SPECIAL COMMUNICATION
Correction for inner filter effects in turbid samples: fluorescence
assays of mitochondrial NADH
Stephanie A.
French,
Paul R.
Territo, and
Robert S.
Balaban
Laboratory of Cardiac Energetics, National Heart, Lung, and Blood
Institute, Bethesda, Maryland 20892-1061
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ABSTRACT |
Fluorescent
determinations of NADH in porcine heart mitochondria were subject to
significant errors caused by alterations in inner filter effects during
numerous metabolic perturbations. These inner filter effects were
primarily associated with changes in mitochondrial volume and
accompanying light scattering. The observed effects were detected in a
standard commercial fluorometer with emission orthogonal to the
excitation light path and, to a lesser extent, in a light path geometry
detecting only the surface fluorescence. A method was developed to
detect and correct for inner filter effects on mitochondrial NADH
fluorescence measurements that were independent of the optical path
geometry using an internal fluorescent standard and linear
least-squares spectral analysis. A simple linear correction with the
inner fluorescence reference was found to adequately correct for inner
filter effects. This approach may be useful for other fluorescence
probes in isolated mitochondria or other light-scattering media.
optical methods; light scattering; mitochondria; linear least
squares; calcium; pig; heart
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INTRODUCTION |
OPTICAL METHODS are extremely useful in the analysis of
mitochondrial biochemical function (4, 7, 14, 16). Mitochondrial swelling and associated light-scattering changes that occur with numerous metabolic transitions represent a significant problem that can
affect absorption and fluorescence spectroscopic measurements (3, 25).
Light-scattering and absorption [so called "inner filter"
(29)] effects in turbid media have primary and secondary components depending on the conditions: the primary effect is defined
as scattering or absorption of the excitation light, and the secondary
effect is defined as scattering or absorption of the emitted light.
Fluorescence studies in scattering media, like mitochondrial
suspensions, require that inner filter effects be minimized or compensated for before a quantitative analysis can be made. These scattering effects can be reduced by working with a light path geometry, in which excitation and sampling occur from the same side of
the chamber (surface fluorescence), which minimizes the overall path
length (6, 11). The reflected excitation light can be monitored to
compensate for primary scattering (30) but cannot correct for secondary
effects. Collecting all the light emitted using an integrating sphere
or most of the light using wide slits and close-coupled optics can
reduce many of the scattering effects (24). Inner filter effects can be
corrected for empirically or with direct calculations if the optical
characteristics of the filter can be quantitatively evaluated (5, 15).
Finally, internal fluorescent standards have been used to correct for
inner filter effects in biological tissues (19-21). This latter
approach can potentially compensate for primary and secondary effects
(20). Rhodamine B is one of the internal reference compounds that has been used; however, this agent can interfere with fat metabolism in rat
liver mitochondria (A. P. Koretsky and R. S. Balaban, unpublished observations).
Although a number of recent mitochondrial NADH fluorescence studies
were performed using the surface reflectance signal with or without
standards to minimize inner filter effects (1, 9, 10, 20, 23), others
have presented experiments uncorrected for scattering with an
orthogonal excitation-to-emission light path (8, 22, 28, 31) that would
predictably maximize the primary and secondary effects.
The purpose of this work was to evaluate the significance of
mitochondrial inner filter effects on NADH fluorescence measurements in
a commercial fluorometer and develop a method to correct for the
primary and secondary components of these effects. Toward this goal,
the use of an internal fluorescent standard with appropriate spectral
fitting routines was evaluated as an approach to detect and correct for
inner filter effects in porcine heart mitochondria. By use of spectral
fitting routines, the magnitude of inner filter effects can be followed
as well as corrected for in mitochondrial fluorescence studies.
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MATERIALS AND METHODS |
Porcine heart mitochondria were prepared as previously described (26).
Mitochondria were isolated from left ventricular tissue and kept at
4°C, except where noted. Approximately 5-g aliquots of ventricle
were finely minced using scissors in 10 ml of buffer
A (0.28 M sucrose, 10 mM HEPES, 0.2 mM potassium EDTA,
pH 7.2). Typically 100 g of tissue were pooled and kept in a total
volume of 250 ml of buffer A. This
suspension was then treated with trypsin (type III bovine pancreas
trypsin, 0.5 mg/g tissue) for 15 min. The supernatant was poured off
the settled mixture and replaced volume for volume with
buffer A containing 1 mg/ml BSA and
trypsin inhibitor (2.6 mg/g tissue). Supernatant was again poured off
the settled mixture and replaced with an equal amount of
buffer A with 1 mg/ml BSA. The
suspension was homogenized with two passes of a 1-mm clearance and five
passes of a 0.2-mm clearance Teflon homogenizer (Thomas Scientific,
Swedesboro, NJ). The heart homogenate was centrifuged at 600 g for 10 min in 40-ml aliquots, and
the pellet was discarded. The mitochondria in the supernatant were
pelleted at 8,000 g for 15 min. The
pellets were pooled, rinsed, resuspended, and repelleted three times
with 80 ml of buffer A (containing 1 mg/ml BSA for the first 2 rinses) at 8,000 g for 10 min each. On each wash the
"buffy" coat was carefully removed from the top of the pellet.
Mitochondria were finally resuspended in 4 ml of
buffer B (137 mM KCl, 10 mM HEPES, 10 µM tetraphenylphosphonium ion, 250 mM
Pi, 2.5 mM
MgCl2, 0.5 mM EDTA, pH 7.2).
Proteins and enzymes were obtained from Sigma Chemical (St. Louis, MO).
Mitochondrial content was determined from the cytochrome
a oxidase concentration (2): 1 nmol of
cytochrome from porcine heart mitochondria was determined to be
equivalent to 1 mg of protein (26).
To observe the effects of Ca2+ on
mitochondria, it was necessary to deplete the mitochondria of
endogenous Ca2+. The mitochondria
were depleted of Ca2+ by
incubation in buffer C (125 mM KCl, 15 mM NaCl, 20 mM HEPES, 1 mM EGTA, 1 mM dipotassium EDTA, 5 mM
MgCl2, 2 mM
Pi, 0.1 mM malate, 10 µM
tetraphenylphosphonium ion, with 3.4 mM disodium ATP added fresh on the
day of the experiment, pH 7.0) in the absence of additional carbon
substrates. Experimental procedures were begun after this
Ca2+ depletion process, when the
membrane potential and NADH fluorescence reached a steady state (~8
min). Free Ca2+ was calculated in
this buffer on the basis of the solution binding constants previously
established (13).
Fluorescence standard loading.
A fluorescein-containing fluorescent probe was chosen to provide a high
fluorescence efficiency with emission and excitation spectral
properties close to NADH.
5(6)-Carboxy-2',7'-dichlorofluorescein diacetate,
succinimidyl ester (CF; Molecular Probes, Eugene, OR) was used because
it was pH insensitive and gave a stable reference signal after ester
cleavage. Mitochondria (15 nmol cytochrome a oxidase/ml) were loaded with CF by
addition of 1 nmol CF/nmol cytochrome
a oxidase and incubated at 25°C
for 20 min in 2.5 ml of buffer B.
Mitochondria were pelleted (8,000 g
for 10 min), and excess CF was washed off by three successive washes
with 30 ml of buffer B. The
fluorescence of the third supernatant at 530 nm (340-nm excitation) was
<0.5% of the CF signal of the loaded mitochondria.
Optical measurements.
Fluorescence emission and excitation spectra were collected using two
methods. The first used a luminescence spectrometer (model LS50B,
Perkin-Elmer, Norwalk, CT) having a standard cuvette holder with a
conventional orthogonal excitation-to-emission light path. In
the second method, excitation and emission light were transmitted
through a single multifiber-optic bundle (Perkin-Elmer). The bundle
abutted a single sapphire window embedded in a custom temperature-regulated chamber in a light-tight box. This arrangement detected the surface fluorescence from the mitochondrial suspension. Mixing was carried out with a magnetic stir bar in both orientations. Mitochondria (500 nmol cytochrome a
oxidase/ml) were incubated in solution
B (buffer B without
EDTA) or in buffer C when the Ca2+-depletion process was carried
out. Excitation was at 340 nm with a 350-nm cutoff filter and 10-nm
slit. Emission scans were collected with a 15-nm slit at 200 nm/min
from 360 to 660 nm. All experiments were conducted at 37°C.
Transmission optical absorbance measurements were performed in a
dual-chamber spectrometer (Lambda 3B, Perkin-Elmer).
Data processing.
All linear regressions were performed using resident programs in Sigma
Plot (version 3.06) on the basis of the Marquardt-Levenberg algorithm
with the following conditions:
20 iterations, a step size of 1, and a
tolerance
0.001 (Jandel Scientific, San Rafael, CA). Student's
t-tests were also run on Sigma Plot
with significant differences taken at
P
0.05. Where appropriate, values
are presented as means ± SD.
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RESULTS |
Spectral characteristics of mitochondrial scattering effects.
The effects of glutamate, Ca2+,
and the uncoupler FCCP on the absorbance spectrum of mitochondria are
shown in Fig.
1A.
Uncoupler and, to a lesser extent,
Ca2+ caused a wavelength-dependent
decrease in absorbance consistent with an increase in mitochondrial
volume. The absorbance effect increased with decreasing wavelength,
which is shown in the difference spectra presented in Fig. 1,
B and
C, and is consistent with data previously reported for liver (17).

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Fig. 1.
Frequency-dependent changes in absorbance (A) of porcine heart
mitochondria with various additions.
A: absorbance spectra of 0.5 nmol
cytochrome a oxidase/ml mitochondria
from 400 to 600 nm with no substrate (mito), 5 mM glutamate (Glu), 0.67 mM Ca2+ (calculated 0.7 µM free
Ca2+) (Ca), and 125 nM uncoupler (FCCP).
B and
C: difference spectra between one
state and previous state showing effect of
Ca2+ and uncoupler, respectively,
on mitochondrial absorbance. OD, optical density.
D: absorbance spectra of 0.087 nmol
cytochrome a oxidase/ml mitochondria
from 330 to 350 nm with same additions as
A.
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The absorbances around 340 nm (Fig.
1D) and 450 nm (Fig.
1A) suggest that significant
primary (340 nm) and secondary (450 nm) effects will be caused by
Ca2+ and other agents.
Ca2+ caused a change of 0.90 and
0.06 optical density units · nmol cytochrome
a
oxidase
1 · ml
1
at 340 and 450 nm, respectively. To correct for the inner filter effects, it was hypothesized that an inert internal fluorescent standard, excited by the same excitation light as NADH, would provide
an adequate correction. The reference emission amplitude will be
linearly related to the excitation amplitude correcting for primary
effects. If the frequency of the internal standard emission is close to
that of NADH, then both will experience a similar secondary inner
filter effect, providing a correction for this problem as well.
Internal reference.
CF was chosen as an internal standard for several reasons. The
fluorescence of CF is maximal at 530 nm, which is close to the emission
frequency of NADH fluorescence (450 nm) but distinct enough to resolve
the two emission spectra (see below). Providing a reference frequency
measurement as close as possible to the NADH signal is important to
minimize the spectral dependence of the inner filter effects. The
fluorescence intensity of CF is pH insensitive and varies <5% over a
pH range 6.5-7.5 and is also insensitive to other inorganic ions
(20). The CF ester is a lipophilic compound that readily loads into the
mitochondria, where the ester is cleaved by nonspecific esterases,
allowing the CF to bind to structures within the mitochondria. Its net negative charge also retards its release from the matrix, resulting in
a stable fluorescence reference in the same macroscopic compartment (i.e., the mitochondrial matrix) as NADH. This provided a stable fluorescence reference for the entire mitochondrial preparation. Loading mitochondria with CF had no significant effect on the absolute
maximum rate of ADP/Pi-driven
respiration (P > 0.2) or the ratio
of state 3 to state 4 respiratory rates compared with mitochondria not
treated with CF: 8.4 ± 1.8 for CF mitochondria and 8.3 ± 2.5 for unloaded mitochondria taken through the incubation and extra washes
of the CF-loading procedure. In addition, spectral fitting experiments
(see below) revealed no effects of CF on NADH fluorescence intensity or
spectral characteristics. Thus CF seemed to provide an appropriate
inert internal fluorescence standard.
Spectral fitting.
As discussed above, CF was used because its emission frequency was
close to but distinct from NADH. However, because there is considerable
spectral overlap between the NADH and CF emission spectra, as well as
other potential sources of light, spectral fitting was required to
isolate each element's contribution to the total mitochondrial
emission spectrum. This is illustrated in the spectra shown in Fig.
2, where glutamate/malate (5 mM/5 mM) and
ADP (0.7 mM) were serially added to previously carbon substrate-depleted mitochondria. There is a significant overlap between
NADH and CF emission. The overlap was necessary to keep the emission
frequencies close together, providing an adequate correction for
secondary inner filter effects. Interestingly, not all the spectral
characteristics seen in Fig. 2 can be attributed to NADH or CF. Below
~420 nm there was a gradual increase in light not associated with
NADH or CF that varied with the scattering nature of the medium. On the
basis of the slit width and scattering media dependence (Figs.
3), this component was ascribed to the excitation source bleed through (EBT). Changing the excitation filter
to 390 nm did not eliminate the contribution of EBT and reduced the
signal-to-noise ratio. Minimizing the excitation slit width (Fig. 3A) also decreased EBT
but again reduced the signal-to-noise ratio. Because EBT was directly
dependent on the sample scattering (Fig. 3) and any attempts to
eliminate it reduced the signal-to-noise ratio, EBT was left as a
component in the spectrum. EBT also gave a second measure of
mitochondrial scattering. The resulting three components, EBT, NADH,
and CF fluorescence, were used for spectral fitting.

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Fig. 2.
Emission spectra of porcine mitochondria. Spectra collected from
5(6)-carboxy-2',7'-dichlorofluorescein diacetate,
succinimidyl ester (CF)-loaded mitochondria (0.5 nmol cytochrome
a oxidase/ml) fully oxidized (mito)
and in state 4 with substrate combination 5 mM glutamate-5 mM malate
(G/M) and state 3, 1.3 mM ADP. FL, fluorescence intensity (in arbitrary
units). All subsequent figures were generated from orthogonal light
path geometry data except where noted.
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Fig. 3.
Effects of turbidity on emission spectra in absence of mitochondria.
A: excitation bleed through (EBT) with
40 mg/ml Sephadex at 5-, 10-, and 15-nm excitation slits (ex slit).
B: EBT with 0.0167 and 0.033% milk in
water.
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A least-squares linear fit of EBT, NADH, and CF fluorescence was
adequate to simulate the mitochondrial fluorescence spectra with
minimal residuals. The three individual components of the CF-loaded
mitochondrial emission spectrum (Fig.
4A) were
determined experimentally on each day's preparation. Sephadex beads (1 mg/ml, 40- to 120-µm Sephadex G-10, Sigma Chemical) were used to
model the EBT (MEBT). The
Sephadex spectra were very similar to maximally oxidized mitochondria
(i.e., ADP without exogenous carbon substrate). Sephadex was used
instead of the oxidized mitochondria to ensure that no intrinsic
fluorescence from the mitochondria was in the MEBT component. Dilute colloidal
milk suspensions also gave spectra similar to Sephadex (Fig.
3B). The NADH fluorescence model
(MNADH) was obtained by taking
the difference spectrum between glutamate/malate-supplied mitochondria
in the presence and absence of ADP (Fig. 2). The CF fluorescence model
(MCF) was the difference
spectrum of mitochondria with and without CF carefully matching the
concentration of mitochondria using the cytochrome
a oxidase content. By use of these
components, the entire emission spectrum of the tissue could be fitted
using the following linear equation
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(1)
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where
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(2)
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(3)
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(4)
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The
coefficients INADH,
ICF, and
IEBT were determined in the
spectral fitting program to estimate the contribution of each component
to the spectrum. FNADH,
FCF, and
FEBT are the portions of a
spectrum being contributed by each component. Equation 1 was used to fit each mitochondrial fluorescence
spectrum using the linear least-squares algorithm previously described.
The fitting routine took <8 s to run on a Pentium 200-MHz processor
running Windows 95.

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Fig. 4.
Model spectra and linear least-squares fit for mitochondrial emission
spectrum. A: isolated spectra of 3 components composing every scan:
MEBT is modeled by 20 mg/ml
Sephadex; MNADH is a difference
spectrum between 0.5 nmol cytochrome a
oxidase/ml reduced and oxidized mitochondria in absence of CF;
MCF is a difference spectrum
between 0.5 nmol cytochrome a
oxidase/ml reduced mitochondria with CF and a mitochondrial scan
matched for quantity of mitochondria without CF.
B: spectral fitting results of 3 components (fit) to a scan of CF-loaded mitochondria (data) with
residuals (residual).
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A typical fit is shown in Fig. 4B with
associated residuals. The average
R2 was 0.993 ± 0.005 with a minimum of 0.980. A simulation was performed to
ensure the lack of contamination between components in the fit.
Simulated spectra used the model components discussed above with the
MNADH multiplied over a range in
excess of 10-fold to simulate specific increases in NADH alone. Fitting
this series of spectra revealed a <0.02% error in the determination
of the INADH,
ICF, or
IEBT, suggesting that the spectral
fitting routine can adequately resolve the EBT, NADH, and CF
contributions from the combined spectra.
Addition of detergent dramatically reduces the contribution of
mitochondrial scattering by solubilizing the membranes. As shown in
Fig. 5A,
the addition of the detergent polidacanol (Sigma Chemical) to the
mitochondrial suspension studied with the orthogonal light path
resulted in a large decrease in EBT, which is consistent with a
decrease in scattering (Fig. 3B).
The CF signal increased as the excitation light penetrated farther into
the sample with the reduction of light scattering. There was a 5-nm
shift in the emission frequency of CF from 530 nm in mitochondria to
525 nm in the presence of detergent. The CF emission maximum in free solution was 525 nm. Detergent added to a pure cleaved CF solution had
no effect on the emission wavelength or maximum. Altering the
scattering properties of the medium had no effect on the CF emission
frequency. These data suggest that the interaction of CF with the
matrix resulted in the spectral shift observed. The decrease in NADH
fluorescence with detergent was likely due to the oxidation of NADH in
this uncoupled environment as well as the solubilization of the bound
NADH complexes responsible for the fluorescence enhancement (11).

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Fig. 5.
Effect of detergent on mitochondrial emission spectrum.
A: 0.5 nmol cytotochrome
a oxidase/ml oxidized mitochondria
loaded with CF in absence of carbon substrate (mito), reduced by
addition of 5 mM glutamate and 5 mM malate (G/M), and then solubilized
with 0.2% polidacanol (polid) in an orthogonal light path geometry.
B: same as
A, except with use of surface
fluorescence.
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When the surface fluorescence was sampled with fiber optics, the CF
decreased with detergent solubilization (Fig.
5B), suggesting that the excitation
light penetrated farther into the sample, away from the detection
system. The EBT in surface-monitoring orientation was dominated by the
light scattering in the fiber optics and was not affected by detergent
added to the sample chamber. NADH behaves similarly to the orthogonal
light path orientation. Detergent results demonstrated that orthogonal
and reflected light path geometries were influenced by light-scattering
effects on mitochondrial fluorescence measures.
To correct for the inner filter, it was assumed that the effects on the
excitation and emission light for NADH and CF were linear. Both
molecules are being excited by the same source, such that any primary
effects would be the same for both. The secondary effects of scattering
are very similar between 450 and 530 nm (Fig. 1). Thus a simple ratio
of INADH to
ICF should be adequate to correct
for inner filter effects. To test this hypothesis, a milk suspension
was used as a stable model of mitochondrial scattering to evaluate the
effects of changes in scattering on IEBT,
INADH, and
ICF in solution. These results are
presented in Figs. 6 and 7.
Consistent with increased scattering,
IEBT increased 10-fold with
increasing milk concentration, whereas
INADH and ICF fluorescence significantly
decreased. However, the
INADH-to-ICF (INADH/ICF)
ratio was constant. These results demonstrated that the quotient of
INADH/ICF
will correct INADH for sample
scattering.

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Fig. 6.
Effect of increased turbidity on NADH and CF fluorescence in absence of
mitochondria. Spectra were collected of 3 µM NADH, and 1 µM CF with
0.067 or 0.36% milk was added to increase turbidity. Scans were
performed with an orthogonal light path geometry.
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Fig. 7.
Effect of scattering changes on
ICF,
IEBT,
INADH, and
INADH/ICF,
where I represents contribution of each component to spectrum. Data are
from experiment with various amounts of milk
(n = 5). NS, not significant.
* P 0.05.
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Effect of metabolic perturbations.
The effects of several metabolic perturbations on EBT and CF (light
scattering) and NADH levels in isolated porcine mitochondria using a
conventional orthogonal optical path are shown in Fig. 8. The addition of
Ca2+ to
Ca2+-depleted mitochondria caused
a decrease in IEBT and an increase in ICF, consistent with a decrease
in light scattering and an increase in mitochondrial volume.

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Fig. 8.
Effects of glutamate and Ca2+ on
mitochondrial emission spectra. Glu, addition of 5 mM glutamate; Ca,
addition of 0.67 mM Ca2+ total
(calculated: 0.7 µM free); mito, 0.5 nmol cytochrome
a oxidase/ml oxidized mitochondria in
absence of carbon substrate.
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Other perturbations (anoxia, ADP, and carbon substrates) also caused
significant changes in ICF and
IEBT (Fig.
9), suggesting that light-scattering
changes can significantly influence mitochondrial NADH fluorescence
measures if not properly corrected.

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Fig. 9.
Summary of metabolic transition on model parameters. Three components
of curve-fitting program: INADH
(A),
ICF
(B), and
IEBT
(C). G, addition of 5 mM glutamate;
M, addition of 5 mM malate; ADP, addition of 1.3 mM ADP; anox, sample
at anoxia; see Fig. 8 legend for definition of other abbreviations. All
data were normalized to malate condition. * Significantly
different from previous condition.
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The
INADH/ICF
correction gave values for the relative NADH levels that were
statistically different from the simple amplitude of fluorescence at
450 nm (standard method) and INADH
alone (correcting for spectral influences of
IEBT and
ICF; Fig.
10).

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Fig. 10.
Summary of methods for determining NADH fluorescence amplitude.
Uncorrected 450-nm values (traditional method, raw450) are compared
with NADH quantity determined by curve-fitting program
(INADH) and corrected NADH value
(INADH/ICF).
* Significantly from
INADH/ICF
obtained under same substrate conditions.
a n = 6;
b n = 3. All data were normalized to malate condition. See legends to Figs.
8 and 9 for concentrations and definition of abbreviations.
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DISCUSSION |
It is well established that light-scattering changes occur within
mitochondrial suspensions during metabolic perturbations. In the
present study it was confirmed that associated inner filter effects can
influence the measurement of mitochondrial NADH fluorescence in porcine
heart mitochondria using a conventional spectrofluorometer.
A strategy using a fluorescence standard within the mitochondrial
matrix was presented to compensate for the primary and secondary effects of the inner filter in mitochondrial NADH fluorescence measurements. The CF fluorescent probe was used because its emission and excitation spectra are similar to NADH. The CF 530-nm emission, however, was sufficiently different from the NADH 450-nm emission to
resolve the two peaks.
The CF emission should be linearly related to the amplitude of the
excitation light. Because CF and NADH are in the same compartment, the
amplitude of the CF fluorescence should provide a reasonable compensation for the primary inner filter effects. Emitted light from
the CF is similar in wavelength to NADH and will experience nearly
identical secondary inner filter effects (Fig.
1A). Because of the frequency
dependence of the inner filter effects (Fig. 1A), the use of CF is closer to
NADH emission frequencies than previously used probes such as rhodamine
B (19), which emits in the red. Fluorescence energy transfer from NADH
to CF is possible, because the CF excitation spectrum overlaps the NADH
emission spectrum and both are trapped in the mitochondria. However, no evidence for fluorescence energy transfer between NADH and CF was
found. For example, in Fig. 9 the addition of malate increased INADH, whereas ICF
decreased. This occurred with minimal scattering effects (i.e.,
constant IEBT). Thus NADH
fluorescence does not significantly support CF fluorescence.
By using a linear least-squares fit with appropriate model spectra, the
two major spectral components, NADH and CF, of the mitochondrial
fluorescence spectrum can be resolved. This approach used the empirical
line shapes (18) of NADH and CF in the mitochondria as model components
to fit to the mitochondrial fluorescence spectrum. The use of natural
line shapes provides an internal correction for instrument and sample
modifications of the spectral components. An example of a sample
modification was the 5-nm shift in CF observed within the mitochondria.
In addition to NADH and CF, the effects of the scattered excitation
light bleeding into the fluorescence spectrum (EBT) needed to be
corrected for. Most scattering models such as Sephadex or milk were
adequate to simulate mitochondria-induced EBT. In the commercial
fluorometer used in these studies, EBT could be eliminated only by
reducing the slits to which the signal-to-noise ratio became a serious
limitation. In addition, no more information was provided using these
narrow slit widths (i.e., spectral resolution) with the rather broad
emission characteristic of NADH. By use of the increased
signal-to-noise ratio of the larger slit widths and addition of an
appropriate model component to the spectral fits, EBT effects on NADH
signals were eliminated. Because EBT was scattering dependent, its
amplitude also helped confirm the direction of scattering changes with
different experimental perturbations. EBT alone could not, however, be
used to correct for inner filter effects, since the fit of this
component was heavily dependent on light below 420 nm, where the inner
filter frequency dependence was nonlinear.
NADH fluorescence could be resolved from EBT and CF fluorescence using
a linear least-squares fitting routine. Small residuals were obtained
with this approach in these well-defined spectra. Simulations varying
the NADH component alone over an order of magnitude suggest errors of
<0.1% under idealized conditions. With use of this approach, the
emission data could be broken down into three elements, the weighted
contribution of which (INADH, IEBT, and
ICF) corresponds to the
amplitude of the NADH, EBT, and CF signals.
Several results suggest that ICF
tracks changes in sample scatter: 1)
Reduction of scattering with detergent resulted in appropriate directional changes in ICF with
orthogonal light path geometry (Fig.
5A) and surface fluorescence (Fig.
5B).
2)
ICF decreased when the turbidity
of the medium was increased in solutions and suspensions with the
orthogonal light path geometry (Fig.
9B). 3)
ICF paralleled changes in
absorbance in the mitochondria (Figs. 1 and
9B).
4)
ICF was inversely related to the
scattered excitation light, IEBT,
in the orthogonal geometry (Fig. 9, B
and C).
As discussed earlier, we proposed to use the simple
INADH/ICF
ratio to correct for inner filter effects. The
INADH/ICF
ratio remained constant with large variations in sample scattering and associated changes in INADH,
ICF, and
IEBT (Fig. 7). These results are
consistent with INADH and
ICF simply tracking the excitation light amplitude as the result of primary light-scattering effects. Any
secondary effects were assumed to be the same for
ICF and INADH because of the similar
emission frequencies.
Errors in NADH determination approached 20% for
Ca2+ and substrate additions (Fig.
10) without scattering corrections. This large error suggests that
correction for inner filter effects is essential for mitochondrial NADH
fluorescence assays. Inner filter effects were observed in orthogonal
and surface fluorescence light path geometries, suggesting that light
path geometry alone may not be adequate to correct this problem.
With regard to primary or secondary effects, the absorbance changes
with Ca2+ and uncoupler were
10-fold higher at 340 nm (primary) than at 450 nm (secondary; Fig. 1).
These results suggest that primary effects dominated the inner filter
observed in this study.
There are numerous limitations to the approach presented. It is
critical that the model spectra collected for the linear least-squares fit represent only the elements present. Great care was taken to ensure
the purity of the model spectra by using internal difference spectra
where possible. The
R2 and residuals
provided an objective measure of the quantity of the models and fit.
The spectral fitting technique requires the entire fluorescence
spectrum to be collected. A significant amount of time and data storage
capacity is also needed. Rapid scanning spectrophotometric techniques
may be most appropriate under these conditions, inasmuch as this
collects the entire spectrum in one acquisition. For the secondary
inner filter effects, the frequency differences between CF and NADH
were assumed to be insignificant. For the perturbations evaluated in
this study, this seems to be a reasonable assumption. However, there
are examples in the literature with highly frequency-dependent inner
filter effects (24), which would make the choice of a reference
compound difficult.
Absolute NADH concentrations would be available from direct analytic
assays after extraction (12, 31, 33) or using absorption in intact
suspensions with an estimated extinction coefficient (11). The
extraction methods suffer from the need to extract this highly active
species and make the analysis of time courses difficult. The absorption
methods suffer similar scattering problems, low signal-to-noise ratio,
and difficulty in determining the extinction coefficient under the
rather unique binding conditions in the mitochondria. Absolute NADH
concentrations are difficult to calculate using the fluorescence
methods and were outside the scope of this study. Quantitation of NADH
with fluorescence is complicated by the ill-defined fluorescence
enhancement occurring with NADH in the mitochondrial matrix (11).
However, NADH/NAD levels can be estimated by determining the
INADH/ICF
ratio under maximally oxidized and reduced conditions to provide NAD
and NADH levels, respectively. This approach of quantitating the
NADH/NAD ratio was tested by comparing the
-hydroxybutyrate
dehydrogenase (HBADH) equilibrium within the mitochondria with
literature values. This was accomplished by varying the acetoactate
(AA)-to-
-hydroxybutyrate (HBA) ratio in state 4 mitochondria with
minimal respiratory activity to ensure equilibrium of the HBADH
reaction. These data are shown in Fig. 11. The HBADH
equilibrium
|
(5)
|
where
[NADH],
[H+],
[AA], [NAD], and [HBA] are NADH,
H+, AA, NAD, and HBA
concentrations, was determined at the estimated midpoint of the NADH
fluorescence level between fully oxidized and reduced conditions. The
resulting HBADH equilibrium constant was 3.6 × 10
9, with the assumption of
a matrix pH of 7.7. This compares favorably with literature values for
mitochondrial HBADH equilibrium constant of 1-4.9 × 10
9 at 38°C (27, 32).
This suggests that using the maximally reduced and oxidized levels
provides a reasonable estimate of the NAD and NADH levels for
calculating the NADH/NAD ratio.

View larger version (15K):
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|
Fig. 11.
Effect of acetoacetate-to- -hydroxybutyrate ratio (AA/HBA) on
mitochondrial NADH fluorescence. Mitochondria were incubated at 1 nmol
cytochrome a oxidase/ml at 37°C.
Endogenous substrates were depleted with 200 nmol of ADP; mitochondria
were reduced fully with 10 µmol of HBA, and AA was added by titration
to determine AA/HBA at NAD/NADH = 1. INADH/ICF
values are shown for mitochondria with various AA/HBA. When 50% of
NADH was oxidized to NAD, AA/HBA was estimated to be 0.18.
|
|
The computational requirements for this approach were minimal and not
viewed as a major limitation. Indeed, such routines could be directly
programmed into the spectrophotometer.
The use of an internal fluorescent standard to correct for NADH
fluorescence in isolated mitochondria was presented. This approach is
applicable to other fluorescence probes in mitochondria (e.g., pH and
membrane potential) or any fluorescence study in turbid medium. The
approach is independent of the light path geometry but requires that
accurate model spectra are collected for the components of interest.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. S. Balaban, Laboratory of Cardiac
Energetics, National Heart, Lung, and Blood Institute, Bldg. 10 Rm.
B1D-161, Bethesda, MD 20892-1061.
Received 13 January 1998; accepted in final form 1 June 1998.
 |
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