Decreased chemotactic and of leukocytes during Intralipid
random migration infusion12
.J#{244}rgen Nordenstr#{246}m,3 M.D.,
and Anna
ABSTRACT subjects
The for
2 hr.
leukocytes
was
induced. caused
Migration an impairment
Nutr.
Connie
soybean
During
noted. was
32: 2416-2422,
Jarstrand,4 M.D.,
oil
the
infusion
It was
correlated
emulsion
The lipid
and
dose
given
and
given
intravenously
chemotactic to the
and degree
to random
12
healthy
migration
of
of hypertriglyceridemia added pattern.
in vitro Intrahipid Am. J. Clin.
1979.
stearic acid 4.9%, and hinolenic acid
Subjects
and
general
oleic acid 29.7%, 8.1%.
outline
linoleic acid 46.0%,
of the investigation
Twelve healthy subjects (ages 22 to 42 years) were continuously infused with Intrahipid 20% for 2 hr. Eight subjects were infused at the rate of 100 ml/hr, two subjects received 200 ml/hr and another two received 50 ml/hr. Serum triglyceride (TG) and cholesterol concentrations, granulocyte count, chemotactic and random migration of leukocytes were measured immediately before and after the 1st and 2nd hr of Intralipid infusion, as well as 22 hr after the termination of the infusion.
Chemotaxis
under
agarose
Chemotaxis and random migration were measured by a modification of the method described by Nelson et al. (8). Within 1 hr after sampling, heparinized blood was applied in 5-ml portions above 4 ml of a mixture of methyl cellulose and sodium metrizoate (9) in a plastic test tube (160 X 16 mm). Erythrocytes aggregated and sedimented to the bottom of the tube. After 30 mm, the cells from the top layer of plasma were washed in Trisbuffered Hanks’ balanced salt solution. The granulocytes in a portion of this suspension were counted in a B#{252}rker chamber after staining with crystal violet. The remaining
methods
emulsion
Intrahipid (Vitrum AB, Stockholm, Sweden) is an emulsion of soybean oil (20%) with eggyolk phosphohipids (1.2%) as emulsifying agent, and glycerol (2.5%) in sterile water (pH 7.5). The fatty acid composition of the soybean triglycerides is as follows; palmitic acid 11.3%,
2416
to the
was of the
fully restituted 22 hr after the infusion. Also when of leukocyte motility that followed a dose response
The lipid emulsion Intralipid is widely used for parenteral nutrition. Since the occurrence of septicemia is a common complication in patients given total parenteral nutrition (1), it is noteworthy that infusion of fat emulsions has been reported to impair phagocytosis (2, 3). An accumulation of fat in the cells of the reticuloendotheial system and a reduction in antibody formation have been held in part responsible for this increased proneness to septicemia (4). In experimental animals some lipids intravenously infused have been reported to enhance, others to depress the function of the reticuloendotheial system (3). Freund et al. (5) have reported a fatal case of septicemia attributed to the administration of Intralipid. However, the endotoxin clearance in rabbits has been found to be enhanced by Intralipid (6). Since chemotaxis is an important function in the phagocytic system and as defective granulocyte chemotaxis predisposes to infection and septicemia (7) we have tested the influence of Intralipid on leukocyte migration.
Materials
Intralipid
an impairment
Wiernik,4 Ph.D.
The American
Journal
of Clinical
Nutrition
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/12/2416/4692395 by University of Glasgow user on 02 August 2018
‘From teriology, Stockholm, 2Address Department
the Departments of Surgery and Clinical BacKarolinska institutet at Serafimerlasarettet, Sweden. reprint requests to: Dr. Connie Jarstrand, of Clinical Bacteriology, Serafimerlasarettet, Box 12700, S-112 21 Stockholm, Sweden. Department of Surgery. Department of Clinical Bacteriology.
32: DECEMBER
1979,
pp.
24 16-2422.
Printed
in U.S.A.
MIGRATION
OF
LEUKOCYTES
DURING
cells were suspended 24 x 10’ granulocytes
in Eagle’s medium to a density of per milliliter. One-hahfgram ofagarose Indubiose A 37 (L’Industrie Biologique Francaise S.A., Gennevilhiers, Seine) was dissolved in 10 ml of distilled water by boiling on an electric heater. After cooling in a 52 C water bath, the agarose solution was mixed with 40 ml of a solution containing 20 ml of 0.0125% bovine albumin, 5 ml of “lOx tissue culture medium 199” (Flow Lab, U.K.), 5 ml 6.2% of Hepes buffer and 10 ml ofdistilhed water and adjusted to pH 7.4. Then, 5 ml of this agarose medium was poured on 60 x 15 tissue culture dishes and allowed to harden at room temperature. The dishes were transferred to a refrigerator for 60 mm. Wells with a diameter of 4 mm and spaced 3 mm apart were cut out. Each plate contained six series of three wells. The center well of each series received 20 sh of the heukocyte suspension, the outer well 20 jzl of an Escherichia coli (P. Ward) culture filtrate acting as a chemotactic factor and the inner well 20 l of Eagle’s medium. The dishes were incubated for 3 hr at 37 C in a humidified 5% CO2 atmosphere. Thereafter, the plates were fixed for 30 mm with methanol and for another 30 mm with formahin. The agarose gel was removed and the plates were treated with Wright’s stain. The chemotactic and random migrations
were
measured,
magnification In
were
of
this
study
used
for
values
obtained
Other
laboratory
using
an
enlarging
projector
at
a
x40. two
each
plates,
each
experiment
was
with
and
six
the
series
mean
of
wells,
of the
12
calculated.
Leukocytes were counted in a Biirker chamber. Differential counts were made on stained smears. Triglyceride och cholesterol in serum were determined by enzymatic methods (10).
effect
of Intralipid
on leukocyte
To five out of six plastic test tubes, each with 0.5 granulocyte suspension, 0.5, 0.4, 0.3, 0.2, and 0.1 ml of Intrahipid, respectively, was added. The volumes in all six tubes were adjusted to 1 ml with Hanks’ solution. The tubes were incubated with shaking in a 37 C water bath for #{189} hr. Thereafter, the cells were washed twice in Hank-Tris and finally suspended in 0.5 ml Eagle’s medium. Since the molecular weight of the TG components in the soybean oil emulsion has been calculated to be 87412
the
TG
9.2, 6.9, 4.6, otaxis under
Statistical
concentrations
2.3, and agarose
in
0 nmole/hiter, was carried
the
test
tubes
respectively. out as described
were
11.5,
Chemabove.
of
regression
was
used.
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/12/2416/4692395 by University of Glasgow user on 02 August 2018
2417
Results Effect of Intralipid random migration
on chemotactic of leukocytes
and
Decreased leukocyte migration was observed in all subjects during infusion of Intralipid. Values of chemotactic and random migration of leukocytes from persons given 100 ml of Intraipid per hour (Fig. lb) were lower during the infusion than immediately before and 22 hr after the infuson (P < 0.001 in both cases). There was no significant difference between values obtained 1 and 2 hr after the infusion. The impairment of leukocyte migration was more pronounced when 200 ml/hr was given and less when the dose was 50 ml/hr (Fig. la and c). Further, a correlation was obtained between the dose rate and the effect, calculated as the mean decrease after 1 and 2 hr in each person. Correlation coefficients were 0.65 for chemotaxis and 0.69 for random migration, both corresponding to P < 0.05.
lipids
The plasma TG concentrations were higher during the Intralipid infusion (100 ml/hr) than before and 22 hr after the infusion (P
random migration infusion12
.J#{244}rgen Nordenstr#{246}m,3 M.D.,
and Anna
ABSTRACT subjects
The for
2 hr.
leukocytes
was
induced. caused
Migration an impairment
Nutr.
Connie
soybean
During
noted. was
32: 2416-2422,
Jarstrand,4 M.D.,
oil
the
infusion
It was
correlated
emulsion
The lipid
and
dose
given
and
given
intravenously
chemotactic to the
and degree
to random
12
healthy
migration
of
of hypertriglyceridemia added pattern.
in vitro Intrahipid Am. J. Clin.
1979.
stearic acid 4.9%, and hinolenic acid
Subjects
and
general
oleic acid 29.7%, 8.1%.
outline
linoleic acid 46.0%,
of the investigation
Twelve healthy subjects (ages 22 to 42 years) were continuously infused with Intrahipid 20% for 2 hr. Eight subjects were infused at the rate of 100 ml/hr, two subjects received 200 ml/hr and another two received 50 ml/hr. Serum triglyceride (TG) and cholesterol concentrations, granulocyte count, chemotactic and random migration of leukocytes were measured immediately before and after the 1st and 2nd hr of Intralipid infusion, as well as 22 hr after the termination of the infusion.
Chemotaxis
under
agarose
Chemotaxis and random migration were measured by a modification of the method described by Nelson et al. (8). Within 1 hr after sampling, heparinized blood was applied in 5-ml portions above 4 ml of a mixture of methyl cellulose and sodium metrizoate (9) in a plastic test tube (160 X 16 mm). Erythrocytes aggregated and sedimented to the bottom of the tube. After 30 mm, the cells from the top layer of plasma were washed in Trisbuffered Hanks’ balanced salt solution. The granulocytes in a portion of this suspension were counted in a B#{252}rker chamber after staining with crystal violet. The remaining
methods
emulsion
Intrahipid (Vitrum AB, Stockholm, Sweden) is an emulsion of soybean oil (20%) with eggyolk phosphohipids (1.2%) as emulsifying agent, and glycerol (2.5%) in sterile water (pH 7.5). The fatty acid composition of the soybean triglycerides is as follows; palmitic acid 11.3%,
2416
to the
was of the
fully restituted 22 hr after the infusion. Also when of leukocyte motility that followed a dose response
The lipid emulsion Intralipid is widely used for parenteral nutrition. Since the occurrence of septicemia is a common complication in patients given total parenteral nutrition (1), it is noteworthy that infusion of fat emulsions has been reported to impair phagocytosis (2, 3). An accumulation of fat in the cells of the reticuloendotheial system and a reduction in antibody formation have been held in part responsible for this increased proneness to septicemia (4). In experimental animals some lipids intravenously infused have been reported to enhance, others to depress the function of the reticuloendotheial system (3). Freund et al. (5) have reported a fatal case of septicemia attributed to the administration of Intralipid. However, the endotoxin clearance in rabbits has been found to be enhanced by Intralipid (6). Since chemotaxis is an important function in the phagocytic system and as defective granulocyte chemotaxis predisposes to infection and septicemia (7) we have tested the influence of Intralipid on leukocyte migration.
Materials
Intralipid
an impairment
Wiernik,4 Ph.D.
The American
Journal
of Clinical
Nutrition
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/12/2416/4692395 by University of Glasgow user on 02 August 2018
‘From teriology, Stockholm, 2Address Department
the Departments of Surgery and Clinical BacKarolinska institutet at Serafimerlasarettet, Sweden. reprint requests to: Dr. Connie Jarstrand, of Clinical Bacteriology, Serafimerlasarettet, Box 12700, S-112 21 Stockholm, Sweden. Department of Surgery. Department of Clinical Bacteriology.
32: DECEMBER
1979,
pp.
24 16-2422.
Printed
in U.S.A.
MIGRATION
OF
LEUKOCYTES
DURING
cells were suspended 24 x 10’ granulocytes
in Eagle’s medium to a density of per milliliter. One-hahfgram ofagarose Indubiose A 37 (L’Industrie Biologique Francaise S.A., Gennevilhiers, Seine) was dissolved in 10 ml of distilled water by boiling on an electric heater. After cooling in a 52 C water bath, the agarose solution was mixed with 40 ml of a solution containing 20 ml of 0.0125% bovine albumin, 5 ml of “lOx tissue culture medium 199” (Flow Lab, U.K.), 5 ml 6.2% of Hepes buffer and 10 ml ofdistilhed water and adjusted to pH 7.4. Then, 5 ml of this agarose medium was poured on 60 x 15 tissue culture dishes and allowed to harden at room temperature. The dishes were transferred to a refrigerator for 60 mm. Wells with a diameter of 4 mm and spaced 3 mm apart were cut out. Each plate contained six series of three wells. The center well of each series received 20 sh of the heukocyte suspension, the outer well 20 jzl of an Escherichia coli (P. Ward) culture filtrate acting as a chemotactic factor and the inner well 20 l of Eagle’s medium. The dishes were incubated for 3 hr at 37 C in a humidified 5% CO2 atmosphere. Thereafter, the plates were fixed for 30 mm with methanol and for another 30 mm with formahin. The agarose gel was removed and the plates were treated with Wright’s stain. The chemotactic and random migrations
were
measured,
magnification In
were
of
this
study
used
for
values
obtained
Other
laboratory
using
an
enlarging
projector
at
a
x40. two
each
plates,
each
experiment
was
with
and
six
the
series
mean
of
wells,
of the
12
calculated.
Leukocytes were counted in a Biirker chamber. Differential counts were made on stained smears. Triglyceride och cholesterol in serum were determined by enzymatic methods (10).
effect
of Intralipid
on leukocyte
To five out of six plastic test tubes, each with 0.5 granulocyte suspension, 0.5, 0.4, 0.3, 0.2, and 0.1 ml of Intrahipid, respectively, was added. The volumes in all six tubes were adjusted to 1 ml with Hanks’ solution. The tubes were incubated with shaking in a 37 C water bath for #{189} hr. Thereafter, the cells were washed twice in Hank-Tris and finally suspended in 0.5 ml Eagle’s medium. Since the molecular weight of the TG components in the soybean oil emulsion has been calculated to be 87412
the
TG
9.2, 6.9, 4.6, otaxis under
Statistical
concentrations
2.3, and agarose
in
0 nmole/hiter, was carried
the
test
tubes
respectively. out as described
were
11.5,
Chemabove.
of
regression
was
used.
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/12/2416/4692395 by University of Glasgow user on 02 August 2018
2417
Results Effect of Intralipid random migration
on chemotactic of leukocytes
and
Decreased leukocyte migration was observed in all subjects during infusion of Intralipid. Values of chemotactic and random migration of leukocytes from persons given 100 ml of Intraipid per hour (Fig. lb) were lower during the infusion than immediately before and 22 hr after the infuson (P < 0.001 in both cases). There was no significant difference between values obtained 1 and 2 hr after the infusion. The impairment of leukocyte migration was more pronounced when 200 ml/hr was given and less when the dose was 50 ml/hr (Fig. la and c). Further, a correlation was obtained between the dose rate and the effect, calculated as the mean decrease after 1 and 2 hr in each person. Correlation coefficients were 0.65 for chemotaxis and 0.69 for random migration, both corresponding to P < 0.05.
lipids
The plasma TG concentrations were higher during the Intralipid infusion (100 ml/hr) than before and 22 hr after the infusion (P
