CLIN. CHEM. 28/5,
1198-1200
(1982)
Use of Glycerol-Cryoprotected
Assay of Folic Acid
Lactobacillus casei for Microbiological
Susan D. Wilson1 and Donald W. Home”23
A simple procedure for preparing glycerol-cryoprotected
Lactobacillus casei cultures has been developed. L. case!
grown in medium supplemented with low concentrations
of folic acid (0.3 tg/L) is diluted with an equal volume of
glycerol (800 mL/L) and stored at -20 #{176}C.
Growth response of the glycerol-cryoprotected
L. case! to low
concentrations
of folic acid exceeded that of cultures
maintained by monthly agar stabtransfer.
Also, growth for
the zero-folate
blanks was considerably
less for the
cryoprotected
cultures. Assay of folate in several rat
tissues correlated well (r = 0.999) with the standard microbiological assay. The growth rate of the culture depends
on the inoculum size, and a heavy inoculum of cryoprotected L. case! may be used to complete the assay after
only an overnight incubation.
Microbiological assays of folic acid and its derivatives have
been used for many years to estimate concentrations of folate
in blood and other tissues (1-5). Recently Grossowicz et al.
(6) described the use of glycerol-cryoprotected Lactobacillus
casei for folate assays. Their procedure involved growing L.
in medium supplemented
with minimal concentrations
of folic acid, dilution of this culture into a larger volume of
folate-free
medium with further incubation to deplete the
folate in the culture, washing the bacteria to remove the last
traces of free folate, and finally resuspension of the bacteria
into glycerol (400 mL/L), with storage as a liquid suspension
at -20 #{176}C.
Because
the bacteria
could be grown in large
quantity, stored in a freezer, and used for long periods of time,
this procedure
is of great interest to investigators and clinicians involved
in estimating
folate in research
and clinical
laboratory
settings.
We describe here a modification
of this
procedure
that is simpler, faster, and allows the use of a larger
inoculum, which shortens incubation
times in the assay from
30-48 h to 18 h.
casei
Materials and Methods
Materials.
Lyophilized
cultures
of L. casei subspecies
rhamnosis (ATCC 7469) were obtained from the American
Type Culture Collection, Rockville, MD 20852. L-Ascorbic
acid, sodium salt, and folic acid were from Sigma Chemical
Co., St. Louis, MO 63178. Glycerol was obtained from Fisher
Scientific Co., Pittsburgh, PA 15219. 2-Mercaptoethanol was
from Eastman Kodak Co., Rochester, NY 14650. Assays were
done in 13 X 100mm glass culture tubes covered with aluminum foil, and absorbance (at 540 nm) was determined directly
in these tubes with a Bausch and Lomb Spectronic 20 spectrophotometer. Lactobacilli Broth AOAC, Lactobacilli Agar
AOAC, and Folic Acid Casei Medium were from Difco Laboratories, Detroit,
MI 48232, and were used according to
l Biochemistry
Research Laboratory, Veterans Administration
Medical Center, Nashville, TN 37203.
2 Department
of Biochemistry, Vanderbilt University School of
Medicine, Nashville, TN 37203.
Address
VA Medical
Received
correspondence
to this author: Research
Service (151),
Center, 1310 24th Ave., South, Nashville, TN 37203.
Jan. 22, 1982; accepted
Feb. 16, 1982.
1198 CLINICALCHEMISTRY,Vol. 28, No. 5, 1982
package inserts. De-ionized,
glass-distilled
water was used for
all solutions. Cultures were maintained
by monthly agar stab
transfer.
Glycerol-cryoprotected
L. casei. Inoculum
cultures were
prepared in one of two ways. In the first procedure
1.0 mL of
Lactobacilli broth (to suspend the bacteria) was added to the
lyophilized vial of L. casei. We inoculated 10 mL of broth with
0.5 mL of the suspension, then incubated at 37 #{176}C
for 7 h. In
the second procedure, 10 mL of Lactobacilli broth was inoculated from an agar stab culture and incubated for 24 hat 37
#{176}C.
We added 0.5 mL of either inoculum culture to Folic Acid
Casei Medium prepared as follows: dissolve 9.4 g of powdered
medium and 50mg of sodium ascorbate in 200 mL of distilled
water, add folic acid (0.3 zgfL), and autoclave the flask at 121
#{176}C
for 10 mm. We incubated the inoculated flask for 17-18 h
at 37 #{176}C,
then cooled it in an ice bath. We added the chilled
culture to an equal volume of cold, sterile glycerol (800 mL/L),
and stored 2-mL aliquots in sterile screw-top vials at -20
#{176}C.
Folic acid assay protocol. Prepare Folic Acid Casei Medium
as described in the package insert. Dispense 1.5 mL into 13
X 100 mm culture tubes,add to each tube 0 to 1.5 ng of folic
acid in potassium phosphate buffer (50 mmolIL, pH 6.1,
containing 1.5 g of sodium ascorbate per liter) in duplicate, and
adjust the volume to 3 mL with the above buffer. Autoclave
the tubes at 121 #{176}C
for 3 mm, cool, and inoculate with 50 tL
of a 25-fold dilution (in 9 g/L NaC1) of the cryoprotected L.
casei. Incubate at 37 #{176}C
for approximately 18 hand measure
the absorbance at 540 nm.
The relation AMO vs ng of folic acid from the standard curve
could be fitted to the equation
A
=
k1m
-
k2A,/m
+ k3
(1)
where k1, k2, and k3 are constants, m is folic acid in nanograms, and A is the absorbance at 540 nm. We obtained the
values reported here for tissue folates by multiple linear regression of the standard curve on a Model 9830B desk-top
computer (Hewlett-Packard,
Corvallis, OR 97330), using
equation 1.
Preparation
of rat serum
containing
conjugase
(EC
3.4.22.12,
‘y-glutamyl
hydrolase):
Blood from male Sprague-Dawley
rats was allowed to clot at room temperature.
The clot was removed and the serum was centrifuged at 5000
X g for 10 mm. About 10 mL of serum was dialyzed at 4#{176}C
for
18 h vs 1.0 L of 0.1 molIL potassium phosphate buffer, pH 7.0,
containing 2 g of acid-washed charcoal per liter, to remove
endogenous folates. The dialyzed serum was stored in 0.5-mL
aliquots at -20 #{176}C
for later use. L. casei assay showed no detectable folates.
Preparation
of tissue extracts. A male Sprague-Dawley
rat
was anesthetized
with sodium
pentobarbital.
The bile duct
was cannulated
with polyethylene tubing (PE 10; Clay Adams,
Parsippany, NJ 07054) through a midline abdominal incision.
Body temperature was maintained at 37 #{176}C
with a heat lamp.
Bile was collected for 30 mm into tared tubes containing 15
L of 2-mercaptoethanol. Blood was drawn into a heparinized
syringe,
sufficient
sodium
ascorbate
solution
added to make the blood 1 g/L in ascorbate,
(10 g/L)
was
and the blood was
-
A540
0. I
0
0.2
0.4
0.6
FOLIC
0.8
ACID
1.0
I2
1.4
0.2
.6
0.4
0.6
(ng)
Fig. 1. Standard curves for microbiological folate assay with
use of glycerol-cryoprotected
L. case! (-#{149}-) (n = 5) and
conventional L. case! assay (- - 0 - -) (n = 4)
BarsIndicatestand
deviation. The curves were fitted to equatIon 1 by multiple
linear regression, which gave regression coeffIcients >0.99 for each curve.
Values on the abscissa are nanograms of folate per tube
centrifuged for 10 mm at 5000 X g to obtain plasma. The liver,
kidneys, and brain were removed, minced, and placed in 1.5
volumes (liver) or three volumes (kidneys and brain) of a 20
g/L solution of sodium ascorbate in a boiling water bath for
10 mm, cooled in an ice bath, and homogenized with a Polytron homogenizer (Brinkmann Instruments,
Westbury, NY
15590) at a setting of 10 for 15s. A stream of nitrogen was directed into the tube, to help prevent air oxidation of reduced
folates. The extracts were centrifuged at 4000 X g for 20 mm.
The supernates
were removed and stored in Thunberg tubes
(Kontes, Vineland, NJ 08360), under nitrogen, at -20 #{176}C.
Conjugase
treatment
of extracts.
We added 75 AL of rat
serum conjugase and 5 AL of 2-mercaptoethanol
solution (575
mmolJL) to 500 AL of each extract (liver, kidney, and brain).
A drop of toluene was added to each tube and the tubes were
incubated at 37 #{176}C
for 6 h, then for 5 mm in a boiling water
bath, and centrifuged to remove precipitated
protein;
the
supernates
were stored at -20 #{176}C
in Thunberg
tubes, under
nitrogen, until assayed.
0.8
.0
.2
.6
.4
FOLIC ACID (ng)
Fig.2. Effectof inoculumdilution
on growthofglycerol-cryoprotected L. case! folate assay
Inoculum diluted 12.5-fold (-#{149}-),
25-fold (.- 0--), and 50-fold (Values on abscissa are nanogramsof folate per tube
-
A
- -).
in Figure 2. We followed the assay procedures described
above,
except that in preparing the inoculum we diluted the cryoprotected L. casei by 12.5-, 25-, and 50-fold. Use of a heavier
inoculum
produced
an increased growth response with essentially no greater growth of the zero-folate blanks. We tested
three different
preparations
of cryoprotected
L. casei, with
essentially
the same results as reported above.
We assayed the same rat-tissue extracts and fluids seven
times by use of the cryoprotected
L. casei and three times by
the conventional microbiological assay. Results of the assays
involving cryoprotected
L. casei compared favorably with
those of the conventional assay, with no significant difference
in folate concentrations
as estimated by either assay. Linear
regression gave a correlation coefficient of 0.999 for values
found by the two procedures (Table 1).
Discussion
The use of cryoprotected
L. casei obviates the need for
maintaining
cultures by serial transfer because lyophilized
cultures, readily available from the American Type Culture
Collection, may be used. This should virtually eliminate the
loss of assay time caused by culture reversion to non-folate-
Results
Figure
1 shows the standardcurves for assay of folic acid
with cryoprotected L. casei and with bacteria maintained by
serial agar-stab cultures. These assays were done during three
months. The assay results with cryoprotected L. casei show
a lower zero blank and an increased growth response to low
concentrations
of folic acid. In addition, as indicated by the
standard-deviation
bars, assays in which cryoprotected
bacteria were used were generally more reproducible from one
assay to the next. In initial experiments,
2 mL of the cryoprotected
L. casei suspension was diluted to 10 mL, washed
twice by centrifugation with 10 mL of sterile isotonic saline
(NaCl, 9 g/L), and finally resuspended in 2 mL of sterile saline
before the assay. Subsequent experiments (data not shown)
indicated that this washing procedure was unnecessary, and
we no longer use it.
The effect of inoculum dilution on growth response is shown
Table 1. Folate Content of Various Rat-Tissue
Extracts a and Fluids
M.an foIst. (and SEM)
concn, pg/L, In L cas.I assay
Cryoprot.ct.d
Conventional
(n=7)
Liver
2.54(0.12)
KIdney
1.17(0.11)
0.16 (0.01)
0.16(0.06)
4.72 (0.16)
Brain
Plasma
Bile
(n3)
2.56(0.38)
1.17(0.06)
0.13 (0.01)
0.16(0.02)
4.89 (0.58)
p
>0.9
>0.6
>0.1
>0.8
>0.7
Treated with rat serum conjugase (see text). n is the number of assays
performed on the same sample on dIfferent days.
CLINICAL CHEMISTRY,
Vol. 28, No. 5, 1982
1199
dependent
growth.
In addition, little microbiological expertise
is needed in preparing
the cryoprotected
cultures. A heavier
inoculum
may
be used without increasing
the growth of
zero-folate
blanks, and this allows the assay to be completed
after only about 18 h of incubation
instead of the 24-h incu-
Grossowicz
bation recommended
for the standard
assay or the 30-48 h
used by Grossowicz et al. (6) in their assay in which they used
cryoprotected
L. easel.
Our procedure
for preparation
of cryoprotected
L. casei
involves only two manipulations:
growth
of a culture
in a
medium with low folic acid content and dilution into glycerol.
In contrast, the procedure of Grossowicz et al. (6) requires
repeated manipulation
of the bacterial culture: (a) growth on
minimal folate, (b) dilution with folate-free
medium and incubation
until the culture approaches
the stationary
phase,
(c) centrifugation,
(d) a washing step, (e) resuspension
of
pellet and its dilution in folate-free
medium,
and (f) dilution
with glycerol.
Grossowicz
et al. (6) observed
with their preparation
of
cryoprotected
L. easel that increasing the size of the inoculum
resulted
in decreased growth. They attributed
this effect to
over-aeration
or production
of toxic substances
during preparation of the folate-depleted
cultures.
They also suggested
that L. easel may contain some folate-splitting
enzyme, which
could inhibit growth if a heavy inoculum were used. As Figure
inoculum.
2 illustrates, however, our preparation exhibited increased
growth with a larger inoculum.
The growth
inhibition
seen by
CLIN. CHEM. 28/5,
1200-1203
have
from the production
of
the period of folate starvation.
Folate starvation
of the culture
clearly
is not a prerequisite to low zero-folate
blank values in our procedure,
and
this substantially
simplifies
preparation
of the cryoprotected
et al. may
an inhibitory
substance(s)
resulted
during
This work was supported by the Veterans Administration
Public Health Service Grant No. AM-15289.
and U.S.
References
1. Teply, L. J., and Elvehjem, C. A., The titrimetric determination
of “Lactobacillus casei” factor and “folic acid.” J. Biol. Cheni. 157,
303-309 (1945).
2. Bakerman, H. A., A method for measuring the microbiological
activity
of tetrahydrofolic
acid and other labile
derivatives.
Anal. Biochem. 2, 558-567 (1961).
reduced
folic acid
3. Herbert, V., The assay and nature of folic acid activity in human
serum. J. Clin. Nutr. 40,81-91(1961).
4. Grossowicz, N., Mandelbaum-Shavit,
F., Davidoff, R., and Aronovitch, J., Microbiological determination
of folic acid derivatives
in blood. Blood 20,609-616 (1962).
5. Scott, J. M., Ghanta, V., and Herbert, V., Trouble-free
microbiological serum and red cell folate assays. Am. J. Med. Technol. 40,
125-134 (1974).
6. Grossowicz, N., Waxman, S., and Schreiber, C., Cryoprotected
Lactobacillus
easel: An approach to standardization
of microbiological assay of folic acid in serum. Clin. Chem. 27, 745-747 (1981).
(1982)
Improved Liquid-Chromatography of Aspirin, Salicylate, and Salicyluric Acid
in Plasma, with a Modification for Determining Aspirin Metabolites in Urine
Jean N. Buskin, Robert A. Upton,1 and Roger L. Williams
Detailed performance specifications are given for a specific and sensitive liquid-chromatographic assay for aspirin,
salicylic acid, and salicyluric acid in plasma. The sensitivity
of this method for aspirin (50 tg/L) is 10-fold that of previous methods, so that concentrations of aspirin in plasma
can now be followed for about four or five half-lives after
the peak plasma concentration
arising from a single
650-mg dose. In addition, this assay is as sensitive for
salicylic and salicyluric acid in plasma as any hitherto. A
modification permits measurement of gentisic acid, salicylic acid, and salicyluric acid in urine; further modifications allow indirect measurement of conjugated gentisate
and salicylate in urine.
AddItIonal
Reports
Keyphrases:pharmacokinetics
on the pharmacokinetics
of aspirin
drug assay
and salicylate
and their metabolites continue to appear in the literature
(1,
2). Known metabolites of aspirin (acetylsalicylic acid) include
salicylic acid, gentisic acid, and glucuronic acid conjugates of
School of Pharmacy, University
of California,
San Francisco,
Francisco,
CA, 94143.
To whom correspondence
should be addressed.
Received
1200
Dec. 3, 1981; accepted
CLINiCAL
CHEMISTRY,
Feb. 16, 1982.
Vol. 28, No. 5, 1982
San
salicylic and gentisic acids, and the glycine conjugates
of salicylic and gentisic acids, salicyluric
acid and gentisuric
acid
(2). Many methods have been reported
for measuring
one or
more of these compounds,
including
methods
based on ultraviolet
absorbance,
fluorescence,
or formation
of visible
complexes
(3). These nonchromatographic
methods generally
lack specificity, although specificity can be improved by using
different techniques
of sample preparation
and (or) different
spectral
characteristics.
Greater specificity
is afforded with
liquid-, gas-, paper-, or thin-layer
chromatography.
Modern “high-pressure”
liquid-chromatographic
(HPLC)
methods
offer superior
convenience,
specificity,
and sensitivity, and many such methods have been reported for aspirin
and related compounds
in pharmaceutical
preparations
and
in biological samples. Of the latter group, some are not sensitive enough for concentrations
below the usual clinical or
toxic concentrations,
while others
(4-9) are suitable
for
pharmacokinetic
studies with moderate
doses of drug. The
method we present is approximately
10-fold more sensitive
for aspirin in plasma than any of the other available HPLC
assays but retains the sensitivity
for salicylic acid and salicyluric acid of the most sensitive of the previously
reported
assays. This advantage
in aspirin sensitivity
is highly useful,
because aspirin disappears from the bloodstream
very rapidly.
By enabling observation
of plasma concentrations
of aspirin
for at least four or five half-lives after the peak concentration,