CLIN.CHEM.22/10, 1618-1622 (1976)
Comparison of Procedures for Extracting Free
Amino Acids from Polymorphonuclear Leukocytes
Yves Houpert, Plerrette Tarallo, and Gerard Slest
We studied five methods for extracting amino acids from
human polymorphonuclear leukocytes. Both the use of cell
lysis and of a deproteinizing agent interfere with quantitative determination of the amino acids, basic amino acids
being the most sensitive to the extraction procedure.
Among the methods used, disruption of the cells by
freezing-thawing is the best method for extracting all the
amino acids. Taurine is the only amino acid extracted in
the same amount by all the methods studied, and it represents half of the intracellular pool.
All mammalian tissues so far studied, including leukocytes (1, 2) have a similar amino acid composition (3,
4). Enzyme deficiencies in amino acid metabolism (5)
and the accumulation of cystine during cystinosis have
been demonstrated
in leukocytes
(6, 7). The intragranulocytic amino acid pool is related to nutritional
factors, so that in hypoproteinemia
there is a marked
decrease in the concentrations
of some amino acids (8).
Data on free amino acids in leukocytes could thus reflect
their variations in other tissues of the organism under
various conditions that cause changes.
Methods for extracting amino acids vary with the cell
type, even from the same organism
(9, 10). Moreover,
the leukocytes cannot always be consistently lysed (11)
and the chemicals used for the amino acid extraction
may themselves interfere in the analysis when an ionexchange procedure is used (12, 13). All these considerations led us to compare the different methods for
extracting
amino acids from leukocytes
and to determine their concentrations
in these cells as measured
after extraction.
Materials and Methods
Materials
Chemicals.
Dextran T-500 was supplied by Pharmacia, Uppsala,
Sweden. The ion-exchange
resin
(Chromobeads
C2), the norleucine
standard
(2.5
mmol/liter),
and the surfactant
(“Brij 35”) were obtained from Technicon Instruments
Corp., Tarrytown,
N. Y. 10591. Lithium tricitrate, lithium chloride, lithium
hydroxide, ninhydrin, hydrazine sulfate, Methyl Cellosolve (2-methoxyethanol),
anhydrous
sodium acetate,
5’-sulfosalicylic
acid, methanol, ammonium chloride,
trichloroacetic
acid, and polyvinylpyrrolidone
(mol wt,
25 000-30 000) were provided by E. Merck, Darmstadt,
Germany. The amino acid standards came from Nutritional Biochemicals Corp., Cleveland, Ohio 44128.
Reagents. A 30 g/liter solution of Dextran T-500 and
a 10 gfliter solution of polyvinylpyrrolidone
were made
up in Krebs-Ringer
solution (14). The ninhydrin and
hydrazine sulfate solutions were prepared according to
the methods described in the Technicon reports (15,
16). The pH 2.75, 2.875, 3.10, and 3.80 buffers contained
19.74 g of lithium
tricitrate
tetrahydrate,
2.76 g of lithium chloride,
1 ml of thiodiglycol,
and 10 ml of a 100
g/liter solution of Brij 35, all diluted to 1 liter with distilled water. The pH 6.5 buffer contained 19.74 g of
lithium
tricitrate
chloride, and
with distilled
were adjusted
acid, and they
10 ml
water
with
were
tetrahydrate,
54.68
g of lithium
of the Brij 35 solution, all diluted
to 1 liter. The pH’s of the buffers
1.0 and 0.1 mol/liter hydrochloric
stored at 4 #{176}C.
Procedure
Centre de M#{233}decine
Preventive (Directeur: Prof. R. Senault),
Avenue du Doyen Jacques Parisot, 54500 Vandoeuvre-les-Nancy,
France.
Received April 26, 1976; accepted
July 6, 1976.
1618 CLINICALCHEMISTRY,Vol. 22, No. 10, 1976
2,
Granulocyte
separation.
The granulocytes
were
isolated according to methods described elsewhere (17).
Venous blood, 20 ml, was collected (lithium heparinate
anticoagulant)
from fasting subjects; two volumes of
blood were added to one volume of Dextran T-500 solution, 30 g/liter. The erythrocytes were allowed to settle
at ambient temperature
for 40 mm. The supernate was
decanted into previously chilled tubes. The cell pellet
obtained after 5 mm of centrifugation
at 150 X g and at
4#{176}C
was resuspended in 10 ml of 154 mmol/liter NH4C1
and left for 20 mm at 4 #{176}C.
The cells were then washed
with Krebs-Ringer
solution. This isolation method gave
a cellular suspension,
85-95% of which consisted of
polymorphonuclear
leukocytes, the ratio of thrombocytes to leukocytes being about one to two. No erythrocytes were microscopically
detectable.
Leukocytes
were counted (in an automatic
cell counter; Coulter
Counter
Model
F, Coultronics
France
S.A., Margency,
95580 Andilly, France).
Amino acid extraction.
The cell pellet was resuspended in 1 ml of the extraction fluid containing 250
nmol of norleucine, which acted as an internal standard
for control of both extraction
and chromatographic
analysis. The solutions for extracting amino acids were
the following: 250 ml of absolute ethanol mixed with 750
ml of concentrated
hydrochloric
acid, a 5 gfliter tnchloroacetic acid solution, a 3 g/liter 5-sulfosalicylic acid
solution, a 5 mI/liter Triton X-100 solution, and distified
water. The cells were resuspended
in each extraction
fluid, and lysis was completed by mechanical grinding
with an homogenizer
(Ultra-Turrax,
Paris Labo, Vincennes, France). In the case of distilled water, we lysed
the cells by three cycles of freezing and thawing; the cells
were frozen at -25 #{176}C
for 15 mm and thawed either at
4 #{176}C
for 60 mm or at ambient temperature
for 30
mm.
The proteins were denatured
with crystalline 5-sulfosalicylic acid in a final concentration
of 3 g/liter. The
precipitates were removed by centrifugation
(3000 X g,
10 min, 4#{176}C).
The pH’s of the supernates were adjusted
to 1.5 by addition of crystalline lithium hydroxide or
5-sulfosalicylic
acid. The extract obtained with the
ethanol/hydrochloric
acid mixture was evaporated, the
residue redissolved in 1 ml of 10 mmol/liter HC1, and the
pH adjusted to 1.5 before the chromatographic
analysis.
Total amino acid analysis. Total amino acids were
measured with an AutoAnalyzer
(Technicon
Instru-
ments Corp., Tarrytown, N. Y. 10591), with the ninhydrin/hydrazine
sulfate reaction. A standard solution of
norleucine (25 mmolfliter) was used. The final product
was measured at 570 nm.
Chromatographic
amino acid analysis. We used a
Technicon thermostatically
controlled glass column (0.6
X 75 cm). The resin, initially
in the sodium form, was
put into the lithium form by washing it first with acetone and then with three alternating
washes with 6
mol/liter nitric acid and 3 mol/liter lithium hydroxide.
The resin was then washed with a 0.3 mol/liter lithium
hydroxide solution, and finally with a pH 3.10 lithium
buffer. It was packed in five stages in the column. The
lithium elution gradient was composed of three buffers,
of pH 2.75, 2.875, and 3.8, respectively,
all with 0.2
mol/liter, and one buffer of pH 6.5 with Li 1.5 mol/
liter. The pH 2.75 buffer contained methanol (70 ml/
liter). Until citrulline was eluted (150 mm), the temperature was set at 39 #{176}C
and afterwards at 60 #{176}C.
The
flow-rate of the column was 45 mi/hour. After each
analysis, the resin was regenerated
with 0.3 mol/liter
lithium hydroxide (20 mm) and equilibrated
with the
0.2 mol/liter lithium buffer, pH 3.10(40 mm). The use
of lithium salts causes a great contraction
of the ionexchange resin. To maintain good resolution, the resin
must be removed from the column and repacked after
each 20 analyses.
Results and Discussion
Amino acids were extracted from the isolated cells in
two stages: first, the cells were lysed and secondly the
proteins were eliminated, in order to obtain an extract
suitable for chromatographic
analysis. With currently
used reagents such as picric acid or 5-sulfosalicylic acid
we could perform these two stages simultaneously.
We
compared the total amino acids in granulocytes treated
by various methods (Table 1). Extracting amino acids
with ethanol/HC1
mixture, trichloroacetic
acid (5 g/
liter), 5-sulfosalicylic
acid (3 g/liter), or Triton X-100
surfactant
(5 ml/liter and 10 ml/liter) gave similar results, but values obtained with use of distilled water
were markedly higher. The cells suspended in distilled
water were lysed by three cycles of freezing and thawing,
whereas in the other methods mechanical cell homogenization was used. The variations observed may have
Table 1. Comparison of Results of Different Methods for Extracting Amino Acids from Leukocytesa
Ethanol!
HCI,
25/75 by vol
Trichioroacetic acid,
50 g/Ilt.r
Experiment
no
5-sulfosaIlcylic acid,
30 9/titer
Norleucine
Triton X-100,
5 mi/lIter
Iisn,
nmol/10
Triton X-100,
10 mI/lIter
Distilled
water
cells
1
10.5
9.5
10.0
9.0
9.0
16.3
2
7.4
9.5
10.5
8.8
11.0
15.1
3
Mean
7.9
8.6
9.3
10.2
8.1
8.2
9.4
10.2
8.6
9.4
15.7
15.7
SD
1.5
0.3
0.2
0.4
1.5
0.44
Each assay was performed
on an aliquot of the same batch of cells (40 X 106 to 50 X 106 per milliliter).
CLINICAL CHEMISTRY, Vol. 22, No. 10, 1976
1619
Table 2. Chromatographic Analysis of the Amino
Acids in the Granuiocytic Extracts a
Ethanol/
HCI,
25/75 by vol
Trichloroacetic acId,
50 9/lIter
TrItOn X-100,
5 mI/lIter
Distilled
water
celis
Amino acId
17.46
18.01
17.54
18.10
0.74
0.49
1.05
1.40
1.38
1.20
0.21
Serine
Glutamic
0.97
0.43
0.70
trace
0.32
1.10
1.62
0.86
1.15
Glyclne
Alanlne
VaIlne
Methionine
soleucine
Leucine
Tyrosine
Phenylala-.
Lysine
Hlstidine
Arginlne
nmol/106
trace
0.43
0.37
0.28
0.66
0.39
0.37
trace
-
Glutamic
acid
Glutamine
-
94.1
92.9
92.1
92.7
97.8
94.8
97.2
97.1
88.0
98.3
98.3
94.2
98.2
-
-
-
-
-
1.68
0.41
-
-
-
0.63
-
-
-
-
22.06
20.40
21.89
90.7
97.7
112.4
96.9
97.9
94.1
-
-
-
94.6
98.3
109.6
95.6
89.0
-
-
108.i
96.2
94.3
0.64
-
103.1
98.7
98.9
0.57
-
-
99.8
92.6
97.9
0.22
-
96.6
98.8
98.5
-
-
116.4
Methionine
Isoleucine
Leudne
1.23
-
108
114.2
Cysteine
0.27
-
10 /llter
103.5
Citrulline
-
-
Alanine
3 g/llter
acid
1.21
1.31
2.21
0.19
2.25
0.65
trace
36.35
cells
a
Taurine
Aspartic acid
Threonine
SOrine
Glycine
nine
Ornlthine
5-Sulfoealicyllc
Amino acid
Taurlne
Aspartlc
acId
Threonine
acid
Glutamine
Table 3. Influence of 5-Suifosalicylic Add
Concentration on Analytical Recovery of Amino
Acids a
Valine
Tyrosine
Phenylalanine
Ornithine
Lysine
Histidine
Arginine
a
A pool of plasma was spiemented
recovery was calculated as follows:
Recovery = (value of loaded plasma
with 125 nmol of each amino acid. The
-
value of nonloaded
plasma/125) X
100
Each result is the average of three determinations.
been due either to the low extraction obtained for all the
amino acids or to only one group (Table 2). The acidic
compounds (taurine, aspartic, and glut.amic acids) and
threonine, senine, and glutamine were extracted by all
the methods studied. However, the values obtained for
these compounds depend on the method used, except
for taunine, which is always extracted in similar quantities.
The extracting agents alter the results for some amino
acids. Trichloroacetic
acid gave the lowest values for
asp#{224}rtic
acid, glutamic acid, and glutamine, whereas for
thr#{232}onine
and serine the lowest concentrations
were
obtained with use of Triton X- 100.
Cell lysis by freezing and thawing is the only method
that liberated all the intracellular
amino acids. Homogenization,
used in all the other methods for extracting amino acids, was less effective, even when
Triton X-100 was added, even though in our assays, the
concentrations
of it we used completely
lysed the
granules in the granulocytes. Indeed, at a concentration
of 150 mg of Triton X-100 per liter, they are completely
activated
(18). On the other hand, liver lysosomes (19)
as well as bone lysosomes (20) require twice this concentration for complete lysis. A higher concentration
of Triton X-100 did not increase the extraction. This
1620 CLINICALCHEMISTRY,Vol. 22, No. 10, 1976
fact has been similarly described for proteins, which are
extracted very quickly with small amounts of detergent.
At higher concentrations,
lipid constituents
are extracted (21). The large amounts of amino acids extracted by freezing and thawing could in part have
originated
from partial
proteolysis, because granulocytes contain many proteases;
therefore
we tried
thawing at various temperatures
(results not shown).
The values for the amino acids measured after thawing
at ambient temperature
and at 4 #{176}C
were similar;
thawings at 37 #{176}C
and at 56#{176}C
provoked an important
formation of peptides, which interfere in the chromatographic analysis and thus prevent quantitative
measurement of the amino acids.
Proteins were denatured by addition of 5-sulfosalicylic acid (final concentration,
30 g/liter). A higher
concentrations,
the chromatographic
analysis is disturbed (22) Therefore, a final concentration
of 100 mg
of sulfosalicylic acid per milliliter of extract modifies the
quantitative
data of all the amino acids (Table 3). The
percentage recoveries for taurine, citrulline, and aspartic
and glutamic acids were nearly 110%, whereas for serine,
glycine, methionine,
leucine, tyrosine,
and phenylalanine they were near 95-100%. The concentrations
of
aspartic acid and citrulline remained high, while those
of phenylalanine
stayed low.
Table 4. Quantitative Determination of Amino
Acids in Human Granulocytesa
Mean
AmIno acid
Taurine
Aspartic acid
Threonine
Serlne
Asparagine
Glutamic acid
Glutamine
Glycine
Alanine
Citrulline
Valine
nmol/106
SO
cells
15.47
3.1
1.52
0.25
1.00
1.29
trace
1.29
0.37
0.40
1.10
1.11
1.51
0.29
0.37
0.42
trace
-
0.46
-
0.59
0.24
Methionine
0.28
0.10
Isoleucine
Leucine
Tyrosine
Phenylalanine
Ornithine
0.34
0.96
0.30
0.37
0.32
0.14
0.31
0.12
0.13
0.11
1.36
0.23
trace
0.40
0.11
Lysine
Histidine
Arginine
a
-
Blood from 20- to 40-year-old men. Each result is the mean of 10 determi-
nations.
ulocytes also contain arginine, but this is difficult to
measure, because they also possess an arginase (32).
Asparagine,
which is eluted before glutamic acid, is
found in too small a quantity to be measured under our
conditions. Taurine makes up half of the intragranulocytic aminQ acid pool and it is very slowly metabolized
by the cells (33). It is the only amino acid that is present
almost entirely in the free state, and is probably localized in the periphery of the cell, as would appear from
results of other amino acid extractions
by different
methods. This might, perhaps, explain its role in ion
movements, especially of calcium (34-36). The presence
of taurmne in large quantities in all tissues may allow us
to use it as an index to membrane integrity. In all the
syndromes
accompanied
by cell membrane
lesions,
there is an increase in the amount of taurine in urine (37,
38). The elimination
of the membrane lesions normalizes the urinary excretion of taurine. The study of
granulocytes should allow us to show that the release of
taurine from cells is a result of lesions produced at the
cell membrane level rather than to a modification
of
sulphur amino acid metabolism, as has been suggested
(38).
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