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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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.

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
F = F<SUB>NADH</SUB> + F<SUB>CF</SUB> + F<SUB>EBT</SUB> (1)
where
F<SUB>NADH</SUB> = I<SUB>NADH</SUB> ⋅ M<SUB>NADH</SUB> (2)
F<SUB>CF</SUB> = I<SUB>CF</SUB> · M<SUB>CF</SUB> (3)
F<SUB>EBT</SUB> = I<SUB>EBT</SUB> · M<SUB>EBT</SUB> (4)
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).

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.

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.

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.

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.

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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -hydroxybutyrate dehydrogenase (HBADH) equilibrium within the mitochondria with literature values. This was accomplished by varying the acetoactate (AA)-to-beta -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
<IT>k</IT> = [NADH][H<SUP>+</SUP>][AA]/[NAD][HBA] (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.


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Fig. 11.   Effect of acetoacetate-to-beta -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(3):C900-C909