0031-3998/85/1912-1278$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 12, 1985 Copyright O 1985 International Pediatric Research Foundation, Inc. Printed in U.S.A. Neutrophil Myeloperoxidase Concentration: Changes with Development and during Bacterial Infection ROBERT D. CHRISTENSEN AND GERALD ROTHSTEIN Departments of Pediatrics and Internal Medicine, University of Utah School ofMedicine, Salt Lake City, Utah 84132 ABSTRACT. In experimental animals, the quantity of neutrophil MPO during recovery from infection is the result of myeloperoxidase (MPO) in a volume of whole blood, and deleting one myelocyte division. the neutrophil concentration in that same volume, were determined and the results expressed as units of MPO MATERIALS AND METHODS (lO-')/neutro~hil- Two situations are reported in which the MPO quantiJiCation.Our standard method for the measure- MPo/neutrOphil was found change; ment of myeloperoxidase has been published previously (3, 4). during the growth and and during Briefly, leukocytes, separated from whole blood on discontinuous bacterial infection. Premature and newborn rats had only gradients, are washed and suspended in M~c~~'~5A medium, 25% of the MPO/neutrophil found in adults. One to three after which contaminating erythrocytes are subjected to hypo- wk O1ds had the enzyme During tonic lysis. Final cell concentrations are determined by electronic fatal bacterial infection, MPO/neutrophil fell rapidly, often counting (Coulter Electronics, Hialeah, FL), and cell differential to undetectable levels, but during sublethal infections, fol- counting. Myeloperoxidase is extracted from cells by suspending lowing a 24-h lag period in and a 48-h lag in them in 0.5% hexadecyltrimethylammonium bromide (Sigma neonates, the concentration increased to twice normal. Chemical co., st. ~~~i~,MO) in 50 m~ potassium phosphate (Pediatr Res 19: 1278-1282,1985) buffer, pH 6.0. Specimens are subjected to sonication in an ice bath for 10 s (Heat Systems-Ultrasonics, Plainview, NY), fol- Abbreviations lowed by freeze-thawing and repeat sonication. MPO is assayed spectrophotometrically; 0.1 ml of the material to be measured is MPO, myeloperoxidase CFU, colony forming unit mixed with 2.9 ml of 50 mM phosphate buffer, pH 6.0, contain- ing 0.167 mg/ml o-dianisidine dihydrochloride (Sigma Chemical CFUc, colony forming unit in culture Co.) and 0.0005% hydrogen peroxide (Mallinckrodt, Paris, KY). The change in absorbance at 460 nm is measured with a Beck- man DU spectrophotometer (Beckman Instruments, Fullerton, CA). One unit of MPO activity is defined as that amount MPO is an antimicrobial enzyme located within the primary degrading one micromole of peroxide per minute at 25" C (5). granules of mammalian neutrophils (1, 2). In this study we The micromethod differs from the standard method in that employed a method for quantification of MPO using only 50 pl neutrophils are not separated from the blood. Instead, 50 PI of of blood, thus enabling its determination in very small subjects. blood are drawn into a heparinized microcapillary tube (Dade We assessed two situations in which we suspected that the Diagnostics, Inc., Miami, FL) and transferred to a test tube concentration of MPO in neutrophils might vary; during devel- containing 2 ml Hanks' balanced salt solution. Hypotonic lysis opment and during bacterial infection. First, blood samples from of the erythrocytes is accomplished by adding 6 ml distilled water rats at various stages of pre- and postnatal development were and mixing for 2 min, after which 2 ml of 3.5% NaCl is added. tested. In premature and newborn animals, the quantity of MPO The mixture is then centrifuged at 1200 x g for 5 min. The per neutrophil was only 25% of that found in adults, while supernatant is removed and 0.5 ml hexadecyltrimethylammon- neutrophils from 1 to 3 wk olds contained 50% of the adult ium bromide buffer (50 mM phosphate with 0.5% hexadecyltri- enzyme concentration. Next, the effect of bacterial infection on methylammonium bromide) added. The sample is then frozen neutrophil MPO content was measured. During fatal infections, at -70" C and assayed for MPO as described above. In both the concentration of MPO fell significantly, often to undetectable methods, the mean neutrophil MPO value was calculated by levels. In contrast, during sublethal infections mean neutrophil dividing the units of MPO from a known quantity of blood by myeloperoxidase did not fall, but rather, increased to twice the number of neutrophils measured in that same quantity of normal concentrations following a 24-h lag period in adults and blood. Values are expressed as units MPO (10-')/neutrophil. a 48 hour lag in neonates. We speculate that: 1) the low MPO Animals. Sprague-Dawley rats (Simonson Laboratories, Gil- concentration in the neutrophils of neonates is a result of addi- roy, CA) of various ages were studied. Groups of 10 or more tional myelocyte divisions, 2) the fall in neutrophil MPO during animals were taken prematurely at 19 and at 20 days gestation lethal infection is due to MPO exocytosis, and 3) the increase in (term is 21 days) by performing an hysterotomy on timed- pregnant females. Other animals were studied late on the first Received December 7, 1984; accepted July 23, 1985. day of life (6- 18 h old), on the 2nd day, between the 7th and 9th Address reprint requests to Dr. R. D. Christensen, Division of Hematology, day, at 3, 4, 6, 9, and 12 wk of age. Animals were weaned from University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132. their mothers when were between 3 and wk old. At that Supported by Public Health Service Grant HD-14419 and a grant from the time they were caged in groups of five to eight and allowed water Thrasher Research Fund. and rat food ad libitum. 1278 NEUTROPHIL MPO DURING DEVELOPMENT AND INFECTION Blood samples were obtained in a manner previously described (6). Briefly, the animals were first subjected to inhalation anes- thesia (Methoxyflurane, Abbott Laboratories, N. Chicago, IL) and then, in those 2 days old or younger, the external jugular vein was incised with scissors. Blood was collected directly into heparinized capillary tubes for MPO determination and elec- tronic cell counting. In older animals, blood was drawn by venipuncture from the inferior vena cava into a syringe contain- ing heparin (Panheparin, Abbott Laboratories) in a concentration of 10-20 U/ml blood. A 50-p1 aliquot was taken for the micro- method determination using a heparinized capillary tube. In all animals, a blood smear was prepared, stained with Wright stain, and a 100-200 cell differential performed. The accuracy and 3.0 reproducibility of neutrophil enumeration by this method have previously been established (7). 2 3.0 4.0 5.0 8 6.08 8 7.03 8 8.0o I 9.08 I 10.01 Bacteria. Type I11 group B streptococci, isolated from a human 4'1..MACRO METHOD neonate, were used to produce either lethal or sublethal infec- MPO UNITS (10-VNEUT tions. The organism was identified by the precipitin method Fig. 1. This study compares MPO/neutrophil values performed by using rabbit antisera (8) and grown overnight at 37" C in Todd- two differentmethods. Seventeen blood samples, each represented by a Hewett broth. After washing with phosphate-buffered saline, dot, were tested by the standard (gradient sedimentation) method and aliquots were frozen at -70" C. Before administering bacteria to plotted on the abscissa, and also by the micromethod, plotted on the animals, the aliquots were thawed and grown overnight in fresh ordinate. The correlation coefficient for the two methods was 0.9430. Todd-Hewitt broth. Bacteria were then sedimented by centrifu- gation and the concentrated organisms were washed three times in phosphate-buffered saline. The organisms were then diluted in phosphate-buffered saline to an optical density (Spectronic 20, Bausch and Lomb, Inc, Rochester, NY) corresponding to a final concentration of either lo", lo9, or lo7 CFU/ml. One microliter of bacterial suspension per gram of body weight was then used for animal inoculation. In 4-wk-old animals, a lethal infection was produced by an intraperitoneal inoculation of lo8 CFU/g body weight. Sublethal infection was produced in 4 wk olds by inoculation of lo6CFU/g. Lethal infection was produced in newborn animals by an intraperitoneal inoculation of lo6 CFU/g body weight, while sublethal infection was produced by inoculation of 1O4 CFW/g. Statistical analysis. Single variable linear regression analysis was used to compare the two myeloperoxidase determinations. All other data were analyzed by means of Student's t test. RESULTS 1/2 4 6 8 10 12 14 16 18 1st SAMPLE Testing the micromethod. To test the reliability of the micro- MPO UNITS (IO-')/NEUT method for MPO measurement, the MPO of neutrophils pre- Fig. 2. The intratest variation of the micromethod for measurement pared by gradient sedimentation was compared with values on of MPO/neutrophil was assessed by performing duplicate tests from blood from the same samples, using the micromethod. Blood for single blood samples. Each sample is represented by a dot. Values for the testing was obtained from 12 adult humans and five adult rats. first micromethod determination are plotted on the abscissa and from MPO by the standard method ranged from 3.6 to 8.5 x U/ the second on the ordinate. The correlation coefficient for the duplicate neutrophil (mean = 5.9), and by the micromethod from 4.0 to determinations was 0.978. 8.8 x U/neutrophil (mean = 6.1) (Fig. 1). The correlation coefficient for the two methods was 0.9430. Intratest variation of the micromethod was assessed by obtain- Mean neutrophil MPO during development. Blood neutrophil ing duplicate 50-p1 samples of venous blood, and performing concentration and MPO/neutrophil determinations were ob- MPO analysis on each (Fig.