Oxidation-reduction reactions involving
ascorbic acid and the
hexosemonophosphate shunt in
corneal epithelium
E. I. Anderson and Abraham Spector
A dehydroascorbic acid reductase and a glutathione peroxidase have been shown to be
present in corneal epithelium. Ascorbic acid (ASC), dehydroascorbate (DHA), or hydrogen
peroxide (HtOt) can be coupled to the glutathione reductase system to effect nicotinamide
adenine dinucleotide phosphate (NADPH) oxidation. A coupling of the hexosemonophosphate (HMP) shunt via the pyridine nucleotide, NADPH, to DHA has been demonstrated.
The suggestion is made that the rate of ASC oxidation in vivo is probably adequate to
reoxidize a significant fraction of the NADPH generated by the shunt. ASC, glutathione
(GSH), HsOi, and total soluble thiol concentrations of corneal epithelium were determined.
At levels of HtOt within the observed physiological limit, endogenous catalase is inactive.
Key words: dehydroascorbic acid reductase, glutathione peroxidase, oxidation-reduction,
corneal epithelium, ascorbic acid, glutathione, hexosemonophosphate shunt.
_1_ he participation of ascorbic acid (ASC)
and glutathione (GSH) as hydrogen carriers in a mammalian electron transport system involving substrates oxidizable by nicotinamide adenine dinucleotide (NADP)
has been a recurrring speculation.1'L>! 3
Such a system, diagramed in Fig. 1,
could be linked to the hexosemonophosphate (HMP) shunt- by the NADP-
specific dehydrogenases of glucose-6phosphate and 6-phosphogluconic acid
(G-6-P and 6-PG). In plant tissue, the
transfers of hydrogen from ASC to molecular oxygen, from GSH to dehydroascorbic
acid (DHA), and from reduced NADP
(NADPH) to oxidized GSH (GSSG) are
enzymically mediated.'1 Of the three requisite enzymes, only glutathione reductase
is widely distributed in animal tissues.
As the oxidation of ASC and the reduction of DHA by GSH can occur spontaneously, some difficulties attend the
demonstration of the respective catalytic
activities. Ascorbic acid oxidase is reportedly not present in animal tissue5 and the
existance of a dehydroascorbic acid reductase6' 7 has not been previously confirmed.
An alternate scheme shown in Fig. 1
From the Department of Ophthalmology, College
of Physicians and Surgeons, Columbia University, New York, N. Y.
This work was supported by grants from the
National Eye Institute of the Department of
Health, Education and Welfare, United States
Public Health Service.
Manuscript submitted Nov. 9, 1970; manuscript
accepted Nov. 30, 1970.
41
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42 Anderson and Spector
Investigative Ophthalmology
January 1971
DHA
G-6-P
NADP
GSH
GSSG
6-PG
REDUCTASE
NADPH
GSSG
R-5-P
Fig. 1. Schematic representation of pathways by which ASC oxidation products may be
coupled to HMP intermediates.
associates ASC with the HMP shunt 3 ' s
through the formation of H2O2. Enzymatic
oxidation of ASC does not yield detectable
H2Oo9; nonenzymatic oxidation catalyzed
by heavy metal ions9-10> n or riboflavin12
in the presence of ultraviolet light will do
so. Although H2O2 may arise during the
oxidation of other tissue metabolites, ASC
could be a significant contributor to the
H2O2 pool in ocular tissues such as cornea
and lens which are normally exposed to
ultraviolet light and possess relatively high
ASC levels.13 In the presence of low levels
of H2Oo which are anticipated to prevail
under steady-state conditions, and in the
absence of other oxidants, the sluggish
oxidation of GSH14 would be potentiated
in a tissue possessing glutathione peroxidase. Coupling the GSSG generated by
either oxidation product of ASC to the
reoxidation of NADPH by glutathione reductase would provide a supply of NADP
which is critical to maintenance of the
HMP pathway.
For a tissue such as the corneal epithelium with a capacity to metabolize glu-
cose via the HMP shunt to the extent of
an estimated 35 per cent in bovines15 and
70 per cent in rabbits,10 the reoxidation of
NADPH is of fundamental importance. In
the present study, the existence in corneal
epithelium of the pertinent enzymes glutathione peroxidase and dehydroascorbie
acid reductase has been shown clearly.
Furthermore, a direct demonstration of the
capacity of this tissue to couple a product
of ASC oxidation to the oxidation of an
HMP shunt intermediate has been accomplished with in vitro techniques.
Methods and materials
Freshly collected calf corneal epithelium was
ground in a chilled mortar with a small amount
of sand and extracted with cold 0.05M phosphate
buffer (pH 6.8, 1:2, w/v). The supernatant,
subsequent to centrifuging for 15 min. at 27,600
xg, dialysis with agitation at 5° C. for 19 to 22
hr. against 200 Vol. of buffer, and recentrifugation, served as extract in the enzymatic assays.
The protein concentration of the dialyzed soluble
fraction was estimated17 and amounted to 7.4
per cent of the wet epithelial weight; wet
epithelial weight averaged 48.7 rhg. per cornea.
ASC was determined spectrophotometrically ac-
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Volume 10
Number 1
cording to the method of Roe18 on protein-free
2 or 3 per cent metaphosphoric acid (MPA) extracts adjusted to pH 3.5. A correction factor for
dye reduction in the presence of GSH was determined by assaying each of three concentrations
of ASC (0.019, 0.038, and 0.076 /tmoles ml.-1)
in the presence of GSH at 0.5, 1, and 2 times
the respective levels of ASC. Recoveries of ASC
from epithelial extracts ranged between 96.3
to 101.4 per cent with an average of 98.9 per
cent of theoretical.
GSH was determined either on pH 6.8 extracts directly according to the method of Jocelyn19 for nonprotein thiol or on neutralized 3 per
cent MPA extracts according to the modification
of Beutler and associates.20 Recovery of GSH
added at 0.5, 1 or 2 times the endogenous level
to extracts at pH 2.5 or pH 6.8, averaged 100.8
and 99.1 per cent of theoretical, respectively.
Total thiol was determined by the method of
Jocelyn.19
H-jOij was determined according to the experimental design of Mapson21 as employed by
Pirie.3 The HzO* concentration was estimated
using a E*/o for 2,6-dichlorophenolindophenol of
21,000.22 An interval of about 1 hr. was required
for sample preparation.
Glutathione reductase activity was determined
spectrophotometrically.23 The presence of ethylenediaminetetraacetic acid (EDTA) was found
necessary to achieve full activation. Activity of
glutathione peroxidase was measured essentially
according to the assays of Paglia and Valentine21
and Hochstein and Utley25 which couple glutathione peroxidase activity to glutathione reductase
activity. Separate controls for the following mixtures were included: GSH and NADPH, corneal
extract, and NADPH in the presence and absence
of either H2O2 or GSH. Various samples of GSH
contained 2 to 5 per cent GSSG and this was
converted to GSH in situ before introduction of
H2Oi. Nonenzymatic oxidation of GSH by H2O2
was measured under identical conditions with
exogenous glutathione reductase.
Dehydroascorbate reductase activity and the
oxidation of ASC in the presence of GSH were
determined with the same protocol used for the
assay of glutathione peroxidase with the exception that DHA or ASC replaced H2O2. Comparable
controls were included and nonenzymatic oxidation
of GSH was similarly measured with exogenous
glutathione reductase.
Catalase activity was determined before and
after dialysis of extracts by Feinstein's method.20
All spectrophotometric assays were made in a
final volume of 3.0 ml. in quartz cells of 1 cm.
light path in a model DU Beckman spectrophotometer at the temperature prevailing in the cell
compartment. Absorption readings of enzymatic
Oxidation-reduction reactions 43
activities were taken every 15 sec. and reaction
rates were based on the initial maximal rates;
spontaneous interactions were monitored every
30 sec. and reaction rates calculated from the
average linear rate.
Unless specified otherwise, reference to buffer
in the text, tables, and figures indicates phosphate
bufFer prepared from Na2HPO< and KH2PO4 with
a pH of 6.8. When used, EDTA was incorporated
with the bufFer as a preneutralized solution.
Preparations of GSH, CSSG, G-6-P, 6-PG, NADH,
NADPH, NADP, and horseradish peroxidase
were supplied by Sigma Chemical Co. St. Louis,
Mo. Preparations of DHA (K & K Labs., Inc.,
Plainview, N. Y.) were estimated from a Ei^ of
15,2OO27 for ASC to contain 3.4 per cent reduced form. Solutions of potassium cyanide and
sodium azide, as well as the organic substrates
and coenzymes, were adjusted to pH 6.8 immediately before assay. NADPH was 96 per cent
pure and had a E*r0 of 6160. NADP was 98 per
cent pure and a solution of 6 mg. ml.- 1 had an
optical density (O. D.) at 340 m/t of 0.434 at
pH 7.0. Glutathione reductase (Mann Research
Laboratories, New York, N. Y. or Sigma Chemical
Co.), obtained as an (NHi)2SC\i suspension, was
dialyzed overnight against 0.05M buffer.
Results
Steady-state levels of ASC, GSH, and
H2O2 in the current source material were
determined as an aid in the selection of
experimental substrate levels. The possibility that reduction of DHA may be effected by protein thiol groups28' -°
prompted estimation of the latter component in the soluble extracts. Table I summarizes the results and includes, for comparative purposes, values for ASC and
GSH taken from the literature. The concentrations of ASC and GSH are nearly
equimolar and average, gr 1 of wet weight
of corneal epithelium, 3.82 and 3.93 /xmoles,
respectively, when MPA extracts were used
for assay. The GSH value determined as
nonprotein thiol directly on pH 6.8 extracts, however, averaged approximately
80 per cent higher than that reported with
the use of MPA extracts. Thiol groups of
proteins not removed from the initial pH
6.8 extracts when acid precipitation was
omitted most probably contributed to the
higher value. Soluble protein thiol groups
were estimated, therefore, as the difference
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44 Anderson and Spector
Investigative
Ophthalmology
January 1971
Table I. Steady-state levels of ASC, GSH, H2O2, and thiol groups in bovine
corneal epithelium
Substance
assayed
ASC
CSH
H2OS
Sample No."
1
2
Foundf
3.79
3.84
3
4
71f
81f
Concentration
(fimoles gmr1 wet weight)
Reported range
Average
3.82
2.67 to 5.35|
3.93
2.54 to 4.88§
4.41 to 5.79||
3.81
4.05
0.22
0.31
0.26
Total thiol
3
5
6
13.64
11.75
15.54
13.64
"For each sample, 10 epithelia were pooled with the following exceptions: No. 4, 13; No. 7 and No. 8, 5.
fEach value is an average of three determinations agreeing with ± 1 . 0 per cent except for H2O2, for which the first two
determinations of five performed sequentially are averaged and agree with ± 8.0 per cent.
t Calculated from data of Pirie. 30
{Calculated from data of Herrmann and Moses31 obtained by assay with nitroprusside.
II Calculated from data of Herrmann and Moses31 obtained by assay with glyoxylase.
^Samples prepared in subdued light as a precautionary measure to minimize photoxidation of ASC and GSH.
between total thiol and the GSH value
determined with the use of MPA extracts
and averaged 9.71 /xmoles gr 1 wet weight.
Because of its relatively low level and the
seeming tendency to rise in the extracts
with time, the absolute concentration of
H2O2 may be more critically dependent
than the other metabolites on the rapidity
with which the tissues are processed and
assayed. The extracts were assayed within
15 min. after preparation. Over the next
45 min., the H2O2 values increased 1.5
times. The reported 15 min. values must
therefore be considered maximum levels.
As all spectrophotometric assays involving the oxidation of GSH were dependent
on the reduction of GSSG by glutathione
reductase and the fact that this enzyme is
endogenous to corneal epithelium,2 the
activity in the dialyzed extracts was assessed. GSSG, 0.0015M, was reduced at the
rate of 0.03 //.moles min."1 mgr 1 protein at
pH 6.8 and 25° C. No activity was observed if NADH was substituted for
NADPH.
In Fig. 2 the spontaneous (Curve I)
and extract-catalyzed (Curve II) oxidations of GSH in the presence of an equimolar amount of ASC are presented. In
this and subsequent figures, the initial fall
in extinction after the addition of GSH
represents the reduction of contaminating
or spontaneously formed GSSG in solution.
The steeper slope for this portion of Curve
I, the control, as compared to Curve II,
offers visual proof that the level of glutathione reductase in the control exceeded
that in the extract and did not limit the
spontaneous reaction. Addition of ASC resulted in observed rates of spontaneous
and extract-catalyzed NADP formation
min.-1 of 0.0015 and 0.0093 /unoles, respectively. From these values, the corrected
rate of NADP formation due to the presence of soluble extract was 0.002 //.moles
minr 1 mg."1 of protein. The total amount
of NADP formed in the presence of extract
corresponded to 99.2 per cent of the
NADPH available. The final absorption of
0.080 O.D. units in Curve II is due to the
extract. Essentially no NADP formation
was observed in controls containing either
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Volume 10
Number 1
Oxidation-reduction reactions 45
MINUTES
Fig. 2. Spontaneous and tissue-catalyzed oxidation of GSH in the presence of ASC. The
assay system contained the following final concentrations: buffer, 0.056M; EDTA, 0.0026M;
KCN, 0.01M; GSH and ASC, each 0.0025M; NADPH, 7.8 x 10-5M. Water was added to a
final volume of 3.0 ml. Curve I, the control, was prepared with 10 fig of glutathione reductase
having a reducing capacity of 1 //mole of GSSG min.-1 at pH 7.6 and 25° C. Curve II was
prepared with 3.79 mg. of soluble epithelial protein. For ease of visual comparison the curves
are shown with a common origin. Curve I actually had an initial O.D. 0.08 units lower.
Results are from one of two experiments performed.
glutathione reductase or extract in combination with NADPH alone or together
with either GSH or ASC.
Oxidation of GSH by DHA is shown in
Fig. 3. In the absence of extract the amount
of NADP formed min."1 as a result of the
reduction of spontaneously formed GSSG
(Curve I) was 0.039 ^moles, whereas in
the presence of extract (Curve II), the
observed rate was raised to 0.075 //.moles.
Supplementing the extract with an amount
of exogenous glutathione reductase in excess of that which would give maximal
activity under the prevailing assay conditions (Curve III) increased the NADP
formed min."1 to 0.252 /xmoles. When corrected for nonenzymatic oxidation of GSH,
the apparent and optimal activities of
DHA reductase under the experimental
conditions were, expressed as NADP
formed min."1 mg."1 of protein, 0.012 and
0.072 /mioles, respectively. No oxidation of
NADPH occurred in the presence of DHA
with either extract or glutathione reductase.
Note that when 99.S per cent of the
available NADPH was consumed, the
curves in Fig. 3 show a slow, steady reversal. This was not observed when ASC
replaced DHA (Fig. 2). DHA is reportedly
transparent,27'32 but in the presence of
ASC undergoing oxidation, peaks of absorption have been observed by Herbert
and colleagues32 to appear at 290 and 340
mix. The phenomenon was investigated by
monitoring the absorption of solutions of
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Investigative Ophthalmology
January 1971
46 Anderson and Spector
M I NUTES
Fig. 3. Oxidation of GSH by DHA in the presence and absence of coineal epithelial extract.
The following final concentrations pertain: buffer, 0.058M; EDTA, 0.003M; GSH, 0.0025M;
DHA, 0.00125M; NADPH, 1.45 x KHM. A final volume of 3.0 ml. was made with water.
Curve I and Curve III were prepared with 16.5 /*g of glutathione reductase having a reducing
capacity of 1.65 jumoles GSSG min.-1 at pH 7.6 and 25° C. Curve II and also Curve III were
prepared with 2.94 mg. of epithelial protein. The results are typical of three experiments performed.
DHA and ASC in the presence and absence of EDTA. Transitions in the absorption profile of freshly solubilized DHA
were detectable almost immediately. Similar results were found with or without
EDTA. The peak at 265 m/x in Fig. 4
(Curve I) indicates the presence of 3.4
per cent contaminating ASC. Within 3 hr.
(Curve II) this absorption is broadened,
flattened, and shifted toward higher wavelenghts. The trend continues with time
(Curve III), producing at 48 hr. (Curve
IV) a continuous absorption with weak
bands at 276, 293, and around 360 m/*. No
detectable new absorption bands accompanied the spontaneous oxidation of comparable amounts of ASC, although disappearance of the characteristic peak at 265
in/!, was observed. One can conclude, therefore, that the observed progressive increases in absorption with reaction mixtures containing DHA in the presence of
of low levels of ASC are attributable to the
formation of undefined molecular species.
The measurement of ASC formed, although less sensitive, is another method
to assay the oxidation of GSH by DHA
and is reported in Table II. The ASC produced within 1 and 5 min. of spontaneous
interaction are, respectively, 108 and 97
per cent of the maximal amounts calculated from the rate of GSSG formation observed in Fig. 3. In the succeeding 10,
20, and 30 min. periods of reaction, the
amount of ASC formed falls progressively
to 89, 78, and 45 per cent of the calculated
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Volume 10
Number 1
Oxidation-reduction reactions 47
240
360
WAV
EL E N G T H ,
400
MJJ
Fig. 4. Absorption characteristics of a DHA solution with time. At the following times after
dissolution of DHA (0.001GM) in buffer (0.05M) the curves were prepared: Curve I,
immediately; Curve II, 3 hr.; Curve III, 22 hr.; Curve IV, 48 hr.
levels. In the presence of extract, the
quantities of ASC formed within 1, 5, and
10 min. intervals correspond to 69, 57, and
45 per cent of those estimated from GSSG
rates.
The functionality of the scheme shown
in Fig. 1 can be demonstrated spectrophotometrically with 6-PG as hydrogen donor,
NADPH as pyridine nucleotide, and DHA
as final hydrogen acceptor. Such a demonstration requires that the rates of reduction of both DHA to ASC and GSSG to
GSH exceed the rate of 6-PG to ribulose5-phosphate (R-5-P). In the three curves
of Fig. 5 the GSH level was varied while
the concentrations of all other components
were held constant. As inherent GSSG in
the GSH preparation is proportionally increased as the level of GSH is raised, the
first parts of each curve show a proportional
increase in the amount of NADPH oxidized.
When the reduction of contaminating GSSG
is complete, addition of 6-PG immediately
institutes the restoration of NADPH to its
Table II. Detection of ASC during
spontaneous and extract-catalyzed
reaction of DHA and GSH0
Total ASC formed
(fimoles)
Incubation period
(min.)
Without
extract
With
extract
0.084
0.150
1
0.378
0.636
5
1.002
0.696
10
1.214
1.441
20
1.416
1.769
30
'Incubation mixtures, prepared in tubes preflushed with
nitrogen gas, had the following final concentrations:
buffer, 0.04M; EDTA, 0.0042M; GSH, 0.0025M; DHA,
0.00125M; extract equivalent to 6.20 mg. of protein and
distilled water to give a final volume of 6.0 ml. The air
space was again replaced with nitrogen gas before the
tubes were stoppered and incubated at room temperature. Portions, taken after time intervals indicated, were
treated with 2 per cent MPA, centrifuged, and analyzed.
original level. The reaction can go no
further as there is no system for reoxidizing the totally reduced pyridine nucleotide. At this point, addition of DHA starts
the cycling of GSH to GSSG and, in the
presence of NADPH, the reformation of
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48 Anderson and Spector
Investigative Ophthalmology
January 1971
24
28
32
MINUTES
Fig. 5. Spectrophotonietric demonstration of DHA sparking the oxidation of 6-PG. The 3.0
ml. assay system contained the following final concentrations: buffer, 0.058M; EDTA, 0.003M;
NADPH, 1.06 x 10-*M; 6-PG and DHA, each 0.001M; and 3.14 mg. of epithelial protein.
The GSH levels in Curves I, II, and III were, respectively, 2.0 x 10~4M, 1.0 x 10~»M, and
0.5 x 10-"M.
GSH. This is reflected at first as a fall in
absorption as NADP is generated. The
curves gradually reach a minimum at which
time the capacity of the system to generate
NADPH begins to outpace its capacity to
reoxidize the NADPH. It should be noted
that the small but continuous increase in
absorption at 340 m/x due to the phenomenon described earlier with DHA solutions
must be superimposed on the curves. This
can be seen as a slight break in the terminal
linear portions of Curves I and II in Fig.
5 where the levels of NADPH (corrected
for the extraneous absorption) are approximately 88 and 93 per cent, respectively, the levels when DHA was introduced.
Oxidation of GSH by a low level of H2O2
is shown by the coupled assay for glutathione peroxidase in Fig. 6. Spontaneous
oxidation of GSH by H2O2 at substrate
levels within the physiological limit (Curve
I) amounted to 0.006 ^moles of NADP
min."1 In the presence of extract (Curve
II), the rate of NADP formed min."1 was
increased to 0.110 /xmoles. A further acceleration of the rate to 0.253 /rnioles
min."1 was achieved (Curve III) when
the extract was supplemented with an
excess of exogenous glutathione reductase.
Thus, endogenous glutathione reductase
also limits the enzymatic oxidation of GSH
by H2O2. The apparent and optimal activities of glutathione peroxidase under the
experimental conditions chosen, expressed
as micromoles of NADP formed min."1
mg."1 of protein, were 0.035 and 0.084,
respectively. In various experiments, between 9S.6 and 100.3 per cent of the available H2O2 was accounted for by the total
NADP formed.
Relatively weak catalase activity of bovine corneal epithelium has been reported33
even when the H2O2 level is at the initially
high level of 0.0625N. The extent to which
this enzyme might compete with gluta-
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Volume 10
Number 1
Oxidation-reduction reactions 49
MINUTES
Fig. 6. Glutathione peroxidase activity of comeal epithelial extract and nonenzyniatic oxidation of GSH by H2O2. Reaction mixtures were prepared as described in Fig. 3 with the same
reagents except for the substitution of H2O2, 8.8 x 1(HM, for DHA and the reduction of
NADPH to 1.1 x KHM. The results are representative of five experiments performed.
thione peroxidase at low H2O2 levels is
pertinent to the interpretation of the results in Fig. 6. Preliminary experiments
ascertained that catalase activity of the extracts is unaffected by the dialysis period
normally used in sample preparation. Activity could be suppressed 90 per cent in
the presence of 0.01M potassium cyanide
and 100 per cent with 0.0037M sodium
azide. These concentrations of inhibitors
had no observable effect on either exogenous or epithelial glutathione reductase
activity. The reported stimulation of GSH
oxidation by azide34 was, as also noted
by Paglia and Valentine,24 not significant
in the present experiments. The results
in Table III demonstrate that catalase does
not compete with glutathione peroxidase
at low peroxide levels. The rate curves
prepared from absorption readings taken
at 340 m/x during 15 sec. intervals were
also indistinguishable.
The effect of tissue extract on photooxidation of ASC is shown in Table IV.
Riboflavin was incoqDorated in the mixtures at a concentration comparable to
the in vivo level of 1.8 mg. kg."1 of wet
weight reported35 for comeal epithelium.
Under the prevailing experimental conditions, approximately 52 and 70 per cent
of the ASC was oxidized in 15 and 30
min., respectively. In the presence- of less
than 1 mg. of epithelial protein ml.-1 (Experiments 1 and 2) this oxidation was reduced to about 11 and 20 per cent for the
same respective time intervals. Similar results were observed when the protein level
was reduced to 90 /x.g ml."1 (Experiment
2). At these lower levels of oxidation, the
rates were linear with respect to time for
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Investigative Ophthalmology
January 1971
50 Anderson and Spector
Table III. Activity of glutathione
peroxidase at low levels of H2O2 in the
presence and absence of catalase
inhibitors*
NADP formed
(fimoles)
Concentration
Catalase inhibitor (M x 10-*)
Experiment
1
2
None
0.189
0.274
Potasium cyanide
1.0
.193
.271
Sodium azide
0.37
.273
Sodium azide
1.9
.190
•The 3.0 ml. mixtures contained buffer (0.058M) and
EDTA (0.003M). Final concentrations of the other reactants in Experiments 1 and 2 were, respectively:
GSH,
0.0012M, 0.0025M; NADPH, 1.4
x 10-4M, 1.8 x 10-^1;
H2O2, 6.5 X KHM, 9.2 x 10-5M; and epithelial protein,
3.37 mg., 2.94 mg. Potassium cyanide and sodium azide
were added at the expense of distilled water.
at least 30 min. As the comparable protection afforded by EDTA in Experiment 1
was attributable to removal of heavy metal
ions by chelation, the possibility that protein thiol groups of the extract were involved in a similar mechanism was explored. Based on the maximal amount of
available protein thiol, an 8.5-fold excess
of p-hydroxymercuribenzoate (pHMB)
was preincubated with extract (Experiment 2). The ensuing 15 min. oxidation
of ASC was nearly twice that in the presence of extract alone but still amounted
to less than one half the maximal rate.
H2O2 was detected in all incubation mixtures. Based on the final concentrations
of H2O2, the calculated per cent of ASC
oxidized to this product is difficult to
interpret, as H2O2, once generated, can, in
the presence of orthophosphate buffer,9
be consumed in the further oxidation of
ASC.
Discussion
The scheme presented in Fig. 1 suggests that the NADPH generated in the
HMP pathway can be reoxidized by the
ASC-GSH oxidation-reduction system. The
current experiments demonstrate that in
corneal epithelial extracts, the spontaneous
and enzyme-mediated reactions necessary
to link these systems together are indeed
functional.
ASC, when used at a physiological level
to effect NADPH oxidation, generated
NADP at a rate of 0.15 /xmoles min.-1
Gmr 1 of wet corneal epithelium. In the
presence of DHA, a rate of 0.90 /mioles
of NADP formed min."1 Gmr 1 of wet
epithelium was found. At least two factors
may have contributed to this sixfold difference in rates: (1) From a consideration
of the previously reported KM of 2 x 10~3M
with respect to DHA for the dehydroascorbic acid reductase in plant tissues,30 a
relatively high level of DHA was employed
to demonstrate the presence of this enzyme
activity. The amount of DHA used (1.25
mM) was far in excess of that generated
from ASC. (2) In the presence of heavy
metal ions, riboflavin, and ultraviolet radiation, ASC oxidation was suppressed approximately 75 per cent by the amount of
EDTA used in the experiments to insure
glutathione reductase activity.
Compared to a rate of ASC oxidation
of 0.8 per cent min."1 in the absence of
GSH, a rate of 0.15 /mioles of NADPH
oxidized min."1 Gmr 1 of wet epithelium
by ASC in the presence of GSH suggests a
7.4-fold increase in ASC oxidation. If generation of H2O2 occurred concomitantly
with DHA, this would be reflected as a
greater oxidation of NADPH compared to
ASC. This disparity may also be partially
attributable to the regeneration of ASC
in the presence, but not in the absence,
of GSH and NADPH. A greater oxidation
of ASC would be expected in a system that
could maintain a higher level of substrate.
Kinoshita and Masurat37 reported the
rates of oxidation of G-6-P and 6-PG by
corneal epithelial extracts to be essentially
equal. Each metabolite could yield 0.67
/xmoles of NADPH min."1 Gm."1 of wet
epithelium when the respective substrates
are initially present at 1.66 x 10~3M. At
these levels of substrates, a total regeneration of 1.35 /xmoles of NADP min.-1 Gin."1
of wet weight would be required to maintain the system. From reported estimates
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Volume 10
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Oxidation-reduction reactions 51
Table IV. Effect of epithelial extract, EDTA, and pHMB on riboflavin-catalyzed
photo-oxidation of ASC
ASC oxidized
Addition(s) to reaction mixture*
Final concentration
Experiment
None
EDTA
Extract
3.8 x 10-3M
0.71 mg. of protein/ml.
Within
15 min.
Within
30 min.
48.5
11.4
9.1
65.4
22.0
14.9
75.4
54.7
None
10-'M
75.4
55.1
pHMB
0.90 mg. of protein/ml.
25.4
12.9
Extract
Extract
0.09 mg. of protein/ml.
12.1
24.2
Extract + pHMBf
0.90 mg. of protein/ml. + lO-'M
22.9
37.5
°The 4.0 ml. incubation mixtures were prepared in 14 x 100 mm. tubes and had the following final concentrations: buffer,
pH 7.58, 0.04M; riboflavin, 4.8 x 10-°M; ASC, 9.7 x lO^M in Experiment 1 and 5.8 x 10^M in Experiment 2. Tubes
were illuminated at a distance of 8 cm. from the liquid surface with a Blak-Ray Model UVL-21 lamp having a rated
capacity of 87 uw. per square centimeter of 3,660 A. at 46 cm. At zero min. and after 15 and 30 min. of incubation at
room temperature, portions of all mixtures were treated with 2 per cent MPA, centrifuged, and assayed.
JpHMB was preincubated 15 min. with extract and buffer before other additions.
of G-6-P in kidney,3S it is reasonable to assume that these substrate levels are in excess of those present in vivo by at least
one order of magnitude. The unfavorable
conditions for ASC oxidation and its use at
a level only 66 per cent of that found
physiologically would support the conclusion that the observed rate of NADPH oxidation of 0.15 /xmoles min."1 Gm."1 of tissue
by ASC via DHA-GSH represents a minimum value. With DHA at a level comparable to that of the HMP intermediates
employed by Kinoshita and Masurat,37 as
much as 67 per cent of the NADP requirement of the HMP shunt could be met.
Should HaO2 be generated concomitantly
during the oxidation of ASC, additional
NADP would be made available. Thus,
it is reasonable to conclude that without
involving other pathways that are undoubtedly operational in the tissue, ASC
oxidation could provide a significant proportion of the NADP demanded by the
HMP shunt.
The presence of a dehydroascorbic acid
reductase in corneal epithelium confirms
the finding of Grimble and Hughes7 that
this enzyme is not confined to plant or
bacterial sources. The sixfold increase in
reaction rate of extracts after supplementation with exogenous glutathione reduct-
ase indicates that the activity of dehydroascorbic acid reductase is limited by that
of the inherent glutathione reductase. As
the spectrophotometric assay is essentially
a detection of GSSG formed, the product
of any additional DHA reduction by thiol
groups of the epithelial proteins would not
be detectable. A contribution by protein
thiol groups to DHA reduction under the
prevailing experimental conditions seems
unlikely in view of the low level of protein thiol present with respect to GSH.
Protein disulfide groups in the extracts could not, moreover, have contribbuted to the formation of GSSG in this
reaction. This is deducible from the fact
that no detectable differences in total
NADP formation were observed when
either exogenous glutathione reductase or
extracts were used to effect reduction of
GSSG-contaminated GSH solutions. This
would not have been possible had the level
of GSSG been increased as a result of protein disulfide interaction with GSH.
A separate determination of ASC formed
under conditions similar to the oxidation
of GSH by DHA (except that NADPH
and glutathione reductase were omitted)
gave initial rates for the spontaneous interaction comparable to those calculated from
the determination of GSSG. The rate of
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Investigative Ophthalmology
January 1971
52 Anderson and Spector
ASC generation in the presence of extract, however, was 34 per cent less than
that of GSSG formation for the first minute; the difference increased with time.
An explanation for this discrepancy is not
readily apparent. Possibly the enzymatic
reduction of DHA involves intermediates
which give rise to ASC at a rate much
less than that observed for the oxidation
of NADPH.
Jacob and Jandl39 have presented evidence that ELOa and GSH together can
control the activity of the HMP pathway
in the red blood cell. A similar conclusion
was reached by Benard and DeGroot40
for the thyroid tissue. A 20 per cent
enhancement in 14CO2 production from
glucose-l-14C when physiological amounts
of ASC were added to erythrocytes in
vivo was also noted by Jacob and Jandl.39
As H2O2 arises from the oxidation of
ASC in the presence of oxyhaemoglobin,41
the mechanism by which ASC could affect the HMP shunt in red blood cells
is generally viewed only from the standpoint of the oxidation product, H2O2, and
glutathione peroxidase.
It is not possible to ascertain whether
H2O2 or DHA represents the major metabolite for the reoxidation of NADPH
via ASC in corneal epithelium. Although
the observed activities for DHA reductase
and glutathione peroxidase were nearly
the same when glutathione reductase was
not limiting, the respective conditions of
assay for these enzymes were not equatable. With H2O2 as substrate, a concentration approaching the physiological level
(10"5M) in corneal epithelium was used.
This level is approximately the same as
the reported KM with respect to H2O2 for
glutathione peroxidase isolated from red
blood cells.24 As already noted, DHA was
used at concentrations well above the
assumed physiological level. A valid assessment of the relative contribution of
the two pathways would require the determination of the KM of both comeal
enzymes, as well as the rate of DHA and
H2O2 generation in this tissue.
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