reduction of methemoglobin by tetrahydropterin and glutathione

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Page 1: Reduction of methemoglobin by tetrahydropterin and glutathione

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 179, 456-461 (1977)

Reduction of Methemoglobin by Tetrahydropterin and Glutathione

DORIS TAYLOR AND PAUL HOCHSTEIN

Department ofPharmacology, University of Southern California School of Medicine, 2025 Zonal Avenue, Los Angeles, California 90033

Received June 9, 1976

The reduced pteridine 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine nonenzymatically reduces methemoglobin in solution and in intact erythrocytes. The extent of the reaction in whole cells is markedly increased in the presence of glucose. The stimulating effect of glucose is absent in erythrocytes from individuals deficient in glucose 6-phosphate dehydrogenase. Glucose functions by maintaining levels of reduced glutathione which, in turn, reduce the dihydropterin to the active tetrahydro form. Although it appears unlikely that this mechanism could contribute in more than a minor way to the maintenance of ferrous hemoglobin in vivo, the results suggest that the interaction of glutathione with pterins might be of consequence in the regulation of pterin-dependent pathways in other tissues.

Tetrahydrobiopterin is an unconjugated 2-amino-4-hydroxypteridine (pterin) found in mammalian tissue which serves as a cofactor in the hydroxylation of phenylala- nine (11, tyrosine (2,3), and tryptophan (4, 5). Analogs of tetrahydrobiopterin, such as the 6-methyl and 6,7-dimethyl tetrahy- dropterins, can substitute for the natu- rally occurring compound in these reac- tions (6). During oxidation, tetrahydrop- terms are first converted to the quininoid dihydropterins (7), which are reducible by such agents as NADPH and mercaptoetha- no1 (8) and by a reduced pyridine nucleo- tide-dependent dihydropterin reductase (7). Any quininoid dihydropterin which es- capes reduction isomerizes spontaneously to the 7,8-dihydropterin. Dihydrofolate re- ductase is able to convert the naturally occurring 7,8-dihydrobiopterin to the te- trahydro form, but is not able to do so with the 6,7-dimethyl-7,8-dihydropterin (9). By these mechanisms, pterins are able to cy- cle between their tetrahydro and dihydro forms, donating two electrons with each cycle. Tetrahydropterins oxidize sponta- neously in the presence of oxygen, produc- ing hydrogen peroxide and, under certain conditions, superoxide anion (10).

We have previously shown that the abil- ity of 2-amino-4-hydroxy-6,7-dimethyl-

5,6,7,8-tetrahydropteridine (DMPH,)’ to reduce cytochrome c results in ATP forma- tion over site 3 of mitochondria (11). The ability of cytochrome c to oxidize the te- trahydropterin leads to decreased phenyl- alanine hydroxylation in vitro in the pres- ence of mitochondria (unpublished work). During these investigations on the inter- action of pterins with cellular constitu- ents, we have found that DMPH, is able to reduce methemoglobin in cell lysates and intact red cells and that this reaction is enhanced in whole cells by the addition of glucose in the medium. A preliminary re- port of these findings has appeared (12).

MATERIALS AND METHODS

Hemoglobin in erythrocytes from normal adult volunteers was converted to methemoglobin (MI-RI) by treating fresh, heparinized blood with NaNO, at a final concentration of 1%. After standing for 15 min on ice, the blood was diluted with 4 vol of 0.9% NaCl and centrifuged at 400g for 5 min. The diluted plasma and buffy coat were removed and the red cells were washed four times in 8 vol of 0.9% NaCl. Packed, washed cells were diluted with Krebs- Ringer phosphate buffer, pH 7.4, and kept on ice.

* Abbreviations used: DMPH,, 2-amino-l-hy- droxy - 6,7 - dimethyl - 5,6,7., 8 - tetrahydropteridine; MHb, methemoglobin; GSH, glutathione; DTE, di- thioerythritoh G-8PD, glucose 6-phosphate dehy- drogenase; NEM, N-ethylmaleimide.

456

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0003-9861

Page 2: Reduction of methemoglobin by tetrahydropterin and glutathione

TETRAHYDROPTERIN AND METHEMOGLOBIN 457

This buffer was used in all experiments unless oth- anaerobic with nitrogen. It was neutralized with erwise indicated. KOH and kept on ice, under nitrogen. The concen-

Whole cell experiments were carried out by incu- tration of DMPH, was determined by its extinction bating the MI-&containing cells at a final hemato- at 265 nm (18). crit of 30% in buffer. Other additions are noted in individual experiments. The reaction was started RESULTS

with DMPH, and incubated at 37°C without shak- ing. The absorbance at 630 nm was recorded imme-

When DMPH, was added to a solution of

diately after mixing an aliquot of the incubation purified bovine MHb, a rapid decrease in

mixture in 19 vol of 1 mM potassium phosphate absorbance at 630 nm was seen (Fig. 1,

buffer, pH 6.0, containing 0.1% Triton X-100. curve B), indicating the conversion of fer-

Cell lysates were prepared by freezing and thaw- ric hemoglobin (Ml%) to the ferrous form. ing the washed, MHb-containing cells three times About i min after the addition of DMPH4, and diluting with buffer. reoxidation of the hemoglobin began to oc-

Bovine hemoglobin (Sigma Chemical Co., 2x cur. Inclusion of superoxide dismutase did crystallized) was found to be greater than 90% in the not prevent this reoxidation (curve 0, but ferric form by its absorbance in solution at 630 nm and was used without further oxidation.

catalase did (curve D), indicating, that

Experiments involving lysates or bovine MHb H,O, was the species involved in the oxida-

were carried out at 23°C in a recording spectropho- tion of hemoglobin, not the superoxide

tometer. Reaction components, excluding DMPH,, radical.

were mixed in the cuvette and allowed to equili- If intact human erythrocytes in which

brate. The reaction was started by mixing in the hemoglobin had been converted to MHb DMPH, and was followed at 630 nm. The initial were incubated with DMPHI, the MHb rates of reduction were linear with DMPH, concen- was similarly reduced (Fig. 2, curve B). tration and the reaction was saturated with respect Reoxidation did not begin as soon after to MHb. addition of DMPH, as in the MHb solu-

The amount of MHb reduction in each experi- ment was calculated from the decrease in absorb-

tion, nor was it as rapid once it did begin,

ance at 630 nm. The extinction coefficient for MHb presumably because catalase was present

was determined by oxidizing a solution of human in the cells. Maximal reduction was seen

hemoglobin (Hemoglobin Standard, Sigma Chemi- at about 30 min after addition of DMPH,.

cal Co.) with an excess of potassium ferricyanide The amount of MHb reduced was about (13) and measuring the absorbance at 630 nm. In the half that expected on the basis of 2 mol of pH 6.0 buffer system described above, the extinction heme reduced per mole of DMPH,. Inclu- coefficient was found to be 4.2 mM-‘, and this value was used for the whole cell experiments. A value of 0 J

4.7 rnM-’ was found for MHb in Krebs-Ringer phos- t

A

phate buffer, pH 7.4, and was used to calculate the amount of MHb reduced in the lysate experiments and experiments carried out with the recrystallized MHb.

Superoxide dismutase was prepared from bovine erythrocytes by the method of McCord and Fridovich (14).

Cells containing MHb were depleted of GSH by

B -.l

C

the method of Beutler et al. (15). Equal volumes of packed, MHb-containing cells and 2 mM N-ethyl- -A 01234561 maleimide were mixed and allowed to stand at room MINUTES

temperature for 20 min. The cells were washed four FIG. 1. Reduction of purified bovine methemo- times with 4 vol of buffer. As a control, cells were globin by DMPH,. The l.O-ml reaction mixture con- carried through the procedure in the absence of N- tained 3.2 mg of bovine methemoglobin and 1 pmol ethylmaleimide. Determination of GSH was carried of DMPH, in Krebs-Ringer phosphate buffer at pH out according to Beutler (16). 7.4. The reaction was carried out at 37°C. The arrow

The glucose 6-phosphate dehydrogenase content indicates the addition of DMPH+ Curve A, no of red cells was assayed according to the method of DMPHI; B, DMPH, only; C, 60 pg of superoxide Glock and McLean (17). dismutase added before DMPH,; D, 300 units of cat-

DMPH, (hydrochloride form, Aldrich Chemical alase added before DMPH,. Total reduction of MHb Co.) was prepared fresh each day in buffer made would result in a A A,,, of 0.94.

Page 3: Reduction of methemoglobin by tetrahydropterin and glutathione

TAYLOR AND

“t I 0 30 60 PO vm

IYC”~lTlO” Till ,.l”“III~ FIG. 2. Reduction of methemoglobin in intact

erythrocytes. Hemoglobin was oxidized and incuba- tions were carried out as described under Materials and Methods. The final reaction volume was 2.0 ml. Curve A, 5.5 mM glucose; B, 1 mM DMPH,; C, 1 mM DMPH, and 5.5 mM glucose; D, 5.5 mM glucose and 0.1 mM methylene blue; E, 1 mM DMPH, and 5 mM DTE. Results are the averages of three experiments. The maximum reduction found with DMPH, and DTE (curve E) represents more than a 90% reduc- tion of MHb present.

sion of glucose in the medium resulted in a threefold stimulation in the conversion of MHb to hemoglobin by DMPH, (curve C). About 50% more MHb was reduced in 2 h as that expected on a stoichiometric basis, leading to the conclusion that glucose was causing the pterin to cycle between its oxi- dized and reduced forms. Reduction of MHb by methylene blue in the presence of glucose (curve D) is shown for comparison. The dye reduces MHb and is in turn re- duced by NADPH (19). Dithioerythritol (DTE) can, like its isomer, dithiothreitol, reduce quininoid dihydropterin directly, and it caused a very rapid reduction of MHb by DMPH, (curve E). Curve A shows the effect of glucose alone on MHb reduc- tion. Lack of any reduction of MHb in the presence of glucose alone may indicate that small amounts of sodium nitrite re- mained in the cells. DTE or methylene blue alone did not affect MHb reduction, but these curves have been omitted from the graph for clarity.

Table I shows the effectiveness of var- ious concentrations of DMPH, in reducing MHb in whole cells. Effects could be seen down to 10e6 M when either DTE or glucose was used to regenerate the DMPH,. The concentration of DMPH, effective in this

HOCHSTEIN

system is in the range of concentration of tetrahydropterins in mammalian tissues. For example, concentrations of 0.2 x 10V6 M have been found for 6-hydroxyalkyl pterins in human blood (20), and 1.5 x 1O-5 M has been found in a high-speed superna- tant fraction of rat liver (21). In human liver, tetrahydrobiopterin levels are about 6 X lo-‘j M (22).

In an effort to determine how glucose was exerting its enhancing effect on the reduction of MHb by DMPHI, red blood cells from two individuals deficient in glu- cose 6-phosphate dehydrogenase (G-8PD) were examined. The patients’ cells had less than 10% of the normal G-6-PD activ- ity. The red cells from both patients showed almost no enhancement by glucose of the DMPH,-dependent MHb reduction (Table II). Unlike the other experiments reported in this paper, these studies were carried out with shaking during the incu- bation period. Results for shaking and nonshaking experiments were found to be qualitatively similar, but the amount of DMPH,-dependent MHb reduction was al- ways less when the reaction mixtures were agitated during incubation because of the increased autoxidation of DMPH,.

Because erythrocytes from the individ- uals deficient in G-6-PD did not show a glucose enhancement of DMPH,-depend- ent MHb reduction, it can be concluded

TABLE I

REDUCTION OF MHb BY DMPH, AND GLUCOSE OR

DMPH, AND DTEQ

DMPH, concen- A MHb reduction (pmol) tration (M)

Glucose DTE

0 0.10 t 0.18 0.02 ? 0.21 10-S 3.9 ” 0.3 5.7 2 0.5 10-4 2.4 t 0.3 3.9 k 0.3 10-S 1.0 t 0.2 1.2 2 0.2 10-G 1.0 2 0.4 0.48 k 0.21

a Hemoglobin was oxidized and 30-min incuba- tions were carried out using whole cells, as de- scribed under Materials and Methods. The final con- centration of glucose was 5.5 mM, DTE was present at 5.0 mM, and the concentration of DMPH, was varied as indicated. Results are given as the in- crease in micromoles of MHb reduced in the 2-ml incubation mixture after 30 min over controls con- taining only cells, buffer, and DMPH,.

Page 4: Reduction of methemoglobin by tetrahydropterin and glutathione

TETRAHYDROPTERIN AND METHEMOGLOBIN 459

TABLE II

REDUCTION OF METHEMOGLOBIN IN RED CELLS DEFICIENT IN GLUCOSE 6-PHOSPHATE

DEHYDROGENASE~

Additions to Micromoles of MHb reduced in 60 incubation min

Experiment 1 Experiment 2

Pa- Normal Pa- Normal tient 1 control tient 2 control

DMPH, 0.037 0.010 0.36 0.69 DMPH, + glu- 0.070 0.56 0.43 1.1

case DMPH, + DTE 2.2 2.0 2.0 1.9 Methylene 0.33 1.7 - -

blue + glu- cose

R Hemoglobin was oxidized and incubations and measurement of MHb reduction in whole cells were carried out as described under Materials and Meth- ods. The hematocrit of the incubation mixtures was 18% and the tubes were shaken during the 60-min incubation period. DMPH, concentration was 1 mM, glucose was 5.5 mM, DTE was 5 mM, and methylene blue was 0.1 mM.

that the basis of the glucose effect resides in the pentose phosphate pathway. The most likely possibilities include (1) an en- zymatically linked reduction of dihydrop- term by NADPH similar to the well- known NADPH-methylene blue reductase or (2) a nonenzymatic reduction of dihy- dropterin by GSH or NADPH. Intensive studies have failed to verify an enzymatic basis for the glucose effect. NADH, NADPH, GSH, and DTE were all able to increase the reduction of MHb by DMPH, in bovine MHb solutions and in human MHb-containing cell lysates. Table III shows the difference in effectiveness of these compounds in cell lysates. The con- centration of total triphosphopyridine nu- cleotide in red cells has been shown to be 5-17 PM (23), but NADPH at 10 PM was ineffective in our system. Although not shown in Table III, the inclusion of gener- ating systems for the reduced pyridine nu- cleotides did not enhance MHb reduction. It can be seen in Table III that GSH is an excellent reducing agent for dihydropterin even below levels of GSH normally found in mammalian tissues 11-5 mM, Ref. (2411.

Table IV lends support to the postulate that GSH is important to the enhancing

TABLE III

METHEMOGLOBIN REDUCTION BY DMPH, IN THE

PRESENCE OF VARIOUS REDUCING AGENTS~

Reducing Concentra- Increase in MHb re- agent tion (mbr) duced (nmol)

Lysate Purified MHb

DTE 5 8.7 2 8.7

GSH 5 42 2 31 31* 0.5 20 0.2 2.1* 8.76

NADH 1.0 11 0.1 5.5 0.01 0

NADPH 1.0 10 0.1 4.3 0.01 0

’ Each l.O-ml reaction mixture contained 6 mg of purified bovine MHb or the equivalent of 20 ~1 of MHb-containing packed red cells as cell lysate (see Materials and Methods). Reducing agent and lysate or MHb solution were mixed and the reaction was started with DMPH, (0.5 mM, final concentration). The reaction was carried out for 8 min at 25” in Krebs-Ringer phosphate buffer, pH 7.4. Results are given as the increase in nanomoles of MHb reduced compared to the control containing lysate or MHb and DMPH,. Results are the averages of duplicates or triplicates.

b Reaction mixture contained 300 units of cata- lase.

effect of glucose. N-Ethylmaleimide se- questers much of the GSH in the cell, and little GSH reappears during the 30-min incubation period. Reduction of MHb by DMPH, alone or with glucose was less in GSH-depleted cells than in cells with nor- mal GSH levels. Presumably, even the low level of GSH that is present in NEM- treated cells is continually regenerated when glucose is included as substrate, thus allowing some stimulation of MHb reduction. This explanation accounts for the minimal MHb reduction by DMPH, alone and the significant amount of activ- ity seen in the presence of DMPH, and glucose in NEM-treated cells. The reason for the slight increase in the amount of MHb present when glucose alone was in-

Page 5: Reduction of methemoglobin by tetrahydropterin and glutathione

460 TAYLOR AND HOCHSTEIN

TABLE IV

REDUCTION OF METHEMOGLOBIN IN RED CELLS DEPLETED OF REDUCED GLUTATHIONE~

Incuba- Additions GSH content (mM) Micromoles of MHb reduced tion

Control cells NEM-treated Control cells NEM-treated

- None 2.1 0.14 -

+ Glucose 2.2 0.20 -0.27 -0.30 + DMPH, 0.37 0.10 1.4 0.69 + Glucose + DMPH, 1.5 0.15 2.8 1.8

o All procedures are described in detail under Materials and Methods. Hemoglobin was oxidized in intact cells and glutathione was alkylated with NEM. GSH and MHb levels in the NEM-treated and control cells were measured before and after a 30-min incubation with the designated additions. Glucose concentration was 5.5 mM, and DMPH, was 1.0 mM. Results are averages of duplicate determinations.

eluded in the medium (Table IV) is not clear.

The levels of GSH in the depleted cells were probably high enough to account for some reduction of MHb even in the ab- sence of glucose, as illustrated by the fact that reduction of lysate MHb by DMPH, was increased in the presence of 0.2 M GSH (Table III). It is also clear from Table III that the GSH-DMPH2 interaction is non- enzymatic, since GSH-dependent stimula- tion of MHb reduction was similar for hu- man cell lysates and the recrystallized bo- vine MHb. The low level of GSH found in control cells or NEM-treated cells in the presence of DMPH, alone may be due -m oxidation by dihydropterin or utilization by glutathione reductase for the detoxifi- cation of hydrogen peroxide (25) produced by autoxidation of DMPH+ The lack of glucose would prevent regeneration of the GSH. These results implicate GSH in the glucose stimulation of MHb reduction by DMPHI, but do not rule out the participa- tion of other, as yet unidentified, meta- bolic processes.

DISCUSSION

Until recently, the only known effect of tetrahydropterins on cellular functions was that they acted as cofactors for aro- matic amino acid hydroxylases. We are reporting in this paper another interaction of a tetrahydropterin with a cellular com- ponent, methemoglobin. This DMPH,-de- pendent reduction of MHb is increased in the presence of glucose, and the effect of glucose seems to depend on its ability to generate reduced glutathione. This con-

cept is supported by the fact that red cells from individuals deficient in G-8PD, which have a diminished capacity to gen- erate reduced glutathione, have only a marginal response to glucose. Since GSH will reduce dihydropterin directly and DMPH, can reduce MHb without enzy- matic involvement, the system described does not require any metabolic component other than per&se phosphate shunt activ- ity.

It is not clear from our studies whether or not tetrahydropterins reduce MI-lb in the intact animal. In view of the fact that there are several methemoglobin reduc- tases, but that only one of them is of clini- cal significance in humans (261, it is doubt- ful that the system we describe could con- tribute in more than a very minor way to maintenance of ferrous hemoglobin levels in uiuo. In addition, levels of 6-hydroxy- alkylpterins are low in uiuo, i.e., 0.2 x lo-” M in human whole blood (20).

These results raise the question of the physiological significance of GSH reduc- tion of dihydropterins. Although the te- trahydropterin used in our experiments is a synthetic analog of the naturally occur- ring mammalian compound,’ their biologi- cal characteristics are qualitatively simi- lar in many respects, including suscepti- bility to nonenzymatic reduction by endog- enous agents (6). Kaufman et al. (22) have suggested that endogenous reducing com- pounds such as GSH and ascorbate may affect the activity of the aromatic amino acid hydroxylase in uiao. Our experiments show that, at levels of GSH found in mam- malian tissues, i.e., l-5 mM, dihydropterin reduction can be affected significantly.

Page 6: Reduction of methemoglobin by tetrahydropterin and glutathione

TETRAHYDROPTERIN AND METHEMOGLOBIN 461

ACKNOWLEDGMENT 13. ANTONINI, E., AND BRUNORI, M. (1971) in Hemo- This work was supported in part by Research globin and Myoglobin in Their Reactions with

Grant No. HD08159 from the National Institutes of Ligands (Antonini, E., and Brunori, M., Health. eds.), pp. 41-42, American Elsevier, New

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