chicks during development of virus-induced leu
kemia.

Low SFAA has been found in cases of malnutrition and myeloid leukemia virus (18), were observed for 31 days.
intestinal malabsorbtion (2, 12, 16) and in experimentally Two control groups, consisting of 16 uninoculated 10-day
induced folic acid deficiency (11). Low values have also old chicks, were followed for 22 and 26 days, respectively.
been found in megaloblastic anemia of pregnancy (12, 16)
and in conditions accompanied by increased blood-cell METHODS
formation (5, 19). SFAA.—Ten milliliters of blood was obtained from
Following the report by Hoogstraten et al. (15) of a low each human subject. Serum samples were diluted with
folic acid activity in serum (SFAA) in some cases of 5 X 10-2 M sodium phosphate buffer (pH 6.1), in which
leukemia we undertook a systematic study of the SFAA 50 mg/100 ml of ascorbic acid had been freshly dissolved,
level, determined microbiologically, in normal subjects and and the SFAA was determined microbiologically (1, 12)
those with leukemia, malignant lymphoma and other using Lactobcwilus casei.
tumors. Part of the material of this study has been The serum-buffer solution was autoclaved for 2 min. at
described in a preliminary report (22). 118°C. Coagulated proteins were centrifuged down and
Heilman et al. (9) have recently reported low SFAA in the clear supernatant was diluted 1 : 2.5 with distilled
10 and borderline levels in another 10 out of 35 cases of water. Of the diluted serum 0.5 ml, 1.0 ml, and 2.0 ml
solid tumors. were each added in duplicate to 2.0 ml of the double
strength, basal medium and distilled water was added to
MATERIAL a final volume of 4.0 ml. Tubes were sterilized by auto
Clinical material.—The SFAA was examined in 10 cases claving at 118°C.for 2 mm. and, after cooling, inoculated
of leukemia, 14 of malignant lymphoma (8 of Hodgkin's along with the standard folic acid assay tubes.
disease, 3 of lymphosarcoma, 2 of lymphocyte-lymphoblast L. ca@ei(ATCC 7469) was maintained in a yeast extract
lymphoma, 1 of mycosis fungoides), and 65 of other protease-peptone medium (1) and stored at 4°C. After
malignant tumors. Patients receiving chemotherapy or not more than 1 week 1 drop of the stored liquid culture
antibiotic therapy were excluded. Forty-six healthy was transferred to 10 ml of the fresh maintenance medium,
volunteers served as controls. All the blood samples incubated 18 hr. at 37°C.and stored at 4°C. On the day
were drawn after an overnight fast. prior to the assay one drop of an 18-hr. culture was
Folic acid activity in white blood cells and erythrocytes transferred to 4.0 ml of single-strength basal medium (13),
was determined in 4 cases of acute leukemia and in 6 supplemented with 400 pug of folic acid (FA) and incu
controls. bated at 37°C.for 6 hr. This culture was centrifuged and
Chicks with virus-induced leukemia.—Twelve 10-day-old washed twice with 5.0 ml of freshly prepared 0.9 % saline.
chicks, inoculated with erythroleukemia virus (17), were It was then resuspended in 5.0 ml of saline; 1.0 ml of this
followed for 34 days; 17 10-day-old chicks, inoculated with suspension was diluted 1 : 25 with saline. The assay tubes
were inoculated with one drop of the diluted suspension.
1 This study was aided by a grant from the Swedish Cancer
Society.
A standard FA curve was prepared for each assay:
Received for publication April 28, 1964; revised September 14, 2.0 ml double strength basal medium was dispensed into
1964. test tubes. From 0.2 ml to 2.0 ml of the standard FA
221

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 222 Cancer Research Vol. 25, February 1965

TABLE 1 TABLE 2
SFAA IN THE VARIOUSGROUPSOF SUBJECTS SFAA IN VARIOUSMALIGNANTTUMORS (EXCLUDING
LEUKEMIA AND LYMPHOMA)
No. of SFAA E.@of of
PatientsMean level difference with
the meanSignificance
controlsNormal (m@@g/ml)S. respect to of SFAA E. of
GroupNo. patientsMean
level themean
(mug/sal)S.
controls
Leukemia 40 2.66 0.27 P < 0.001 Radiotherapy 30 3.3 0.34
Malignant 14 2.27 0.14 P < 0.001 No radiotherapy 34 3.5 0.25
lymphoma
Other malignant 644.60 3.420.20 0.21— P < 0.001 ESR@: 0-50 mm/hr. 35 3.6 0.29
tumors46 51—130
mm/hr. 27 3.4 0.30
a S.E., standard error. General condition : good 43 3.5 0.24
poor 19 3.5 0.42

@ solution was added in triplicate to provide 40, 80, 160, Hemoglobin: 12.2 gm/100ml 34 3.Ob 0.26
240, 320, and 400 @wgFA. The final volume was made >12.2 gm/100 ml 28 4.0k 0.31
up to 4.0 ml with distilled water. Blanks with no added
FA were included. The tubes were plugged with cotton Age : 60 years 17 3.2 0.39
and autoclaved for 2 min. at 118°C.,cooled, inoculated, 61—70
years 28 3.5 0.33
and incubated at 37°C.in a water bath for 20 hr. The >70 years 19 3.3 0.40
growth was measured in a Beckman B spectrophotometer
at 660 ma. The uninoculated blank control was set at a The abbreviations used are : ESR, erythrocyte sedimenta

tion rate; S.E., standard error.

100 % transmittance and the relative transmittance of
b The difference between those two groups is almost significant
other tubes determined. Transmittance was plotted (P < 0.05). There was no significant difference between the
against the logarithm of the FA concentration. FA other groups.
activities of serum samples were calculated using a stand
ard curve. Ca8e8 of leukemia.—In this group the values ranged from
Chick blood was drawn from a wing vein and 0.1 ml of 0.8 to 7.0 mpg. The mean SFAA for these patients,
the plasma was similarly assayed. The FAA difference (2.66 m@g/ml) was significantly lower than that found in
for chicks was calculated in relation to the FAA value the controls (P < 0.001; Table 1).
before inoculation, and for the uninoculated chicks in Casesof nw2ig,wntlymphoma.—All
SFAA values in this
relation to the FAA from the first day of the experiment. group fell below the normal mean of 4.6 mpg/mi, the
A positive difference indicated an increase and a negative values ranging from 1.3 to 3.5 mpg/mi. The mean for
difference a decrease of FAA. the group, 2.27 mpg/mi, was significantly lower than that
Separation of cells.—About 9 ml of blood was added to for the normal controls (P < 0.001; Table 1).
1 ml of 6 % dextran (m.wt. 250,000, AB Pharmacia) and Case8 of other malignant tumors.—In this group of 64
0.2 ml of 5 % ethylenediamine tetraacetate (EDTA) in a patients the values ranged from 1.1 to 8.5 mpg/nil. The
heparinized tube. Agglutinated red cells were permitted mean SFAA of 3.4 mug/mI was also significantly lower
to sediment for 45 mm. and the supernatant (51) siphoned than that for the normal controls (P < 0.001 ; Table 1).
off and centrifuged at low speed for 2—3
mm. The second Of these 64 cases 30 had radiotherapy less than 1 month
supernatant (S2) contained mainly white cells and plate before the SFAA determination and the mean SFAA for
lets. This supernatant (52) was recentrifuged for 1 mm. this group was 3.3 m@g/m1; the mean for the 34 not
at high speed (about 5000 rpm). The sediment (S3) was irradiated was 3.5 mpg/mi (Table 2).
studied with a white cell count in the conventional manner, The series was divided into a group of 36 cases in which,
a differential count to determine the contamination with according to clinical and x-ray examination, the tumor
erythrocytes, and a bioassay. From the bottom of the was restricted to the primary site and possibly the area of
first sediment (Si) a sample was obtained consisting of the regional lymph nodes, and a group of 28 with a more
erythrocytes only; the absence of leukocyte contami extensive spread. The means of the two groups were
nation was checked in smears stained by the May-Griine identical (3.4 mug/mi).
wald Giemsa method. In this sample the erythrocyte Grouping with respect to the “degreeof emaciation―
FFA was measured; the value obtained in this manner was gave 44 patients in a good, and 20 in a poor, general
used for correction of the erythrocyte contamination in condition. This grouping was performed by one of the
the white cell sample. No correction for contamination authors at the time of sampling and before the results of
by platelets was made. the study were known. The means for the two groups
were almost identical (Table 2).
RESULTS There was no difference when the grouping was made
Normal human controls.—The SFAA values in 46 normal with respect to the origin of the tumor, but the series is too
fasting controls ranged from 2.5 to 7.5, with a mean of 4.6 small to permit of any definite conclusions on the associ
mug/mi. This is lower than some previous values (10,12) ation between this factor and the SFAA level.
but comparable with others (3, 4, 8). It is important to note that the patients with malig

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 RAO et al.—Folic Acid Activity 223
TABLE 3 not differ significantly from the values for the normal
FOLIc AciD AclTlvrry (FAA) IN LEUKEMIC BLOOD controls.
CELLSC Plasma FA Acilvity in Chinks with Virws-Induced Leukemia
The FA activity in plasma of the uninoculated control
czu.sERYTEROCITESNo.of
Wzrrz
chicks over a period of 3 weeks is given in Chart 1. The
values ranged from 7.6 to 16.5 mpg/ml, and the means
FAA FAA .0 seemed to increase during the age period studied. Corn
level level
meanNormal casesMean
@:;
cell)@O
meanNo.of casesMean
cell).
parative values for the 16 chicks developing leukemia are
given in Chart 1 (7).

controls DISCUSSION
Acute leukemia6 45.4 9.40.7 0. 77 40.5 0. 60.1 0.2
Whether the low SFAA level in cases of malignant
a The FAA in WBC was significantly higher (P < 0.01) in tumors is a consequence of the disease and has no effect
acute leukemia than in normal controls, although the FAA in on the development of the pathologic picture, or whether
serum was significantly lower (P < 0.01, Table 1). it is a factor in the wasting observed in these cases, was
b S.E., standard error. not established by this investigation. A possible expla
nation is a decrease in the intake of folic acid. Some
correlation between folic acid activity and nutritional
state of the patient has been observed by Heilman et al.
(9) in a series of 35 cases of carcinoma. In the present
series, however, there was no relationship between the
E4 degree of wasting and the SFAA (Table 2). An acceler
ation of iron metabolism has been observed in cases of
small malignant tumors (14, 20). The present findings
20 25 indicate decreased production, or accelerated metabolism,
Days
of another nutrient—folic acid. Although the serum FAA
was significantly lower, the value for WBC from cases of
—0— Uninocutated controls acute leukemia was significantly higher than for such cells
— A — Chicks with erythroleukemia
from normal controls. The increase in FAA in white cells
— . — Chicks with myeloic teukemie
was based on only a few observations, and may be due to
CHART 1.—Plasma FAA in chicks with virus-induced leukemia. differences in cell size. The finding is, however, consistent
The FAA difference was calculated in relation to the value before with previous results relating increased Pediococcus cere
virus inoculation, and in uninoculated controls in relation to the visiae activity and decreased uptake of C'4 from formate
value on the first day of the experiment. A positive difference
points to an increase and a negative one to a decrease of FAA. into ribonucleotides of leukemic cells (6, 23).
The FAA values for the untreated controls increased slightly
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Folic Acid Activity in Leukemia and Cancer
P. B. Rama Rao, B. Lagerlöf, J. Einhorn, et al.

Cancer Res 1965;25:221-224.

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