OF BIOCHEMISTRY AND BIOPHYSICS 188, No. 1, May, pp. 26-30, 1978
Effect of Galactose
on Free Radical Reactions Leukocytes’ J. LITCHFIELD”
of Riochemi.stry, Received
1, 1977; revised
W. WELLS” Lansing,
To account for impaired bactericidal activity of polymorphonuclear leukocytes during galactosemia, we have investigated the effects of galactose upon free radical reactions associated with the oxygen-dependent killing mechanism of guinea pig polymorphonuclear leukocytes. Millimolar levels of galactose, which are encountered in the circulation of infants with galactosemia, did not affect the chemical reaction of superoxide anion with either ferricytochrome c or nitroblue tet.razolium. Galactose did, however, significantly inhibit the reaction of hydroxyl radical with methional, and we propose that such hydroxyl radical removal would be deleterious to normal bactericidal activity. Additional experiments demonstrated that polymorphonuclear leukocytes incubated in the presence of galactose reduced less ferricytochrome c than polymorphonuclear leukocytes incubated without galactose. These results imply that galactose impairs the cellular release of superoxide anion, which would further disable bactericidal activity.
Millimolar levels of D-galactose significantly inhibit the bactericidal activity of polymorphonuclear leukocytes (PMN? (1, 2). These levels are also encountered in infants with galactose l-phosphate uridylyltransferase (EC 126.96.36.199) deficiency (3, 4), and such infants commonly display enhanced susceptibility to infection (5-8). An important clue to understanding the molecular action of galactose is indicated by the pronounced inhibitory effect of this hexose upon bactericidal rather than phagocytic activity (1). Unlike phagocytosis, the former function depends largely upon cellular oxygen consumption (g-11), and a number of reduced oxygen species, such as superoxide (12, 13), hydrogen peroxide (14-16), and hydroxyl radical (17-19) have been detected during this process. Bactericidal ac-
tivity of PMN, in vitro, can be inhibited in the presence of superoxide dismutase (19), catalase (19, 2O), or hydroxyl radical scavengers such as benzoate, ethanol, mannitol, glucose, or sucrose (17-24). These hydroxyl radical scavengers possess approximately the same bimolecular rate constant for reaction with hydroxyl radical as do all monosaccharides and polyols that have been tested by pulse radiolysis (25-27). In this communication we demonstrate that galactose is a good hydroxyl radical scavenger (k = 1 x 10’” M-’ SK’), and we propose that the inhibitory effect of galactose upon bactericidal activity (1) is a result of this reaction. We also show that galactose does not react directly with superoxide anion. However, galactose does impair the release of superoxide anion from PMN.
’ This investigation was supported by Grant AM 10209, U.S. Public Health Service. Michigan Agricultural Experiment Station Journal Article 7849. ’ Present address: Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19174. .I To whom inquires should be addressed. 4 Abbreviations used: PMN, polymorphonuclear leukocytes; gc, gas chromatography; KRP solution, Krebs-Ringer phosphate solution.
Animals and materials. Adult male guinea pigs were obtained from Elm Hill Laboratories, Cambridge, Mass., and were fed a commercial diet and water with 0.04% ascorbic acid, ad Zibitum. Xanthine, phenazine tetrazolium, methional, methosulfate, nitroblue NADH (grade III), xanthine oxidase (EC 188.8.131.52), and cytochrome c were purchased from Sigma Chemical Co. Catalase (EC 184.108.40.206) and superoxide dismutase
26 ooO3-9861/78/1881-0026$02.00/0 Copyright 0 1978 by Academic Press, Inc All righti of reproduction in any form reserved.
(EC I.15.I.I) were obtained from Worthington Biochemical Co. and Miles Laboratories, Ltd., respectively. All carbohydrates and polyols were purchased from Sigma, Mallinckrodt, or Nutritional Biochemical Co. Polystyrene latex particles were purchased from Dow Diagnostics. Cell preparations. Guinea pig PMN were isolated from casein-injected peritonea as previously described (1, 28). Concentrations of cells in suspension were determined using a hemocytometer under phase optics. Each cell count was performed in quadruplicate. Hydroxyl radical reactions. Reactions between carbohydrates and hydroxyl radicals were determined by monitoring ethylene production from the hydroxyl radical-dependent degradation of methional (29). Hydroxyl radicals were generated nonenzymatically in 1.0 ml of 50 mM potassium phosphate buffer, pH 7.8, containing 1.0 mM H202, 1.0 mM methional, and trace amounts of iron. All reactions were performed at room temperature in 25-ml siliconized Erlenmeyer flasks fitted with gas-tight stoppers. Gas above each reaction mixture was sampled with a gas-tight syringe and was analyzed for ethylene by injecting LO-ml aliquots into a Hewlett-Packard Model 402 chromatograph equipped with a 6-ft glass column of Chromosorb 102 (So-100 mesh), Johns-Manville, Denver, Colo. Column temperature and flame ionization detector temperature were maintained at 90 and 2OO”C, respectively. Since small samples of ethylene were removed from each flask for gc analysis, the total nanomoles of ethylene produced per flask at time ( t) were calculated using the following equation: nmol, = [(R,/&)
X ( V2/K)l
repeated six to nine times using a Gilford Model 3500 computer-directed spectrophotometer thermostated at 25°C. Cellular release of superoxide. The effect of galactose upon the production and release of superoxide from PMN was investigated by following the reduction of extracellular cytochrome c. PMN from two guinea pigs were pooled and suspended in KRP solution containing 10% guinea pig serum. The cell suspension was divided into aliquots of 0.88 x 10’ to 1.07 x 10’ cells/ml, and each aliquot was incubated in the presence or absence of galactose for 2 h at 37°C. This incubation period was chosen to correspond with previously published experimental conditions (1). Following this incubation period, superoxide anion release was measured according to the procedure of Babior el al. (12). All assays were performed in quadruplicate. and dialyzed polystyrene latex particles were employed to stimulate PMN just prior to the superoxide
The production of ethylene from methional in the presence of hydrogen peroxide (Fig. 1) indicated that hydroxyl radical was produced. This nonenzymatic system, containing 1.0 mM HzOz and 1.0 mM methional, depended upon the following four reactions (29, 31) in which iron, contributed as a
+ nmol, I
where K, = detector response with sample injected (cm); R,,,I = detector response with ethylene standard (cm/mob; I/L = volume of flask (ml); VI = volume of sample injection (ml); and nmol, 1 = nanomoles of ethylene lost by analysis of the previous sample volume, V,. Superoxide anion reactions. The possibility that galactose and other carbohydrates could scavenge superoxide was investigated by following the superoxidedependent reduction of both ferricytochrome c and nitroblue tetrazolium. In one set of experiments, superoxide was generated enzymatically in 1.1 ml of 0.05 M sodium phosphate buffer, pH 7.8, containing: 50 pM xanthine, 100 pM EDTA, 10 pM ferricytochrome c, and 3.2 mU of xanthine oxidase. In another set of experiments, superoxide anion was produced nonenzymatically in 1.1 ml of Krebs-Ringer phosphate solution (KRP solution) containing: 78 pM NADH, 23 pM phenazine methosulfate, and 50 pM nitroblue tetrazolium (30). Both sets of experiments were performed in the absence and presence of 27 mM carbohydrate. Initial rates of ferricytochrome c reduction were measured by following absorption at 550 nm, whereas initial rates of nitroblue tetrazolium reduction were determined at 515 nm. All experiments were
FIN:. 1. Inhibition of hydroxyl radical-dependent degradation of methional. All carbohydrate and polyol levels were 40 mM. Benzoate and superoxide dismutase levels were 5 mM and 0.2 mg/ml, respectively.
contaminant of phosphate buffer, was oxidized by Hz02 and reduced by superoxide anion: H,O, + Fe’)+ HaOz+OH 0; CH.,SCHzCH,CHO
+ OH + Fe”,
-OH-+O; + Fe” -
01 + Fe’+,
111 Pl 131
+ OH + r41
The initial lag period in ethylene production indicated that trace amounts of ferric iron rather than ferrous iron were contributed by phosphate buffer. This was verified by stimulating ethylene production without a lag phase by adding 1 nM FeS04 (data not shown) and by prolonging the lag phase in the presence of superoxide dismutase. Ethylene formation was significantly inhibited by 5 mM levels of benzoate and by 40 mM levels of an acyclic polyol, aldohexose, or myo-inositol. Each point in Fig. 1 was obtained by averaging values from four to six determinations, and all differences between control values and values obtained with inhibitors were significant at the P < 0.01 level after 10 min of reaction. Since ethylene formation was not significantly decreased by addition of 0.2 mg/ml of bovine serum albumin (data not shown), the action of superoxide dismutase was taken to represent a specific enzyme effect. Although galactose effectively scavenged hydroxyl radical in the above experiments, it did not significantly affect the reaction of superoxide anion with either ferricytochrome c or nitroblue tetrazolium (Table I). Similar results were obtained with four polyols, including galactitol which is accumulated by PMN during incubation with galactose (2). These results indicated that carbohydrates do not react directly with superoxide anion and that such a reaction would be very unlikely in the presence of another superoxide generating system, the PMN. Galactose did, however, significantly decrease the amount of extracellular cytochrome c reduced by PMN (Table II), and similar results were found in the presence of superoxide dismutase but not catalase. These results indicated that superoxide was produced by PMN and that galactose inter-
WELLS TABLE I EFFECT OF CAHHOHYDKATE ON REACTIONS INVOLVING; SIJPBHOXIDF, ANION RAI)ICAI.” Cytochrome c re- Nitroblue tetrazolium duced” reduced’ (nmol/min) Addition (A,,, ,,,,,/min x 10’) .__ ~~~ 1.49 * 1.61 f 1.52 + 1.55 + 1.55 f 1.57 f
None Galactose Galactitol Sorbitol Mannitol Xylitol
0.07 0.21 0.15 0.18 0.18 0.14
0.95 f !.!I8 f 0.96 f 1.05 + 0.99 f
0.05 0.05 0.06 0.06 0.06
“Each reaction was performed six to nine times using a Gilford Model 3500 spectrophotometer in the general kinetic-l program mode. All values represent average slopes + standard deviation. All carbohydrate levels were 27 mM. ’ Superoxide was generated enzymatically in 1.1 ml of 0.5 M sodium phosphate buffer, pH 7.8, containing: 50 FM xanthine, 100 pM EDTA, 10 pM ferricytochrome c, and 3.2 mU of xanthine oxidase. ’ Superoxide was generated nonenzymatically in 1.1 ml of Krebs-Ringer phosphate solution, pH 7.4, containing: 78 PM NADH, 23 PM phenazine methosulfate, and 50 PM nitroblue tetrazohum (30).
OF GAI.AC.I’OSE ON CYTOCHIWME RErXJgalactose (1, 2), the latter activity is inhibited to a much greater extent (1). This differential action is common among inhibitors of leukocyte function and agrees with previous studies demonstrating that phagocytosis and bactericidal activity are two distinct functions of PMN (9, 10, 32). In this regard, phagocytosis depends primarily upon energy supplied by glycolysis (32), and it has been proposed that galactose directly inhibits this metabolic pathway (1, 28). Bactericidal activity, on the other hand, relies largely upon increased hexose monophosphate shunt activity (33, 34) and upon reduction of oxygen to free radical intermediates (19). Superoxide anion is generated as a result of enhanced NAD(P)H oxidase activity (Eq. ), and hydrogen peroxide is formed by the dismutation of superoxide anion (Eq. ):
scavengers impair bactericidal activity (19)) (ii) that OH. breaks and degrades bacterial DNA (35), and (iii) that OH. leads to premature death of leukocytes following bacterial stimulation (18). As indicated in Fig. 1, 40 mM levels of galactose, glucose, mannitol, and other carbohydrates are as effective in scavenging OH. as are 5 mM levels of benzoate. These reactions are generally written as follows with hydrogen abstraction by the hydroxyl radical (27): GH + OH. + G. + H,O.
These results confirm those obtained by pulse radiolysis of carbohydrate solutions (25, 26) and indicate that all of these scavengers have approximately the same bimolecular rate constant of 10”’ M-’ s-l for reaction with OH ‘. Moreover, these results also imply that in carbohydrate disorders, such as galactosemia, levels of hexose which approach 40 mM (3, 4) may inhibit leukocytic bactericidal activity by scavenging this toxic radical species. The subsequent fate of G ., as exemplified in the case of mannitol radical (36), is determined by the availability of other radicals in the immediate vicinity. Since the most probable dismutation of G occurring at pH 7.4 is with superoxide anion, the steady-state levels of superoxide would be expected to decrease:
G.+()- -GH+Oa H+ VI Decreased levels of intracellular superoxide would result in less superoxide release from PMN, and this could account for the decreased cytochrome c reduction by PMN in 151 the presence of galactose (Table II). This scheme is supported by the failure of galactose (i) to react directly with superoxide O,-+HOzH’HsOr+Oz [al anion (Table I), (ii) to impair hexose monophosphate shunt activity (l), and (iii) to Both superoxide and hydrogen peroxide have been observed in PMN suspensions affect oxygen consumption. Alternatively, galactose could impair su(ll-15), and these molecules are believed peroxide release by limiting the membrane responsible for hydroxyl radical production to superoxide. However, we (Eq. [l]). Hydroxyl radical, which was de- permeability tected in leukocytes by Weiss (17), has been did not find any evidence that membrane implicated as a highly toxic agent to bac- permeability to other molecules, such as 2teria. This implication is based upon nu- deoxyglucose, was affected by galactose merous experiments showing (i) that OH. (28). o,
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