Tumor necrosis factor-enhanced leukotriene B4 generation and chemotaxis in human neutrophils.

In an in vivo study of five normal volunteers infused with endotoxin (20 U/kg of US reference endotoxin lot EC-5), increased neutrophil (PMN) generation of leukotriene B4 and chemotaxis to leukotriene B4 were found concomitantly with elevated plasma tumor necrosis factor (TNF) levels. To clarify the role of TNF in PMN activation, neutrophil responsiveness after in vitro treatment with TNF was examined. Neutrophils from seven normal subjects were incubated with TNF for 30 minutes and tested for chemotaxis to leukotriene B4, formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, or the calcium ionophore A23187 to assess leukotriene B4 generation. A range of 10(-13) to 10(-9) mol/L of TNF was used for these assays. When 10(-9) mol/L of TNF was used, the amount of leukotriene B4 that was produced was significantly greater than in control cells. The effect of TNF on PMN chemotaxis was uniformly inhibitory for the three stimuli at 10(-10) mol/L compared with untreated cells. At a picomolar range, PMN migration to leukotriene B4, but not to zymosan-activated serum or formyl-methionyl-leucyl-phenylalanine, was significantly increased over that of PMNs not exposed to TNF. This suggests that TNF has a specific facilitatory effect on PMN responsiveness for both leukotriene B4 production and chemotaxis to leukotriene B4 and may be the same signal for this phenomenon in endotoxemic patients.

Use of aminophenylboronic acid affinity chromatography to measure glycosylated albumin levels.

A simple technique for the measurement of glycosylated albumin by affinity chromatography on m-aminophenylboronic acid agarose columns is presented. The technique relies on bromcresol green determination of albumin in the nonbound and bound fractions. There is a linear correlation between albumin concentration of the bound fraction and glycohemoglobin values in individuals. A control nondiabetic plasma pool with a glycohemoglobin value of 7.10% +/- 0.05% (mean +/- SEM) had a glycoalbumin value of 1.64% +/- 0.06%, while a diabetic control plasma pool with a glycohemoglobin value of 13.63% +/- 0.07% had a glycoalbumin value of 4.02% +/- 0.12%. Compared with results from the affinity technique, the preponderance of colorimetric reaction determined with the thiobarbituric acid procedure is nonspecific, in that it does not correlate with diabetic status or with values derived by the affinity procedure. The bulk of thiobarbituric acid-reactive material is present in the fraction of albumin that does not bind to aminophenylboronic acid. This nonbound fraction contains plasma glucose, which significantly interferes with thiobarbituric acid determinations but only very slightly interferes with the affinity procedure. Prolonged incubation of plasma with 500 mg/dl glucose dramatically increases affinity-determined glycosylated albumin. Thiobarbituric acid reactivity increases much less, the increase being mainly in the fraction bound to aminophenylboronic acid. The percentage glycosylated albumin determined by the affinity technique in crude plasma samples differs very slightly, if at all, from that determined by purification of the albumin from plasma. The affinity technique appears very promising for eventual clinical applications in the management of diabetes.

Aminophenylboronic acid affinity chromatography and thiobarbituric acid colorimetry compared for measuring glycated albumin.

Two techniques originally developed for measurement of glycated ("glycosylated") hemoglobin but also applicable to determination of glycated albumin are the thiobarbituric acid colorimetric technique (I) and the aminophenylboronic acid affinity chromatographic procedure (II). The latter reliably distinguishes diabetics from nondiabetics, and concentrations of glycated hemoglobin and glycated albumin are linearly correlated. I is nonspecific; it neither correlates with diabetic status nor with values derived via the affinity technique. Most of the chromogenic material is present in the fraction of albumin that does not bind to aminophenylboronic acid. Glucose interferes significantly with I but only slightly with II. Prolonged incubation of plasma with glucose dramatically increases the II-determined glycated albumin. Reactivity with thiobarbituric acid increases much less, and mainly in the II-bound fraction. This fraction contains a high proportion of nonspecifically reactive material. The percentage of glycated albumin determined in crude plasma samples by II differs only slightly from the value determined by purifying the albumin from the plasma. This technique appears more promising than I for eventual clinical applications.

Elevated production of neutrophil leukotriene B4 precedes pulmonary failure in critically ill surgical patients.

Leukotriene B4, a potent neutrophil chemotactic factor, is also made by the neutrophil. Neutrophil function was studied in 12 patients at risk for the development of adult respiratory distress syndrome (ARDS) after admission to the surgical intensive care unit (ICU) to test the hypothesis that increased generation by the neutrophil generation of this mediator precedes the development of pulmonary failure. Peripheral blood neutrophils were tested for chemotaxis to f-met-leu-phe (fMLP) and leukotriene B4 (LTB4) and the generation of LTB4. Plasma was collected simultaneously for assay of C3a desArg levels. Five patients had ARDS a mean of 2.2 +/- 0.25 days after admission to the ICU. Neutrophil generation of LTB4 was significantly enhanced on ICU day 1 in these patients as compared with patients at risk for ARDS but not developing the syndrome (119.4 +/- 6.1 versus 101.0 +/- 5.1, per cent control, p less than 0.05). Chemotaxis to fMLP and LTB4 was significantly reduced in both groups of patients. However, neutrophil chemotaxis improved in patients who did not have pulmonary failure during the time in the ICU, whereas neutrophil chemotactic responsiveness worsened in patients who did have pulmonary failure. Plasma C3a desArg levels were significantly elevated over normal laboratory values on ICU day 1 in the ARDS patients (317.2 +/- 74.0 versus 132.0 +/- 16.0 milligrams per milliliter, p less than 0.01). These data indicate that LTB4 production by the neutrophil occurs concomitantly with complement activation, is a predictor of subsequent ARDS and may play a significant role in the development of pulmonary failure in critically ill surgical patients.

Differential neutrophil activation before and after endotoxin infusion in enterally versus parenterally fed volunteers.

This study was done to determine whether or not increased susceptibility to infection seen in enterally versus parenterally fed patients was caused by altered neutrophil (PMN) responsiveness. To determine the differential effects of route of feeding on human PMN activation, plasma C3a levels, circulating PMN counts, PMN migration to leukotriene B4 (LTB4), the peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and zymosan activated serum (ZAS) and generation of LTB4 were assayed before and after and infusion of endotoxin. Nine normal volunteers were enterally (n=4) or parenterally (n=5) fed a diet sufficient to maintain body weight for seven days prior to a standard challenge of endotoxin. Samples were taken prior to the infusion and hourly thereafter for six hours. Prior to the injection of endotoxin, significant differences were seen in the two feeding groups. Plasma C3a levels, absolute circulating PMN counts and chemotaxis to LTB4 were all significantly (p less than 0.02) elevated in the enterally fed group. Generation of LTB4 was higher in the intravenously fed group at base line than the orally fed group (p less than 0.05). Plasma C3a levels rose in the enterally fed group, but not in the intravenously fed group, at two hours after infusion. Neutrophil counts rose in both feeding groups after endotoxin infusion; but the change in percentage was greater in the enterally fed group than in the intravenously fed group. Chemotaxis to FMLP and ZAS was not different during the study and did not differ between the two feeding can have significant impact on neutrophil function and that parenteral nutrition may impair host responsiveness.