- Email: [email protected]
In vitro effect of intralipid on polymorphonuclear leukocyte function in the neonate
7 10
Clinical and laboratory observations
included milk that contains a bacterial colony count of <103 C F U / m l with "no demonstrable pathogens ''4 or a total count of < 105 C F U / m l with no detectable Staphylococcus aureus, coliforms, pneumococci, group B streptococci, or gentamicin-resistant gram-negative rods. 12 By way of comparison, regulations for cow milk after pasteurization require total counts of < 3 • 104 C F U / m l with <10 coliforms/ml) 3 Our data suggest that feeding premature infants human milk with -->103 gram-negative bacilli per milliliter is associated with an increased incidence of feeding intolerance, and at higher levels (-> 10~/ml) with suspect sepsis. Indeed, such high levels of contamination may overcome any putative protective effect of raw human milk. Because continuous nasogastric feeding requires holding milk at room temperature for prolonged periods, which may allow small inocula to grow to these unacceptable levels, we have now eliminated our use of this feeding technique in favor of intermittent feeding. We also have discontinued use of donor milk, reemphasized an asepsis education program for mothers and nursery personnel: and established a policy of microbiologic screening of mother's milk before first use. Only milk containing <103 C F U / m l potential pathogens is considered acceptable; higher concentrations of normal skin flora, such as coagulase-negative staphylococci and diphtheroids, are also accepted. After initiation of feeding, we have continued microbiologic surveillance of milk at biweekly intervals. On the basis of our experience, use of unpasteurized human milk in premature nurseries without a screening program appears inadvisable. We thank Jan Tyrrell for technical assistance; and Sandra Wilks, R.N., and the other Special Care Nursery personnel.
The Journal of Pediatrics October 1986 REFERENCES
1. Canadian Paediatric Society. Statement on human milk banking. Can Med Assoc J 1985;132:750-2. 2. Larson E, Zuill R, Zier V, Berg B. Storage of human breast milk. Infect Control 1984;5:127-30. 3. Breastfeeding. In: Forbes GB, Woodruff CW, eds. Pediatric nutrition handbook, 2nd ed. Elk Grove Village, Ill.: American Academy of Pediatrics, 1985:2-15. 4. Sauve R, Buchan K, Clyne A, McIntosh D. Mothers' milk banking: microbiologic aspects. Can J Publ Health 1984; 75:133-6. 5. Meier P, Riordan J. Breast-feeding support in the high-risk nursery and at home. In: Riordan J, ed. A practical guide to breast-feeding. St. Louis: CV Mosby, 1983:237-74. 6. Fomon SJ, Ziegler EE, Vasquez HD. Human milk and the small premature infant. Am J Dis Child 1977;131:463-7. 7. Ryder RW, Crosby-Ritchie A, McDonough B, Hall WJ III. Human milk contaminated with Salmonella kottbus: a cause of nosocomial illness in infants. JAMA 1977;238:1533-4. 8. Stiver HG, Albritton WL, Clark J, Friesen P, White FMM. Nosocomial colonization and infection due to E. coil 0125:K70 epidemiologicallylinked to expressed breast-milk feedings, Can J Publ Health 1977;68:479-82. 9. Donowitz LG, Marsik FJ, Fisher KA, Wenzel RP. Contaminated breast milk: a source of Klebsiella bacteremia in a newborn intensive care unit. Rev Infect Dis 1981;3:716-20. 10. Stocks RJ, Davies DP, Allen F, Sewell D. Loss of breast milk nutrients during tube feeding. Arch Dis Child 1985;60: 164-6. 11. Schreiner RL, Eitzen H, Gfell MA, et al. Environmental contamination of continuous drip feedings. Pediatrics 1979; 63:232-7. 12. TysonJE, Edwards WH, Rosenfeld AM, Beer AE. Collection methods and contamination of bank milk. Arch Dis Child 1982;57:396-8. 13. Kruse CW. Sanitary control of milk. In: Last JM, ed. Maxcy-Rosenau public health and preventive medicine, 1lth ed. New York: Appleton-Century-Crofts, 1980:920,49.
In vitro effect of Intralipidon polymorphonuclear leukocyte function in the neonate Shalsta S. Usmani, M.D., Rita G. Harper, M.D., Concepcion G. Sia, M.D., Kamala Suntharallngam, M,A., and William R. Robeson, M.A. From the Division of Perinatal Medicine, Department of Pediatrics, and the Department of Medicine, North Shore University Hospital, Manhasset, N e w York, and Cornell University Medical College, N e w York; and the Department of Pediatrics, State University of N e w York-Downstate Medical Center, Brooklyn
Intralipid is an intravenously administered emulsion of soybean oil stabilized with 1.2% egg phospholipids and Submitted for publication Feb. 7, 1986; accepted May 9, 1986. Reprint requests: Shaista S. Usmani, M.D., North Shore University Hospital, 300 Community Drive, Manhasset, NY 7_1013.
rendered isotonic with 2.5% glycerol. This fat emulsion is used for parenteral nutrition in neonatal intensive care HBSS PMN
Hanks' balanced salt solution Polymorphonuelearneutrophil leukocyte
I
}
I
Volume 109 Number 4
Clinical and laboratory observations
711
Table. In vitro effect of various Intralipid concentrations on P M N function Intrallpld concentrations (mg/ml) PMN source
Chemiluminescence (cpm • 103) Adults (n = 6) Neonates (n = 12) Random motility Adults (n = 6) Depth Cells Neonates (n = 12) Depth Cells Chemotaxis Adults (n = 6) Depth Cells Neonates (n = 12) Depth Cells
0
144 136
_+ 6.6 _+ 21.7
25
148 173
'100
50
+ 8.6 _+ 25.5
147 191
+ 7.7 _+ 37.6
145 189
P (ANOVA)
-+ 13.0 + 37.6
NS NS
68.3 +__ 4.0 7.7 _+ 0.3
69.3 _+ 3.3 8.2 _+ 0.3
63.3 + 2.1 7.5 _+ 0.6
64.3 + 3.7 7.5 _+ 0.3
NS NS
49.1 _+ 4.6 4.7 +_ 0.8
55.0 _+ 5.6 5.8 _+ 0.9
58.1 _+ 5.5 7.8 +_ 0.7
58.5 - 6.7 7.2 _+ 0.9
NS NS
108.3 _+ 4.0 26.3 +_ 0.7
108.3 _+ 3.0 26.5 _+ 0.4
103.3 _+ 2.1 28.5 +_ 1.1
105.0 _+ 2.0 21.3 +-- 1.24
NS <0.02*
83.7 _+ 6.4 21.9 _+ 1.7
98.4 _+ 7.9 29.2 +_ 1.8
95.0-!-- 7.9 29.9 _+ 2.6
81.1 -+ 10.5 25.1 _+ 1.6
<0.02t <0.03t
Values represent mean _+SEM. *Cell chemolaxiswith 100 mg lntralipid vs control. ~'Celland depth chemotaxiswith 25 and 100 mg Intralipid vs control.
units to provide adequate nutrition and increased caloric intake. Both depression and stimulation of polymorphonuclear leukocyte function after Intralipid infusion have been reported in adults, l3 Wheeler et al.' recently reported that chemiluminescent and chemotactic activities of P M N s in cord and adult blood were not altered when incubated with 10 m g / m l Intralipid. Studies to determine the effect of higher concentrations of Intralipid on chemiluminescence and chemotaxis of P M N s from neonates have not been performed, although higher concentrations of Intralipid are usually recommended for use in neonates) We report the effect of Intralipid on in vitro oxidative and chemotactic functions of P M N s from healthy neonates. METHODS The study group comprised 12 healthy full-term newborn infants (mean + SD birth weight 3.32 -+ 0.32 kg, gestational age 39.5 _ 0.82 weeks) between 12 and 72 hours of age. All infants were delivered spontaneously by the vaginal route after an uncomplicated pregnancy and labor. There was no evidence of neonatal asphyxia, infection, or congenital malformations. The study was approved by the institutional research and publications review committee for research on human subjects. Informed parental consent was obtained. For comparison studies, P M N s from six normal adult volunteers were obtained. P M N functional studies. Five milliliters (10 U / m l ) of heparinized blood was obtained and tested immediately after collection. Total and differential white blood cell
counts were performed manually on each whole blood sample. P M N s were separated according to the method of B6yum 6 and washed twice in Hanks balanced salt solution without Ca ++, Mg ++, and phenol red. After isolation the cell suspension contained 85% PMNs, and the cells were adjusted to concentration of 10 • 106 P M N s / m l in HBSS. One-half milliliter of cell suspension (PMNs 5 • 106) was placed in each of four plastic tubes, and 25, 50, or 100 mg 20% Intralipid (Cutter Laboratories, Emeryville, Calif.) was added to three of the four tubes, which represented an Intralipid-triglyceride concentration of 2.9, 5.7, and 11.5 mmol/L, respectively. (The usual plasma-triglyceride concentration is 1 mmol/L. Therefore the three concentrations we used represent hypertriglyeeridemia). The volume in all four tubes was then adjusted to 1.0 ml with HBSS. The tubes were incubated with gentle shaking in a 37 ~ C water bath for 30 minutes. The cells were then washed twice before the assays were performed. Trypan blue dye exclusion showed >95% P M N viability of each cell preparation. P M N concentration was adjusted with HBSS to 1.0 • 106 cells/ml for chemiluminescence studies, and with 2% bovine serum albumin to 2.0 • 106 cells/ml for chemotaxis studies. Luminol-enhanced (Sigma Chemical Company, St. Louis) chemiluminescence, an assay of oxidative and metabolic function of phagocytizing PMNs, 7,g was measured in a liquid scintillation system (Beckman LS-230, Beckman Instruments, Fullerton, Calif.) using latex particles of 0.797 #m diameter (Seragen Diagnostics, Indianap-
7 12
Clinical and laboratory observations
olis) as stimulant. Samples were tested in duplicate, and peak levels in counts per minute (cpm x 103) for 1.0 X l & PMNs were recorded. Chemotactic activity was tested by a modified Boyden technique using blind-well chambers (Neuro-Probe Inc., Bethesda, Md.) and 5 /~m pore cellulose nitrate filters (Millipore Corporation, Bedford, Mass.)? Pooled human serum actived by Zymosan (Sigma) was used as a chemoattractant, and tissue culture medium 199 (Difco Laboratories, Detroit) for random motility. Samples were tested in duplicate after incubation of cells for 2 hours at 37 ~ C. Chemotactic and random motility of PMNs was determined in two ways: 1. The depth in micrometers at which the leading front of PMNs was in focus (• objective) was measured in one microscopic field. Measurements were made at five locations, and the averages of all determinations were recorded. 2. A count of the leading front of PMNs that migrated to the lower surface of the filter was noted, and the averages of five microscopic fields recorded. Statistical analysis. Analysis of variance with the Dunnett-Newman-Keuls test was used to evaluate all data. Each infant served as his or her own control to minimize day-to-day environmental influences on neonatal P M N function. The chemiluminescence and chemotaxis data obtained without treatment was compared with those obtained with 25, 50, and 100 mg/ml Intralipid treated PMNs. RESULTS Incubation with Intralipid in vitro had no effect on the chemilumineseent activity of PMNs from 12 healthy neonates <72 hours of age and from six adults, at any of the three concentrations (Table). For chemotaxis, data were available for only 10 infants; two blood samples yielded too few PMNs to assess chemotactic function at the three Intralipid concentrations. Preincubation of PMNs with Intralipid did not impair the random or chemotactic motility of PMNs from 10 healthy neonates and six adults compared with cells incubated without Intralipid (Table). On the contrary, in neonates ehemotactic motility of PMNs was enhanced on incubation with 25 or 50 mg/ml Intralipid compared with the control cells (P <0.05). DISCUSSION This study indicates no impairment of oxidative metabolic or chemotactic functions when neonatal and adult PMNs are exposed to 25, 50, or 100 mg/ml concentrations of Intralipid. Past in vitro studies on PMNs from adults have shown markedly decreased chemotactic activity after
The Journal of Pediatrics October 1986
incubation with 12.5 to 100 mg/ml Intralipid. 1.2 Cleary and Pickering I~ noted that Intralipid, acting as a particulate stimulus, activated and exhausted metabolic pathways involved in the respiratory burst and phagocytosis; they suggested that impairment of P M N movement and oxidative metabolic functions could be responsible for the increased susceptibility to infections seen in patients receiving parenteral nutrition. ~.2.lo We, however, found no abnormalities in chemiluminescent and chemotactic activity at any of three Intralipid concentrations. Wheeler et al? recently reported no abnormalities of chemotactic or chemiluminescent activities in cord or adult PMNs incubated with 10 mg/ml Intralipid compared with paired controls incubated with buffer. The reason for diminished in vitro chemiluminescent and chemotactic activities of PMNs as reported by earlier investigators is not clear. Many nonspecific factors, such as pH, ionic composition, and osmolarity, could cause inhibition of dubious physiologic relevance. Further studies are needed to determine whether prolonged Intralipid infusion impairs chemiluminescent and chemotactic activities of PMNs in neonates.
REFERENCES 1. Nordenstrom J, Jarstrand C, Wiernik A. Decreased chemotactic and random migration of leukocytes during Intralipid infusion. Am J Clin Nutr 1970;32:2416. 2. Wiernik A, Jarstrand C, Julander I. The effect of Intralipid on mononuclear and polymorphonuclear phagocytes. Am J Clin Nutr 1983;37:256. 3. English D, Roloff JS, Lukens JN, et al. Intravenous lipid emulsions and human neutrophil function. J PEDIATR 1981;99:913. 4. Wheeler JG, Boyle RJ, Abramson JS. Intralipid infusion in neonates: effects on polymorphonuclear leukocyte function. J Pediatr Gastroentcrol Nutr 1985;4:453. 5. American Academy of Pediatrics Committee on Nutrition: Use of intravenous fat emulsions in pediatric patients. Pediatrics 1981;68:738. 6. B~yum A. Separation of leukocytes from blood and bone marrow. Stand J Clin Lab Invest 1968;21:7. 7. Stevens P, Winston DJ, Van Dyke K. In vitro evaluation of opsonic and cellular granulocytc function by luminol-dcpendent chemiluminescence: utility in patients with severe ncutropenia and cellular deficiency states. Infect Immun 1978;22:41. 8. Horan JD, English D, MePherson TA. Association of neutrophil chemiluminescence with mierobieidal activity. Clin Immunol Immunopathol 1982;22:259. 9. Smith W, Hollers JC, Patrick RA, Hasselt C. Motility and adhesiveness in human neutrophils: effects of chemotactic factors. J Clin Invest 1979;63:221. 10. Cleary TG, Picketing LK. Mechanisms of lntralipid effect on polymorphonuclear leukocytes. J Clin Lab Immunol 1983;11:21.
Clinical and laboratory observations
included milk that contains a bacterial colony count of <103 C F U / m l with "no demonstrable pathogens ''4 or a total count of < 105 C F U / m l with no detectable Staphylococcus aureus, coliforms, pneumococci, group B streptococci, or gentamicin-resistant gram-negative rods. 12 By way of comparison, regulations for cow milk after pasteurization require total counts of < 3 • 104 C F U / m l with <10 coliforms/ml) 3 Our data suggest that feeding premature infants human milk with -->103 gram-negative bacilli per milliliter is associated with an increased incidence of feeding intolerance, and at higher levels (-> 10~/ml) with suspect sepsis. Indeed, such high levels of contamination may overcome any putative protective effect of raw human milk. Because continuous nasogastric feeding requires holding milk at room temperature for prolonged periods, which may allow small inocula to grow to these unacceptable levels, we have now eliminated our use of this feeding technique in favor of intermittent feeding. We also have discontinued use of donor milk, reemphasized an asepsis education program for mothers and nursery personnel: and established a policy of microbiologic screening of mother's milk before first use. Only milk containing <103 C F U / m l potential pathogens is considered acceptable; higher concentrations of normal skin flora, such as coagulase-negative staphylococci and diphtheroids, are also accepted. After initiation of feeding, we have continued microbiologic surveillance of milk at biweekly intervals. On the basis of our experience, use of unpasteurized human milk in premature nurseries without a screening program appears inadvisable. We thank Jan Tyrrell for technical assistance; and Sandra Wilks, R.N., and the other Special Care Nursery personnel.
The Journal of Pediatrics October 1986 REFERENCES
1. Canadian Paediatric Society. Statement on human milk banking. Can Med Assoc J 1985;132:750-2. 2. Larson E, Zuill R, Zier V, Berg B. Storage of human breast milk. Infect Control 1984;5:127-30. 3. Breastfeeding. In: Forbes GB, Woodruff CW, eds. Pediatric nutrition handbook, 2nd ed. Elk Grove Village, Ill.: American Academy of Pediatrics, 1985:2-15. 4. Sauve R, Buchan K, Clyne A, McIntosh D. Mothers' milk banking: microbiologic aspects. Can J Publ Health 1984; 75:133-6. 5. Meier P, Riordan J. Breast-feeding support in the high-risk nursery and at home. In: Riordan J, ed. A practical guide to breast-feeding. St. Louis: CV Mosby, 1983:237-74. 6. Fomon SJ, Ziegler EE, Vasquez HD. Human milk and the small premature infant. Am J Dis Child 1977;131:463-7. 7. Ryder RW, Crosby-Ritchie A, McDonough B, Hall WJ III. Human milk contaminated with Salmonella kottbus: a cause of nosocomial illness in infants. JAMA 1977;238:1533-4. 8. Stiver HG, Albritton WL, Clark J, Friesen P, White FMM. Nosocomial colonization and infection due to E. coil 0125:K70 epidemiologicallylinked to expressed breast-milk feedings, Can J Publ Health 1977;68:479-82. 9. Donowitz LG, Marsik FJ, Fisher KA, Wenzel RP. Contaminated breast milk: a source of Klebsiella bacteremia in a newborn intensive care unit. Rev Infect Dis 1981;3:716-20. 10. Stocks RJ, Davies DP, Allen F, Sewell D. Loss of breast milk nutrients during tube feeding. Arch Dis Child 1985;60: 164-6. 11. Schreiner RL, Eitzen H, Gfell MA, et al. Environmental contamination of continuous drip feedings. Pediatrics 1979; 63:232-7. 12. TysonJE, Edwards WH, Rosenfeld AM, Beer AE. Collection methods and contamination of bank milk. Arch Dis Child 1982;57:396-8. 13. Kruse CW. Sanitary control of milk. In: Last JM, ed. Maxcy-Rosenau public health and preventive medicine, 1lth ed. New York: Appleton-Century-Crofts, 1980:920,49.
In vitro effect of Intralipidon polymorphonuclear leukocyte function in the neonate Shalsta S. Usmani, M.D., Rita G. Harper, M.D., Concepcion G. Sia, M.D., Kamala Suntharallngam, M,A., and William R. Robeson, M.A. From the Division of Perinatal Medicine, Department of Pediatrics, and the Department of Medicine, North Shore University Hospital, Manhasset, N e w York, and Cornell University Medical College, N e w York; and the Department of Pediatrics, State University of N e w York-Downstate Medical Center, Brooklyn
Intralipid is an intravenously administered emulsion of soybean oil stabilized with 1.2% egg phospholipids and Submitted for publication Feb. 7, 1986; accepted May 9, 1986. Reprint requests: Shaista S. Usmani, M.D., North Shore University Hospital, 300 Community Drive, Manhasset, NY 7_1013.
rendered isotonic with 2.5% glycerol. This fat emulsion is used for parenteral nutrition in neonatal intensive care HBSS PMN
Hanks' balanced salt solution Polymorphonuelearneutrophil leukocyte
I
}
I
Volume 109 Number 4
Clinical and laboratory observations
711
Table. In vitro effect of various Intralipid concentrations on P M N function Intrallpld concentrations (mg/ml) PMN source
Chemiluminescence (cpm • 103) Adults (n = 6) Neonates (n = 12) Random motility Adults (n = 6) Depth Cells Neonates (n = 12) Depth Cells Chemotaxis Adults (n = 6) Depth Cells Neonates (n = 12) Depth Cells
0
144 136
_+ 6.6 _+ 21.7
25
148 173
'100
50
+ 8.6 _+ 25.5
147 191
+ 7.7 _+ 37.6
145 189
P (ANOVA)
-+ 13.0 + 37.6
NS NS
68.3 +__ 4.0 7.7 _+ 0.3
69.3 _+ 3.3 8.2 _+ 0.3
63.3 + 2.1 7.5 _+ 0.6
64.3 + 3.7 7.5 _+ 0.3
NS NS
49.1 _+ 4.6 4.7 +_ 0.8
55.0 _+ 5.6 5.8 _+ 0.9
58.1 _+ 5.5 7.8 +_ 0.7
58.5 - 6.7 7.2 _+ 0.9
NS NS
108.3 _+ 4.0 26.3 +_ 0.7
108.3 _+ 3.0 26.5 _+ 0.4
103.3 _+ 2.1 28.5 +_ 1.1
105.0 _+ 2.0 21.3 +-- 1.24
NS <0.02*
83.7 _+ 6.4 21.9 _+ 1.7
98.4 _+ 7.9 29.2 +_ 1.8
95.0-!-- 7.9 29.9 _+ 2.6
81.1 -+ 10.5 25.1 _+ 1.6
<0.02t <0.03t
Values represent mean _+SEM. *Cell chemolaxiswith 100 mg lntralipid vs control. ~'Celland depth chemotaxiswith 25 and 100 mg Intralipid vs control.
units to provide adequate nutrition and increased caloric intake. Both depression and stimulation of polymorphonuclear leukocyte function after Intralipid infusion have been reported in adults, l3 Wheeler et al.' recently reported that chemiluminescent and chemotactic activities of P M N s in cord and adult blood were not altered when incubated with 10 m g / m l Intralipid. Studies to determine the effect of higher concentrations of Intralipid on chemiluminescence and chemotaxis of P M N s from neonates have not been performed, although higher concentrations of Intralipid are usually recommended for use in neonates) We report the effect of Intralipid on in vitro oxidative and chemotactic functions of P M N s from healthy neonates. METHODS The study group comprised 12 healthy full-term newborn infants (mean + SD birth weight 3.32 -+ 0.32 kg, gestational age 39.5 _ 0.82 weeks) between 12 and 72 hours of age. All infants were delivered spontaneously by the vaginal route after an uncomplicated pregnancy and labor. There was no evidence of neonatal asphyxia, infection, or congenital malformations. The study was approved by the institutional research and publications review committee for research on human subjects. Informed parental consent was obtained. For comparison studies, P M N s from six normal adult volunteers were obtained. P M N functional studies. Five milliliters (10 U / m l ) of heparinized blood was obtained and tested immediately after collection. Total and differential white blood cell
counts were performed manually on each whole blood sample. P M N s were separated according to the method of B6yum 6 and washed twice in Hanks balanced salt solution without Ca ++, Mg ++, and phenol red. After isolation the cell suspension contained 85% PMNs, and the cells were adjusted to concentration of 10 • 106 P M N s / m l in HBSS. One-half milliliter of cell suspension (PMNs 5 • 106) was placed in each of four plastic tubes, and 25, 50, or 100 mg 20% Intralipid (Cutter Laboratories, Emeryville, Calif.) was added to three of the four tubes, which represented an Intralipid-triglyceride concentration of 2.9, 5.7, and 11.5 mmol/L, respectively. (The usual plasma-triglyceride concentration is 1 mmol/L. Therefore the three concentrations we used represent hypertriglyeeridemia). The volume in all four tubes was then adjusted to 1.0 ml with HBSS. The tubes were incubated with gentle shaking in a 37 ~ C water bath for 30 minutes. The cells were then washed twice before the assays were performed. Trypan blue dye exclusion showed >95% P M N viability of each cell preparation. P M N concentration was adjusted with HBSS to 1.0 • 106 cells/ml for chemiluminescence studies, and with 2% bovine serum albumin to 2.0 • 106 cells/ml for chemotaxis studies. Luminol-enhanced (Sigma Chemical Company, St. Louis) chemiluminescence, an assay of oxidative and metabolic function of phagocytizing PMNs, 7,g was measured in a liquid scintillation system (Beckman LS-230, Beckman Instruments, Fullerton, Calif.) using latex particles of 0.797 #m diameter (Seragen Diagnostics, Indianap-
7 12
Clinical and laboratory observations
olis) as stimulant. Samples were tested in duplicate, and peak levels in counts per minute (cpm x 103) for 1.0 X l & PMNs were recorded. Chemotactic activity was tested by a modified Boyden technique using blind-well chambers (Neuro-Probe Inc., Bethesda, Md.) and 5 /~m pore cellulose nitrate filters (Millipore Corporation, Bedford, Mass.)? Pooled human serum actived by Zymosan (Sigma) was used as a chemoattractant, and tissue culture medium 199 (Difco Laboratories, Detroit) for random motility. Samples were tested in duplicate after incubation of cells for 2 hours at 37 ~ C. Chemotactic and random motility of PMNs was determined in two ways: 1. The depth in micrometers at which the leading front of PMNs was in focus (• objective) was measured in one microscopic field. Measurements were made at five locations, and the averages of all determinations were recorded. 2. A count of the leading front of PMNs that migrated to the lower surface of the filter was noted, and the averages of five microscopic fields recorded. Statistical analysis. Analysis of variance with the Dunnett-Newman-Keuls test was used to evaluate all data. Each infant served as his or her own control to minimize day-to-day environmental influences on neonatal P M N function. The chemiluminescence and chemotaxis data obtained without treatment was compared with those obtained with 25, 50, and 100 mg/ml Intralipid treated PMNs. RESULTS Incubation with Intralipid in vitro had no effect on the chemilumineseent activity of PMNs from 12 healthy neonates <72 hours of age and from six adults, at any of the three concentrations (Table). For chemotaxis, data were available for only 10 infants; two blood samples yielded too few PMNs to assess chemotactic function at the three Intralipid concentrations. Preincubation of PMNs with Intralipid did not impair the random or chemotactic motility of PMNs from 10 healthy neonates and six adults compared with cells incubated without Intralipid (Table). On the contrary, in neonates ehemotactic motility of PMNs was enhanced on incubation with 25 or 50 mg/ml Intralipid compared with the control cells (P <0.05). DISCUSSION This study indicates no impairment of oxidative metabolic or chemotactic functions when neonatal and adult PMNs are exposed to 25, 50, or 100 mg/ml concentrations of Intralipid. Past in vitro studies on PMNs from adults have shown markedly decreased chemotactic activity after
The Journal of Pediatrics October 1986
incubation with 12.5 to 100 mg/ml Intralipid. 1.2 Cleary and Pickering I~ noted that Intralipid, acting as a particulate stimulus, activated and exhausted metabolic pathways involved in the respiratory burst and phagocytosis; they suggested that impairment of P M N movement and oxidative metabolic functions could be responsible for the increased susceptibility to infections seen in patients receiving parenteral nutrition. ~.2.lo We, however, found no abnormalities in chemiluminescent and chemotactic activity at any of three Intralipid concentrations. Wheeler et al? recently reported no abnormalities of chemotactic or chemiluminescent activities in cord or adult PMNs incubated with 10 mg/ml Intralipid compared with paired controls incubated with buffer. The reason for diminished in vitro chemiluminescent and chemotactic activities of PMNs as reported by earlier investigators is not clear. Many nonspecific factors, such as pH, ionic composition, and osmolarity, could cause inhibition of dubious physiologic relevance. Further studies are needed to determine whether prolonged Intralipid infusion impairs chemiluminescent and chemotactic activities of PMNs in neonates.
REFERENCES 1. Nordenstrom J, Jarstrand C, Wiernik A. Decreased chemotactic and random migration of leukocytes during Intralipid infusion. Am J Clin Nutr 1970;32:2416. 2. Wiernik A, Jarstrand C, Julander I. The effect of Intralipid on mononuclear and polymorphonuclear phagocytes. Am J Clin Nutr 1983;37:256. 3. English D, Roloff JS, Lukens JN, et al. Intravenous lipid emulsions and human neutrophil function. J PEDIATR 1981;99:913. 4. Wheeler JG, Boyle RJ, Abramson JS. Intralipid infusion in neonates: effects on polymorphonuclear leukocyte function. J Pediatr Gastroentcrol Nutr 1985;4:453. 5. American Academy of Pediatrics Committee on Nutrition: Use of intravenous fat emulsions in pediatric patients. Pediatrics 1981;68:738. 6. B~yum A. Separation of leukocytes from blood and bone marrow. Stand J Clin Lab Invest 1968;21:7. 7. Stevens P, Winston DJ, Van Dyke K. In vitro evaluation of opsonic and cellular granulocytc function by luminol-dcpendent chemiluminescence: utility in patients with severe ncutropenia and cellular deficiency states. Infect Immun 1978;22:41. 8. Horan JD, English D, MePherson TA. Association of neutrophil chemiluminescence with mierobieidal activity. Clin Immunol Immunopathol 1982;22:259. 9. Smith W, Hollers JC, Patrick RA, Hasselt C. Motility and adhesiveness in human neutrophils: effects of chemotactic factors. J Clin Invest 1979;63:221. 10. Cleary TG, Picketing LK. Mechanisms of lntralipid effect on polymorphonuclear leukocytes. J Clin Lab Immunol 1983;11:21.