Ganatra, R. D.

Jeejeebhoy, K. N.

Mehan, K. P.

Nadkarni, C. D.

Parekh, D. V.

Pathare, S. M.

Ramanath, P.

English

Publications of the IAS Fellows

JOURNAL OF NUCLEAR MEDICINE 8:40-49, 1967
A Simple Method of Estimating Folic Acid Absorption(AModifiedFaecalExcretionMethod)â€˜
K. N. Jeejeebhoy, M.R.C.P.(Lond)., Ph.D(Lond), P. Ramanath, B.Sc,
K. P. Mehan, B.Sc, S. M. Pathare, B.Sc, DV. Parekh, M.B.B.S.,
C. D. Nadkarni, M.Sc, R. D. Ganatra, M.B.B.S., F.C.P.S.,
M.Sc(Pharmac)
Parel, Bombay
Folic acid absorption can be measured by observing the blood folic acid
levels following an oral load of unlabelled folic acid (1), but this method has
several disadvantages. One disadvantage is that it does not give an accurate idea
of percentage absorption and, secondly the load of folic acid alters the haema
tological status of the patients. Furthermore, since the oral load of folic acid
amounts to a few mgms, it is unphysiological, because it is greatly in excess of
the normal daily intake.
Recently, the method of measuring folic acid absorption by using 3H folic
acid has become popular (2). Absorption with this tracer can be estimated in
one of two ways. First, after an oral dose, absorption can be measured by sub
tracting the amount excreted in feces from the dose given. Second, it can be done
by flushing out the absorbed tracer in the urine with an injection of unlabeled
folic acid, a method similar to the Schilling test for vitamin B,2. The first method
largely overcomes the problems associated with the use of methods utilising un
labeled folic acid, but the second method is semiquantitative and the injection
of folic acid alters the haematological status of the patients.
The faecal excretion method, though technically very satisfactory, has two
main difficulties: first, the preparation of fecal samples for tntium counting re
quires some form of oxidation, either with explosive agents like perchioric acid
(3) or by the use of expensive combustion bombs. Second, collection of feces
for many days is required to ensure complete recovery of the unabsorbed tracer.
The latter process makes the procedure unattractive to the patients and may
necessitate admission to ensure satisfactory collection. Furthermore, complete
collection is possible only with very cooperative patients.
â€˜Radiation Medicine Centre, Medical Division, Atomic Energy Establishment Trombay
Tata Memorial Hospital, Parel, Bombay-12.
40
ESTIMATING FOLIC ACID ABSORPTION 41
These two difficulties have made the fecal excretion methods rather com
plicated. This paper presents a method which largely overcomes these difficulties
and compares it with the results of absorption measured by total fecal collections
done simultaneously in control subjects and patients with various intestinal
conditions.
PRINCIPLE OF METHOD
The preparation of the fecal samples for tritium counting was accomplished
by wet oxidation with acid potassium permanganate and hydrogen peroxide.
This method does not require any elaborate apparatus and there is no danger
of an explosion.
The necessity for collecting feces for many days was overcome by feeding
chromium-51, (as chromic oxide) an unabsorbable substance simultaneously
with 3H folic acid. The absorption was calculated from the ratio of 51Cr to 3H
folic acid in a single stool collection made in the first 24 hours after the dose.
This method has already been used to measure vitamin B,2 absorption (4).
MATERIALS AND METHODS
RADIOACTIVE COMPOUNDS
Hydrogen-3 folic acid was prepared by exchange labeling of pure cyrstalline
folic acid with tritium followed by chromatography, at the Isotope Division of
the Atomic Energy Establishment, Tronibay, a method similar to that described
by Johns et al (5). The specific activity was 250 LC/mg.
900
ELUTION OF CHROMATOGRAPHICALLY
800 PURIFIED FOLIC ACID ON DEAE 150
CELLULOSECOLUMN. 11 140
700 I' I3O@%It
- I' I20'-@
111
100 10
@ 0
D 2 4 6 8 10 121416 18202224262830323436384042444648 SO
TUBE NUMBER
Fig. 1. Elution of chromatographically purified folic acid on a DEAE cellulose column
showing the radiochemical purity of the compound fed. Solid line represents counts per minute
and dashed line microbiological activity.
42 JEEJEEBHOY,RAMANATH,MEHAN, PATHARE,PAREKH,NADKARNI,GANATBA
In order to test purity, a sample of the â€˜Hfolic acid used was subjected to
chromatography on I)EAE cellulose, using a modification of the method de
scribed by Johns el al(s). The elutes were subjected to microbiological assay for
folic acid using Lactobacilius Casei (6) and also counted for â€˜Hactivity.
To ascertain the stability of â€˜Hlabel in the intestine, lOzC of â€˜Hfolic acid
was fed to two rats. After one hour, the rats were sacrificed and the small intestine
was removed and divided into two equal halves. The intestinal contents in each
half was washed out with five nil phosphate buffer 0.O1M, pH 6.8 containing 100
mg% ascorbic acid. An aliquot of the contents were counted as such and then
dried, reconstituted with buffer and again counted. This procedure would indicate
if there was any free â€˜H20produced during digestion. The contents were then
applied to a I)EAE column and chromatographed (5). The elutes were subjected
to microbiological assay for folic acid using Lactobacillus @asei and were also
counted for â€˜Hradioactivity.
Eighty MCof â€˜Hfolic acid was mixed with 10-15 @Cof â€œCrin a waxed paper
cup and fed to fasting patients. The cup was washed twice and the washings were
also fed.
The first 24 hours' fecal sample was collected and a further five daily 24-hour
collections were made after the dose.
LOWER INTESTINAL CONTENTS OF RAT ( AFTER FEEDING PURIFIED
FOLIC ACID) ON DEAE CELLULOSE COLUMN.( 60 MIN.AFTER FEEDING)
Fig. 2. Chromatographic pattern of the lower intestinal contents of a rat after feeding
purified folic acid. The figure shows that the radioactivity was firmly bound to the folic acid
activity during intestinal digestion. Solid line represents counts per minute and dashed line
microbiological activity.
w
â€˜/,
L)
-j
w
0@
z
0
O 2 4 6 810 12 14 16 1820 2224 262830 3234 36384042444648
TUBE NUMBER
43ESTIMATING FOLIC ACID ABSOHPTION
The fecal samples were thoroughly and separately hoinogenised, weighed
and aliquots were taken for counting of hydrogen-3. The â€œCrexcretion in the
first 24-hour collection was also counted.
The first 24-hour fecal collection was counted between two sodium iodide
crystals in Nuclear Chicago Model GA-s multiprobe unit with model 1620 c.
Ratemeter and Model 1810 Radiation Analyser. The counts were referred to a
standard made from the dose fed and expressed as percentage.
A two-gram aliquot of the first 24-hour collection which had been counted
for â€œCrwas taken in a double-necked flask and mixed with 5 cc. of concentrated
sulphuric acid. A reflux condenser was fitted to the central neck and the contents
were stirred continuously with a magnetic stirrer. From the side neck, two grams
of potassium pernianganate were gradually added and a brisk reaction was seen
in the flask. The stirring was continued until the reaction sul)sided. Then 0.9 â€”
1 ml of hydrogen peroxide, 20 volumes per cent was gradually introduced until
0/ ABSORPTION OF 3H-FOLIC ACID.
0
w
I
z
0
I.
-a
0
I..)
-J
Lu
4U.
-J
4
0
@- 10
100
0
0
00
a
a
0
0
â€¢
0
10 20 30 40 50 60 70 80 90
DOUBLE TRACER METHODÂ°4 ABSORBED.
Fig. 3. The results of absorption measured by the double tracer method compared to the
absorption measured by the total faecal collection.
U-,
p.
z
Ui
I
30La.
0
@20
I
z
44 JEEJEEBHOY,RAMANATH,MEHAN, PATHABE,PAREKH,NADKARNI,GANATRA
the solution in the flask became colorless. Fifteen to twenty milliliters of 40%
sodium hydroxide were added to the acid solution until a permanent black pre
cipitate had just formed. The pH at that point was about eight. The whole solu
tion was transferred to a nieasuring cylinder and volume was noted. An aliquot
was centrifuged and 0.5 ml of the clear supernatant was counted in 20 ml of
Dioxane scintillation fluid containing four per cent thixotropic gel. The counts
were referred to an internal standard made up of an aliquot of the dose fed. The
same procedure was adopted with subsequent 24-hour collections. The calcula
tion of tritium excreted is illustrated in the example given below.
Weight of the 24-hour collection
Weight of aliquot taken
Volume of the oxidised faecal mixture
Net counts in 0.5 ml
Net counts in internal standard
Dilution of dose used to prepare@ 2000
internal standards.
100 gms
2gms
3Oml
1000 cpm
35 X 10' cpni
} 1000X30X2X100=3X10@
2
Counts of tritium excreted
in 24 hour collection.
Counts fed to patients. .. 35 X 10' X 2000 = 70X 106
3X 106X 102 â€”30@@
-____ -- 4.3%
70X106 7
% excretionof tritium.
40
% ABSORPTIONOF 3H-FOLIC ACID.
Fig. 4. Distribution of levels of absorption of H3 folic acid.
ESTIMATING FOLIC ACID ABSORPTION 45
Since â€œCris not absorbed, the percentage of this tracer in a random faecal
sample can be used to correct the tritium excretion in the same sample for 100%
excretion of â€œCr,the assumption being that 5'Cr and â€˜Hhave remained intimately
mixed in the gastrointestinal tract. For example, if 10% of the orally fed 5'Cr
was excreted in the 24-hour collection of stool containing 4.3% of 3H folic acid,
4.3X 100
then the total excretion of unabsorbed tritium would be â€”= 43%
10
Hence absorption of â€˜Hfolic acid would he 100 â€”43 = 57%.
Known amounts of â€˜Hfolic acid were added to faeces and the recoveries
were studied after oxidation by the method described above.
There were 16 control subjects of whom 15 were entirely normal and one
was a treated case of iron deficiency anaemia. None of them had gastrointestinal
disease.
Three patients had nutritional megaloblastic anaemia, two had low serum
L. casei (Folic acid) levels and one a low serum vitamin B12 level without evi
dence of clinical or biochemical malabsorption. There were 19 patients with
gastrointestinal disease and 17 of them had tropical sprue with steatorrhoea.
The remaining two patients had ulcerative colitis and partial gastrectomy with
a low serum L.casei (Folic acid) level, respectively.
RESULTS
PURITY OF 3H FOLIC ACID
The results are given in Fig. 1. It will be seen that the radioactivity comes
out as a sharp peak between tubes 34 to 40.
This position corresponded to the elution of a known folic acid standard.
Furthermore, the peak of microbiological activity corresponded to the peak of
3H activity confirming the purity of the 3H folic acid used.
TABLE I
% recoveryof H3folic acid
Experiment No. added to faeces.
1 105.2
2 104.2
3 84.6
4 97.6
98.0
6 100.0
Double
tracer methodTotal collection
46 JEEJEEBHOY,RAMANATH,MEHAN, PATHARE,PAREKH,NADKARNI,GANATRA
TABLE II
CONTROL SUBJECTS
Absorption of 3H Folic acid
% ofdosefed excreted
in the collection used
to calculate absorption
in column 3.
.,
55
78
74
4.3
16.5
55.9
40.6
91.5
16.5
69.9
78.0
84.8
37.3
59.0
100
56.4
21.4
24.6
19.5
No Clinical
Condition
1 2
1. Iron deficiency
(treated)
2. Normal
3. Normal
4. Normal
5. Normal
6. Normal
7. Normal
8. Normal
9. Normal
10. Normal
11. Normal
12. Normal
13. Normal
14. Normal
15. Normal
16. Normal
17.
18.
19.
Mean and standard
deviation of absorp
tion of â€˜HFolic acid
in normal subjects.
% of dosefed
3 4
79 76.9
78 80.3
75.6 69.4
74.2 76.2
73.4 78.2
73.3 72.1
66.8 64.3
93.5 92.7
72.5 93.3
91.5 90.0
86.0 79.7
83.0 83.8
84.7 90.1
95.8 96.3
96.8 95.6
85.0 89.7
PATIENTS WITH NUTRITIONAL MEGALOBLASTIC ANAEMIA
76.4 77.4
52.8 55.6
71.5 75.4
79.44Â± 11.1
Absorption of 3H Folic acid%
ofdosefedexcretedNoClinical
ConditionDouble
Total
tracer method collectionin
the collection used
to calculate absorption
in column3.%
of dosefed1
2 3 4 5
ESTIMATING FOLIC ACID ABSORPTION 47
TABLE II (cont'd)
CONTROL SUBJECTS
PATIENTS WITH GASTROINTESTINAL DISEASES
20. Ulc. Colitis 63. 9 62. 8 3.9
21. Partial gast. 46 44.7 59.5
22.Sprue 36 44.6 21.9
23.Sprue 33.5 43.4 21.6
24. Sprue 94.5 74.8 71.0
25.Sprue 42 46.6 56
26. Sprue 26 27 83.0
27.Sprue 27.8 31.1 17
28.Sprue 33.6 37.4 27.9
29.Sprue 26.6 42.5 72.4
30. Sprue 93.7 83.3 79.4
31. Sprue 91.9 85.9 69.0
32. Sprue 87.8 71.9 63.0
33. Sprue 96.4 96.9 93.0
34. Sprue 82.3 71.8 77.2
35. Sprue 90.8 95.0 49.7
36. Sprue 93.8 92.8 9.8
37. Sprue 28.5 33.5 23.3
38. Sprue 70.5 81.7 62
Mean and standard
deviation of absorp
tion of 3H Folic acid
in patients with
sprue. 62.1 Â±30.3
48 JEEJEEBHOY,RAMANATH,MEHAN, PATHARE,PAREKH,NADKARNI,GANATRA
STABILITY 3H LABEL IN THE SMALL INTESTINE
There was no difference in the counts of an aliquot of the intestinal contents
in the wet and dried state, indicating that no 3H20 was produced during diges
tion. The chromatographic pattern of intestinal contents was identical in upper
and lower small intestine and a representative pattern is seen in Fig. 2. From the
figure, it will be observed that the pattern of elution of folic acid from DEAE
in the intestinal contents was similar to the pattern of the dose fed and showed
no obvious degradation products of folic acid. Furthermore, both microbiological
activity and radioactivity coincided, showing that the label was firmly bound to
the folic acid.
RECOVERY OF ADDED 3H FOLIC ACID
The results are given in Table I. Between 84 and 105% of the added â€˜Hfolic
acid was recovered by the oxidation method described.
COMPARISON BETWEEN ABSORPTION ESTIMATED BY TOTAL SIX DAYS COLLECIION
AND THE 51CR METHOD
The results are given in Table II and Fig. 3. It can be seen that a good cor
relation exists between the results obtained by the methods described and the
results of absorption from total fecal collection in both controls and patients with
gastrointestinal disease. Taken as a whole, the coefficient of correlation was
r = 0.9; however, considering individual results, only in three subjects. (9, 24
and 29) out of 38, the two results were obviously different. Furthemore, good
agreement was obtained even when the amount of 51Cr in the collection taken
to calculate total absorption was as low as 4% (Table II, column 5) indicating
that a small aliquot of stool could be used to measure total absorption.
DISTRIBUTION OF THE LEVELS OF ABSORPTION OF :@J@JFOLIC ACID
The results are given in Fig. 4. It will be seen that the absorption of folic
acid falls in a bimodal pattern of distribution with all controls absorbing more
than 60% of the dose and about half the patients with sprue absorbing less than
50% of the dose. One patient with nutritional megaloblastic anaemia absorbed
52% of the dose; this value for absorption lies between the controls and patient
with obvious malabsorption. This patient had no steatorrhoea and the anaemia
responded to small oral doses of folic acid.
DISCUSSION
The results show that acid KMnO4 and H2O2 is a suitable way of preparing
samples of feces for tritium counting. It compares favorably with the other oxi
dation methods and has the advantage of being both safe and inexpensive. Fur
thermore, by the use of 51Cr, it is possible to estimate absorption from a single
random faecal sample, which makes the procedure feasible in non-cooperative
patients and in busy clinical units without adequate facilities for metabolic
ESTIMATING FOLIC ACID ABSORPTION 49
studies. Since folic acid absorption is rapidly becoming an important tool in the
study of malabsorption syndrome and in megaloblastic anaemias, the technique
mentioned above would be useful in making the study feasible in places with
relatively meagre facilities. The one problem which remains is the necessity of
using an expensive liquid scintillation counter for estimating tritium. Here again,
where such facilities are not available, the oxidized fecal samples could be
easily transported to a central laboratory for counting, a process which would
be of importance in underdeveloped countries.
Using this method, the normal absorption appears to be 60% of the dose
and above. This value for normal absorption with 3H folic acid is very similar
to that observed in controls by Anderson et at (7). Malabsorption appears to be
present when less than 50% of the dose is absorbed. Absorption of between 50
and 60% may occur in patients with nutritional megaloblastic anaemia. A slightly
impaired absorption of folic acid in nutritional megaloblastic anaemia has been
observed by Anderson et al (2).
REFERENCES
1. CHANARIN,I., ANDERSON,B. B., AND MOLLIN, D. L.: The absorption of folic acid.
Brit. 1. Haemat. 4:156, 1958.
2. ANDERSON,B., BELCHER,E. H., CHANARIN,I., AND MOLLIN, D. L.: The urinary and
faecalexcretionfradioactivityaf eroraldosesofH@,folicacid.Brit.J.Haemat.6:439,1960.
3. BELcHER,E. H.:The assayoftritiuminbiologicalmaterialby wet oxidationwith
perchloric acid, followed with liquid scintillation counter. Phys. in Med. & Biol. 5:49, 1960.
4. GANATRA,R. D., SuNDA1@M,K., DE54U, K. B., AND GAITONDE,V. B.: Determination
of absorption of labelled vitamin B1@, by a double tracer technique. burn, of Nuclear Medicine,
6:459, 1965. -
5. JOHNS, D. G., SPERTI, S., ANDBURCEN,A. S. V.: Metabolism of tritiated folic acid in
man, I. Clin. Invest. 40:1684, 1961.
6. WATERS,A. H., ANDMOLLIN,D. L.: Studies on the folic acid activity of human serum.
J. Gun. Path. 14:335-344, 1961.
7. ANDERSON,B., BELCHER,E. H., CHANARIN,I., ANDMOLLIN, D. L.: Urinary and faecal
excretion of radioactivity after oral doses of 3H-Folic acid. Brit. J. Haemat. 6:439, 1960.
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A simple method of estimating folic acid absorption (a modified faecal excretion method)

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A simple method of estimating folic acid absorption (a modified faecal excretion method)

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