Mechanism of inhibitory action of ketone bodies on the production of reactIve oxygen intermediates (ROIS) by polymorphonuclear leukocytes

Life Sciences, Vol. Printed in the USA

51, pp. i13-i18

Pergamon Press

MECHANISM OF INHIBITORY ACTION OF KETONE BODIES ON THE PRODUCTION OF REACTIVE OXYGEN INTERMEDIATES (ROIS) BY POLYMORPHONUCLEAR LEUKOCYTES Nonyukl Sato, Hlroyukl Shlmlzu, Yohnosuke Shlmomura, Kumhlko Suwa, Masatomo Mon and Isao Kobayashl First Department of Internal Medicine, and Department of Laboratory Medicine, Gunma University School of Medicine, Maebashl, Japan (Recelved in final form May 5, 1992)

Summary We determined an effect of acetoacetlc acid (AcAc) and 3-hydroxybutyrate (3-OHB) on the production of reactive oxygen Intermediates (ROIs) m polymorphonuclear leukocytes from healthy volunteers Both AcAc and 3-OHB inhibited the lummol-depcndent chemiluminescence (LDCL) actiwtles assessed with initial slope and the inhibition rates were about 42 %, 44 % respectively by AcAc and 3-OHB when the leukocytes were prelncubated with 10 mM AcAc or 3-OHB for 60 minutes The LDCL activity was reduced by 16 % and 42 % following the addition of lmM and 10 mM AcAc The similar reductmn of the LDCL activity was observed in the addltmn of 3-OHB Either 3-OHB or AcAc failed to show a significant reductmn of myeloperoxldase (MPO) acnvlty However, both 3-OHB and AcAc dose-dependently mhiNted superoxlde amon (02-) productmn, measured by using cytochromc c These data provided evidence that both 3-OHB and AcAc suppress neutrophll oxidative metabohsm with respect with 02- production It is well known that bacterial refection is an Important causc of the morbidity and mortality In poorly controlled diabetic patients [1-4] The polymorphonuclear leukocytes play an critical role in the host defense mechanism against various bacterial Infections Recently, we have demonstrated that hyperglycemia suppressed the activity of the lumlnol-dependent chelmlUmlnescence (LDCL) m the leukocytes stlmulated by opsonlzed zymosan from streptozotocminduced diabetic rats [5] In addition, both the LDCL activity, calculated from imtlal slope gradient of chemiluminescence from the leukocytes stimulated by opsonized zymosan, and superoxlde anion production were reduced in poorly controlled dlabetlc patients [6,7]. However, increased circulating ketone bodies levels are supposed to coexist In poorly controlled diabetic patlents, since the development of [3-oxidation of fatty aods accelerates the production of ketone bodies [8] It is highly possible that Increased circulating ketone bodies may affect the bacterlcldal ability of the leukocyte m diabetic state wlth hyperglycemia, which increases the susceptiblhty to refections.

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Over two decades ago, Bybee and Perllhe found that defective mobilization of leukocytes and impaired phagocytosms were restricted only to the ketoacldotlc state [9,10] However, there have been few reports about a direct involvement of circulating ketone bodies on the leukocyte bacterlcldal function and the exact mechamsm by which ketone bodies distort the leukocyte bactericidal function remains to be established The present studies were designed to determme an influence of the existence of two kinds of well-kown ketone bodies, acetoacetate (AcAc) and 3-hydroxybutyrate (3-OHB), on the productmn of reactive oxygen intermediates (ROIs) In the leukocytes by measuring the LDCL activity after opsonlzed zymosan stimulation In addition, we measured both myeloperoxldase activity and superoxlde anion production to know the mechanism of reduced bactericidal function in detail Materials and methods Leukocytes preparation Whole blood samples were obtained from six healthy volunteers with heparlmzed syringes The leukocytes were prepared using Dextran 70 (6 % dextran, w/v, in 0 9 % NaC1) by the method previously described by Ushljlma and Nakano [11] The obtained cells consisted of 9 0 95 % polymorphonuclear leukocytes The lx106 cells were suspended in 1 ml of Hanks buffer LDCL activity measurement L D C L actlx lty of prepared leukocytes were measured by the method previously described [5,6] Briefly, 100 or 500 gl of the leukocytes-suspended solutmns were picked up into the sample tube The lx105 cells and 100 gl of Lumlnol (final concentrations 50 ~tM) were added in 1800 pA of Hanks buffer After the preincubatlon for 2 mln at 37 C, the reaction was started by the addition of 4 0 mg zymosan, dissolved into 100 ~tl Hanks buffer Chemiluminescence was measured by Luminescence Reader (Aloka, Inc, Model BLP 102, Tokyo, Japan) The lumlnoldependent chemiluminescence (LDCL) activity was assessed with lmtial slope gradient Measurement of myeloperoxldase (MPO) activity MPO activity was measured by the method previously described [12] The prepared leukocyte samples were sonlcated for 5 seconds in an ice bath The 0 2 mM phosphate buffer, 40 mM guayacol and 0 02 M Setaburon solution were added to the lxl04 cells disrupted solution The final assay volume was made up to 2 ml with Hanks buffer Following the addmon of 0 5 mM H202, the changes of absorbances after the production of tetraguayacol were measured using spectrophotometer lntracellular MPO activities were expressed as teraguayacol production ablhty (gmole/mln/106 cells) Measurement of superoxlde anion (02-) accumulation The O 7- accumulation was measured by cytochrome c [13] Briefly, the standard reaction mlxture_~Vhlch consisted of 1 0 7 leukocytes and 50 ~tM ferrlcytochrome c, was agitated in an Incubator The reducer (4 0 mg opsonlzed zymosan) was added at zero time and an ahquot (1 2 ml) was taken Immediately (zero time) Additional sampling was carried out at the times The ahquots were transferred into test tube kept in an ice and at the completion of the incubation, the tubes were cenmfuged at 700xg for 10 mln an the cold room Absorbances of the supernatant solutions at 550 nm were determined spectrophotometrlcally and the results were expressed as n moles of cytochrome c reduced using the extraction coefficient, E550=2 1 x 1 0 4 M-lcm -1 [14] In vitro experiment

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Following the preincubatlon with different concentrations (0.1, 1, or 10 mM) of AcAc or 3 OHB for 0, 10, 20, 30 minutes with normal leukocytes, the LDCL activity and 02- were measured by the method described above Statistical analysis All data are expressed as mean+S E M The statistical analysis of the means was performed by analysis of variance (ANOVA), followed by Duncan's multiple range test for the comparisons for the means Results Effects of the addmon of ketone bodies on the LDCL activity assessed with initial slope gradient were examined by using the leukocytes from six normoglycemlc, healthy volunteers. Fig. 1 demonstrated time-course change of the LDCL actlvlty in the prepared leukocytes In both AcAc and 3-OHB, the LDCL activity was suppressed m a time-dependent manner and the Incubation for 60 mm decreased the LDCL activity by 59 5+6 5 % (AcAc), 57 4+_4 6 % (3OHB) of controls, respectively Viability of the leukocytes incubated with 10 mM AcAc or 3 OHB for 60 mm was over 95 % (determined by Trypan blue staining method)

110

100

~ ; i

Q-- • AcAc O--CJ 3-OHBA

. t0 o

>

90

80 ~0

_..1

60

50 i

i

i

i

i

i

i

0

10

20

30

40

50

60

Incubat=on t=me (mm)

Fig 1 Effects of the time incubation with 10 mM acetoacetate (AcAc) and 3-hydroxybutyrate (3OHB) on the luminol-dependent chemiluminescence (LDCL) activity In the leukocytes from normal, healthy subjects The LDCL activity was expressed In relation to the value of nonketone bodies added controls * p<0 05, **. p<0 01 compared with controls in each period

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Table I Effects of acetoacetate ( A c A c ) and 3 - h y d r o x y b u t y r a t e ( 3 - O H B ) on the l u m m o l - d e p e n d e n t chemalummescence (LDCL) activity m the leukocytes from normal, healthy subjects Control

0 1 mM

1 0 mM

10 0 mM

AcAc

161+09

139+1 2*

118+1 1"

96+09*

3-OHB

161+09

129+12"

11 0+1) 8"

91+1 1"

All data were expressed as mean+S E M (x 102 KC/mln2/10 ~' cells) * p<0 01 compared with the value of control group m each treatment Table II Effects of ketone bodies on m y e l o p e r o x l d a s e (MPO) actl~ aty m the leukocytes from normal, healthy subjects MPO activity (x10 -l gmole/mm/10 ~' cells) control

4 47+0 41

A c A c (10 raM)

4 28+(I 98

3 - O H B (10 raM)

4 30+0 76

All data were expressed as mean+_S E M Table III Effect of acetoacetate (AcAc) and 3-hydroxybutyrate ( 3 - O H B ) on superoxlde anion production m the leukocytes from normal, healthy subjects Control

0 1 mM

1 0 mM

10 0 mM

AcAc

3 20+0 38

3 10+0 40

2 56+1130*

1 83+0 16"

3-OHB

3 16+0 36

2 94+0 4{)

2 68+0 32**

2 01+0 311"

All data were expressed as mean+S E M (nmole/mm/10 6 cells) *:p<0 05, ** p<0 01 compared wxth the value of control group m each treatment

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The influences of ketone bodies concentrations m the incubation mixture were presented m Table I A c A c or 3 - O H B was preIncubated in the reaction mixture for 30 rain before the LDCL assay The LDCL activity was significantly reduced by 16 % and 42 % following the addition of 1 mM and 10 mM A c A c The similar reduction of LDCL activity was found on the addition of 3 - O H B The pH of the incubation mixture was maintained within a physiological range and the pH of the mixture containing 10 mM AcAc and 3 - O H B was 7 38+0 02. 7 39+0 01, respectively (N=4) In the next, the effects of ketone bodies on MPO activity and O, production were examined The Incubation with either AcAc or 3 - O H B for 30 mln failed to dffcct the MPO activity of the leukocytes (Table II) Table III shows the effects of ketone bodies on the O f production A c A c or 3 - O H B was added to the reaction mixture for 30 rain before the measurement of the 02accumulation Both A c A c and 3 - O H B similarly inhibited the 02- accumulation from the l e u k o cytes stimulated by opsonazed zymosan in a dose-dependent manner (Table III) Discussion In the present studies, it was demonstrated that ketone bodies including A c A c and 3 - O H B inhibited the LDCL activity and Oo- accumulation in a d o s e - and time-dependent manner, but failed to suppress the leukocyte MI~O activity Over two decades ago, Bybee et al reported that decreased phagocytic function was observed m diabetic ketoacidotlc patients [9] The present finding that both LDCL and O,- production were decreased by the addition of A c A c or 3 - O H B may explain their chnacal obsefvatmn The physiological AcAc or 3 - O H B concentrations were below 0 05 mM m the serum In the pathological state such as diabetic ketoacldosls [15] or long-term starvation [16], the A c A c or 3 - O H B concentrations were increased more than 1-2 mM The present studies demonstrated that the addmon of 0 1-1 0 mM A c A c or 3 - O H B significantly decreased the LDCL activity and over the dose of i 0 mM A c A c and 3 - O H B suppressed the O~- production The A c A c or 3 OHB concentrations in the medium were compatible with pathophysiologlcal concentrations of ketone bodies In the serum It seems that the obtained results in the present studies may have pathophyslologlcal imphcations in the leukocyte functions of the patients with increased ketone production Recovery from ketotlc state should be necessary for the improvement of leukocyte oxidative metabohsm The L D C L activity appears to correlate well with antlmlcroblal activity Changes in the microbial killing were paralleled with those of chemiluminescence In pharmacologically altered leukocytes [17] Even in myeloperoxldase deficiency, the impaired antlmlcroblal activity is accompanied by reduced chemiluminescence [18] The observed reduction of the LDCL activity by ketone bodies can partially explain the decreased bactericidal activity in ketotlc patients Alterations m both NADPH oxldase and M P 0 activities involve changes of the LDCL activities m the leukocytes Since the present studies demonstrated that the addition of ketone bodies reduced only the 02- production, but not MPO activity, it is possible that the observed reduction of the LDCL activity by 3 - O H B or A c A c may be attributable to the reduced activity of NADPH oxldase N A D P H - o x l d a s e exists in plasma membrane N A D P H - o x l d a s e in plasma membrane is easier to be Inhibited by circulating ketone bodies, compared with MPO which exists in azule granules It is, therefore, supposed that the reduction of leukocyte bactericidal activity In the patients with ketoacldosis may be partially due to the reduction of O.- production through d e , creased NADPH oxldase activity However, there remain another posslblhty that ketone bodies themselves may serve as scavengers of 02- in the present cytochrome C assay system Further studies should be necessary to clarify the exact mechanism by which 02- production was d e creased by the addition of ketone bodies in the present experiments z

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The data obtained herein provided evidence that both 3-OHB and AcAc suppress neutrophi1 oxidative metabohsm with respect to O.- production Improvement from ketoacldotlc state may be indispensable for avoiding severe inaction in dmbetlc subjects References 1 R COOPAN, Infection and diabetes In Joshn's Diabetes Mehltus MARBELL, L P, R F KRALL (eds), 737-747, Lea & Feblger, Philadelphia (1985) 2 J I CASEY, Host defense and infections in diabetes mellitus In Diabetes Melhtus-Theory and Practice, 667-678, Medical Examination Pubhshlng Co, New York (1983) 3 D.M MOLENAAR, PJ PALUMBO, W R WILSON, R E J RITTS, Diabetes 25 880883 (1976) 4 C M NOLAN, N H BEATY, J D BAGDADE, Diabetes 2__7_7889-894 (1978) 5 N SATO, H SHIMIZU, K SUWA, Y UEHARA, Y SHIMOMURA, I KOBAYASHI, Exp Clan Endocranol 99 (1992) (an press) 6 N SATO, K SUWA, Y SHIMOMURA, M TAKAHASH1, H SHIM1ZU, Y UEHARA, K OHSHIMA, I KOBAYASHI, S KOBAYASHI, J Japan DIab Soc 31 585-590 (1988) (Japanese) 7 S V SHAH, J D WALLIN, S D EILEN, J Chn Endocrinol Metab 57 402-409 (1983) 8 M.B DAVIDSON, Diabetic ketoacldosis and hyperosmolar nonketotlc coma In Diabetes Melhtus-Diagnosas and Treatment, M B DAVIDSON, A WILEY (eds), 193-241, Medical Pubhcataon, John Wiley & Sons, New York (1981) 9 J D BYBEE, D E ROGERS, J Lab Clan Med 64, 1-13 (1964) 10 PE PERILLIE, J P NOLAN, S C FINCH, J Lab Chn Med __591008-1015 (1962) 11 Y USHIJIMA, M NAKANO, J. Appl Blochem 2 138-158 (1980) 12 N SATO, X WANG, M A GREER, Am J Med Scl 299309-312 (1990) 13 B M BABIOR, R S KIPNES, J Y CURNUTRE, J Chn Invest 52 741-744 (1973) 14 V MASSEY, Blochem Blophys Acta 34 255-256 (1959) 15 K SUWA, N SATO, Y SHIMOMURA, Y UEHARA, I KOBAYASH1, S KOBAYASHI, J Jap D1ab Soc 32 721-727 (1989) (Japanese) 16 H SHIMIZU, M MIYAZAKI, Y SHIMOMURA, I KOBAYASHI, J Medicine 22 201210 (1991) 17 T D HORAN, D ENGLISH, T A McPHERSON, Chn Immunol Immunopathol 2 2 2 5 9 269 (1982) 18 H ROSEN, S J. KLEBANOFF, J Chn Invest 58 50-60 (1976)