Determination of myoglobin concentration in blood-perfused tissue.

Abstract

The standard method for determining the myoglobin (Mb) concentration in blood-perfused tissue often relies on a simple but clever differencing algorithm of the optical spectra, as proposed by Reynafarje. However, the underlying assumptions of the differencing algorithm do not always lead to an accurate assessment of Mb concentration in blood-perfused tissue. Consequently, the erroneous data becloud the understanding of Mb function and oxygen transport in the cell. The present study has examined the Mb concentration in buffer and blood-perfused mouse heart. In buffer-perfused heart containing no hemoglobin (Hb), the optical differencing method yields a tissue Mb concentration of 0.26 mM. In blood-perfused tissue, the method leads to an overestimation of Mb. However, using the distinct (1)H NMR signals of MbCO and HbCO yields a Mb concentration of 0.26 mM in both buffer- and blood-perfused myocardium. Given the NMR and optical data, a computer simulation analysis has identified some error sources in the optical differencing algorithm and has suggested a simple modification that can improve the Mb determination. Even though the present study has determined a higher Mb concentration than previously reported, it does not alter significantly the equipoise PO(2), the PO(2) where Mb and O(2) contribute equally to the O(2) flux. It also suggests that any Mb increase with exercise training does not necessarily enhance the intracellular O(2) delivery.

Full text

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ORIGINAL ARTICLE
Determination of myoglobin concentration in blood-perfused
tissue
Kazumi Masuda Æ Æ Kent Truscott Æ Æ Ping-Chang Lin Æ Æ
Ulrike Kreutzer Æ Æ Youngran Chung Æ Æ Renuka Sriram Æ Æ
Thomas Jue
Accepted: 13 May 2008/Published online: 31 May 2008
? Springer-Verlag 2008
Abstract
globin(Mb)concentrationinblood-perfusedtissueoftenrelies
on a simple but clever differencing algorithm of the optical
spectra, as proposed by Reynafarje. However, the underlying
assumptions of the differencing algorithm do not always lead
to an accurate assessment of Mb concentration in blood-per-
fused tissue. Consequently, the erroneous data becloud the
understandingofMbfunctionandoxygentransportinthecell.
The present study has examined the Mb concentration in
buffer and blood-perfused mouse heart. In buffer-perfused
heartcontainingnohemoglobin(Hb),theopticaldifferencing
method yields a tissue Mb concentration of 0.26 mM. In
blood-perfused tissue, the method leads to an overestimation
ofMb.However,usingthedistinct1HNMRsignalsofMbCO
and HbCO yields a Mb concentration of 0.26 mM in both
buffer-andblood-perfusedmyocardium.GiventheNMRand
optical data, a computer simulation analysis has identified
some error sources in the optical differencing algorithm and
has suggested a simple modification that can improve the Mb
determination.Eventhoughthepresentstudyhasdetermineda
higher Mb concentration than previously reported, it does not
alter significantly the equipoise PO2, the PO2where Mb and
O2contribute equally to the O2flux. It also suggests that any
Mb increase with exercise training does not necessarily
enhance the intracellular O2delivery.
The standard method for determining the myo-
Keywords
Respiration
Heart ? Muscle ? Hemoglobin ? Oxygen ?
Introduction
A canonical view of biochemistry confers upon myoglobin
(Mb) a role as an oxygen reservoir or as a facilitated
transporter of O2(Wittenberg and Wittenberg 1989; Wit-
tenberg 1970). Even though in vitro experimental evidence
supports such a view, in vivo experiments have produced
mixed results. Some experiments support a prominent
cellular O2role (Wittenberg and Wittenberg 2003). Other
studies, however, have raised questions. In spontaneously
beating rat heart, the Mb O2store and can only prolong
normal heart function for a few seconds (Chung and Jue
1996). Cellular Mb also diffuses too slowly to play a
prominent O2transport role under steady state normoxic
condition (Lin et al. 2007a, b). Even without myoglobin, a
mouse model shows no respiration impairment (Garry et al.
1998; Godecke et al. 1999).
Many ideas on Mb function have emerged from exper-
imentsfollowingMbconcentration
physiological adaptation. At high altitude, Mb expression
increases (Gimenez et al. 1977; Terrados et al. 1990). With
exercise training, some studies show an increase in tissue
Mb (Beyer and Fattore 1984; Harms and Hickson 1983).
Others, however, do not (Masuda et al. 2001; Svedenhag
et al. 1983). The discordant observations may arise from a
dependence on a standard optical technique to determine
the Mb contribution in blood-perfused tissue. As proposed
by Reynafarje, the standard algorithm removes the inter-
fering hemoglobin (Hb) signals by assuming identical
intensities for Hb a (568 nm) and b (538 nm) bands
(Reynafarje 1963). Even though both Mb and Hb exhibit
changeduring
K. Truscott ? P.-C. Lin ? U. Kreutzer ? Y. Chung ? R. Sriram ?
T. Jue (&)
Department of Biochemistry and Molecular Medicine,
University of California Davis, Davis, CA 95616-8635, USA
e-mail: tjue@ucdavis.edu
K. Masuda
Faculty of Human Sciences,
Institute of Human and Social Science,
Kanazawa University, Kanazawa 920-1192, Japan
123
Eur J Appl Physiol (2008) 104:41–48
DOI 10.1007/s00421-008-0775-x

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similar spectral profiles, the signal intensity ratios at 538
and 568 nm differ. Hb displays a ratio of 1, while Mb
shows a ratio of about 0.8. As a consequence, the signal
intensity difference at 568 and 538 nm divided by the Mb
extinction coefficient should yield the Mb concentration in
all blood-perfused tissue. Such spectral differencing strat-
egyunderpinstherecent
neuroglobin concentration (Williams et al. 2008).
The general validity of such optical differencing tech-
nique does raise some concerns. Yet, its simplicity, in
contrast to the alternative, semiquantitative immunohisto-
chemistry or antibody approach, maintains its popularity
(Kunishige et al. 1996; Nemeth and Lowry 1984). Unfor-
tunately, an unmindful application of the Reynafarje
method leads to an erroneous Mb concentration determi-
nation and a concomitant misunderstanding of Mb function
and O2transport. Because the1H NMR spectra of HbCO
and MbCO show distinct CH3Val-E11 signals, an oppor-
tunity now arises to validate the optical differencing
algorithm, present modifications to improve the optical
differencing technique, and establish the basis for an
alternative methodological approach (Ho and Russu 1981;
Kreutzer et al. 1992). Given the results, the analysis sug-
gests that any increase in Mb concentration with exercise
training does not change significantly the equipoise PO2the
PO2where Mb and O2contribute equally to the O2flux,
and militate against a simplistic interpretation of how Mb
increase can influence O2metabolism.
determinationof tissue
Materials and methods
Experimental animals
Male C57/BL6 mice (25–35 g) were used for the present
experiment. All mice were housed in a temperature-con-
trolled room at 23 ± 2?C with a light–dark cycle of 12 h
and maintained on mice chow and water ad libitum. All
procedures performed in this study conformed to the
Guiding Principles for the Care and Use of Animals in the
Field of Physiological Sciences in the University of Cali-
fornia, Davis. All surgical procedures were performed
under pentobarbital anesthesia.
Tissue preparation
Animals were anesthetized by an intraperitoneal injection
of sodium pentobarbital (65 mg/kg) and heparinized
(1,000 U kg-1). The heart was quickly isolated and either
extracted immediately or perfused with buffer. Hearts for
perfusion were placed in ice-cold buffer solution until
aortic cannulation. The heart was then perfused in Lange-
ndorff mode, with Krebs–Henseleit buffer containing (in
mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.8 CaCl2, 20
NaHCO3, 1.2 MgSO4, 15 glucose. The buffer was equili-
brated with 95% O2, and 5% CO2, and passed through 5
and 0.45 lm Millipore filters. A circulating water bath
(Lauda MT-3) and temperature jacketed reservoir and
tubings maintained the temperature at 35?C. A peristaltic
pump (Rainin Rabbit) maintained a constant, non-recircu-
lating perfusate flow of 2.0–2.5 ml min-1.
After isolation or perfusion, the heart was immediately
weighed, thoroughly minced using stainless scissors, and
homogenized in an ice bath with phosphate buffer
(0.04 M, pH 6.6) bubbled with carbon monoxide (CO).
The homogenate was then centrifuged at 14,000g for
30 min at 4?C (Centra-MP4R, IEC, USA). The clear
supernatant was transferred to a small glass tube and again
equilibrated with carbon monoxide to ensure myoglobin
binding with carbon monoxide (MbCO). Tubes were cap-
ped tightly and kept at 4?C until optical spectroscopy and
NMR measurements.
Blood sample
Mouse blood from the abdominal veins of the anesthetized
mouse was taken with a heparinized syringe. The blood
was lysed with H2O, and the resultant sample was cen-
trifuged at 9,000g for 5 min. The supernatant was
transferred to a small tube and was equilibrated with
carbon monoxide (CO) to form HbCO. The HbCO solution
was stored at 4?C until measurement by optical spectros-
copy and NMR.
Optical measurement
Optical measurement (380–900 nm) of Mb and Hb used
either a UVIKON 941 (Kontron Instruments) or an HP8452
spectrophotometer (Hewlett Packard). For MbCO, the
concentration determination used the extinction coeffi-
cients 14.7 9 103cm-1M-1and 12.3 9 103cm-1M-1
for the respective maxima at 540 and 577 nm. Similarly,
hemoglobin concentration was determined based on the
optical density at 540 and 568 nm (extinction coefficient
for HbCO maxima at 540 and 568 nm = 13.4 9 103cm-1
M-1) (Antonini and Brunori 1971). The optical density at
538 nm (b band) and 568 nm (a band) was used for cal-
culation of both myoglobin and hemoglobin concentration
in the heart tissue as described by Reynafarje (Reynafarje
1963).
Reynafarje method
Assuming only two components, HbCO and MbCO, the
following equations describe the signal intensity difference
at 538 nm (b) and 568 nm (a):
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OD538? OD568¼ e538;HbCO? e568;HbCO
þ e538;MbCO? e568;MbCO
where OD is the optical density or absorbance or signal
intensity, e = extinction coefficient, and CMbCO and
CHbCO= concentration of MbCO and HbCO, respectively.
The Reynafarje method assumes an identical Hb
extinction coefficient at 538 and 568 nm, e538;HbCO¼
e568;HbCO; so the first term on the right cancels. In addition,
itassumesfor Mban
e538,MbCO= 14.7 9 103cm-1M-1
11.8 9 103cm-1M-1at 538 and 568 nm, respectively.
As a consequence, the equations for CMbCOand CHbCO
become
??CHbCO
??CMbCO
extinctioncoefficient
e568,MbCO=
of
and
CMbCO¼
OD538? OD568
e538;MbCO? e568;MbCO¼
¼ OD538? OD568
CHbCO¼OD538? e538;MbCOCMbCO
OD538? OD568
14:7? 103? 11:8? 103
Þ ? 3:45? 10?4M;
ð
e538;HbCO
NMR measurement
1H-NMR experiments used an Avance 500-MHz Bruker
spectrometer equipped with a 5 mm tri-axial gradient
proton/broadband probe. The
calibrated against the H2O signal from a 0.1 mM HbCO
solution. A modified 1-s-3-s-3-s-1-pulse sequence sup-
pressed the water signal and excited the Val-E11 c-CH3
signal of HbCO and MbCO at -1.8 and -2.4 ppm,
respectively (Ho and Russu 1981). A typical spectrum
required 16 k scans, 10,000-Hz spectral width, 2,048
data points, and 110-ms repetition time. Zero-filling the
free induction decay(FID)
Gaussian window smoothed the spectra. A spline fit
interpolation improved the baseline for the analysis.
Sodium-3-(trimethylsilyl)propionate-2,2,3,3-d4
served as the chemical shift (0 ppm) reference and the
concentration standard, based on a difference spectrum
before and after the addition of a known amount of TSP
in the sample.
1H 90? pulse was 9.0 ls,
and apodizing witha
(TSP)
Spectral simulation
All analysis and simulation methods used MATLAB, ver-
sion 6.5/Release 13 (The Mathworks, Natick, MA). The
reference spectra comprised of the optical density versus
wavelength data matrix of the MbCO and HbCO spectra
from 500–600 nm. All analysis employed a spline inter-
polation to correctthebaseline.
minimization algorithm then compared the heart homoge-
nate with the reference spectra to optimize the weights
assigned to the MbCO and HbCO components, which
Aleast squares
minimized the residual error and, therefore, gave the best
estimate of the fractional contribution (Press et al. 2007).
Statistical analysis
All data are expressed as means ± SD. To compare the
values among different methods, the repeated one-way
analysis of variance was utilized. Scheffe ´’s post-hoc test
was conducted if the analysis of variance indicated a sig-
nificant difference. Regression and correlation analysis
were performed to evaluate the relationship among vari-
ables. The level of significance was set at P\0.05.
Results
Figure 1 shows the optical spectra of HbCO, MbCO, and a
mixture of MbCO and HbCO. For solution HbCO from
mouse blood, the peak maxima appear at 537 nm (b) and
567 nm (a) (Fig. 1a). For MbCO from buffer-perfused
mouse myocardium, the two maxima appear at 540 nm (b)
and 577 nm (a) (Fig. 1b). The spectrum of tissue homog-
enateobtainedfrom the
containing blood, shows contribution from both Hb and
Mb.
unperfusedmyocardium
490510530 550 570590610
Wavelength, nm
A
B
C
Fig. 1 Visible spectra of HbCO, MbCO, and MbCO/HbCO mixture:
a HbCO extracted from mouse blood; b MbCO extracted from buffer-
perfused mouse heart; c 1/1 mixture of mouse MbCO/HbCO
Eur J Appl Physiol (2008) 104:41–4843
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Figure 2 shows the MbCO and HbCO peak displace-
ment in the expanded spectral region between 500 and
600 nm. The HbCO 537 nm (b) and MbCO 540 nm (b)
peaks do not coincide. Similarly, the HbCO 567 nm (a)
and the MbCO 577 nm (a) do not match up. Moreover, the
HbCO 537 nm (b) and 567 nm (a) peaks do not exhibit
identical maxima intensity.
Figure 3 displays the1H NMR spectra for HbCO solu-
tion, for MbCO from buffer-perfused heart, and for MbCO
and HbCO from unperfused heart. The solution HbCO
spectrum extracted from mouse blood exhibits the ring
current shifted c-CH3Val-E11 signals from the a and b
subunits at -1.72 and -1.92 ppm (Fig. 3a). Because of the
non-uniform pulse excitation profile, the a subunit peak at
-1.72 ppm has slightly lower signal intensity. Figure 3b
displays the1H NMR spectrum of the buffer-perfused heart
homogenate. Only the MbCO c-CH3 Val-E11 signal
appears and resonates at -2.40 ppm. Figure 3c displays
the spectrum of unperfused heart homogenate, which
contains the well-resolved signals of MbCO and HbCO.
NeithertheMbCOandHbCOmaximawavelengthnorthe
extinction coefficients have the same values for all species.
Tables 1 and 2 summarize some reported values in the lit-
erature. Because of the variation, the Reynafarje method
assumption of a constant extinction coefficient at 538 and
568 nmcanleadtosignificanterrorintheMbdetermination.
The data matrix of absorbance versus wavelength for
pure MbCO and HbCO establishes spectra basis sets. A
least squares algorithm minimizes the error in matching the
heart homogenate spectra with different contribution from
the pure HbCO and MbCO spectral components. Using
baseline corrected spectra leads to an improved estimate of
tissue Mb in blood-perfused heart, as displayed in Fig. 4.
Figure 4a shows the actual spectrum from unperfused
myocardium homogenate, containing a mixture of Mb and
Hb, while Fig. 4b shows the computer generated spectrum,
which approximate the Mb and Hb mixture based on
weights assigned to the MbCO and HbCO pure spectra.
Subtracting the two spectra (Fig. 4b - Fig. 4a) yields the
residual spectrum, Fig. 4c.
The standard Reynafarje method yields an Mb concen-
tration of 0.26 ± 0.06 mM from the perfused heart
homogenate and 0.36 ± 0.07 mM from the unperfused
heart homogenate. In contrast, NMR analysis of perfused
and unperfused heart homogenate reveals a consistent tis-
sue Mb concentration of 0.28 ± 0.04 and 0.26 ± 0.03,
respectively. A deconvolution algorithm using baseline
corrected and weights of pure HbCO and MbCO spectra
also yields an Mb concentration for perfused and unper-
fused heart homogenate of 0.26 ± 0.06 and 0.26 ± 0.05.
Table 3 summarizes the results.
Discussion
Mb concentration in mouse heart
Studies have reported tissue myoglobin in many terrestrial
mammals range from 0.2–0.5 mM. The heart contains
0
0.2
0.4
0.6
0.8
1
1.2
1.4
490 510530 550
Wavelength, nm
570 590610630
Absorbance
A
B
Fig. 2 Visible spectra of HbCO and MbCO: a mouse HbCO
extracted from mouse blood; b mouse MbCO extracted from
buffer-perfused mouse heart. For HbCO, the peak maxima appear
at 537 nm (b) and 568 nm (a). MbCO has maxima at 540 and 577 nm
-1.5-2.0-2.5ppm
A
B
C
Fig. 3
mixture: a HbCO extracted from mouse blood; b MbCO extracted
from perfused mouse heart; c 1/1 mixture of mouse MbCO/HbCO.
For HbCO, the peaks at -1.72 and -1.92 ppm arise from the Val-
E11 c-CH3resonances of the a and b subunits. For MbCO, the Val-
E11 c-CH3 resonance appears at -2.40 ppm. The MbCO/HbCO
mixture shows the distinct peaks from Hb and Mb
1H NMR spectra of HbCO, MbCO, and MbCO/HbCO
44Eur J Appl Physiol (2008) 104:41–48
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about 0.2 mM, while skeletal muscle has about 0.4–
0.5 mM. If the cell volume calculation excludes mito-
chondria (35% of cell volume) and the sarcoplasmic
reticulum (4% of the cell volume), the concentration in
heart cell would rise to 0.33 mM (Wittenberg and Wit-
tenberg 2003). Muscle Mb concentration in marine
mammals can rise to 4.5 g per 100 g tissue or about
3.8 mM in the cytoplasm (Ponganis et al. 1993, 2002).
Determining the tissue Mb concentration has relied
heavily on the direct application of visible spectroscopy
methods and a spectral differencing to separate the frac-
tional contribution of Mb and Hb. The algorithm relies on
equal absorbance intensities for the HbCO at 538 and
568 nm and the corresponding unequal intensities for
MbCO at the same wavelengths.
Hearts perfused with saline buffer contain no Hb.
Optical measurements of perfused heart homogenate reveal
a tissue Mb concentration of 0.26 ± 0.06. From unperfused
heart, the homogenate contains both Hb and Mb. The tissue
Mb concentration, however, should remain the same. The
Reynafarje method no longer yields an accurate assess-
ment.Itoverestimatesthe
0.36 ± 0.07 mM corresponding to an error of 38%.
In contrast, the1H NMR signals of MbCO and HbCO do
not overlap and appear in clear spectral region. Specifically,
the HbCO c-CH3Val-E11 signals for the a and b subunits
resonate at -1.72 and -1.92 ppm, whereas the MbCO
c-CH3Val-E11 signal appears at -2.40 ppm. The spectra
show no MbO2 or metMb signals at -2.8 ppm and
-3.7 ppm. Only the MbCO signal appears (Chung et al.
1996; Kreutzer and Jue 2004). Similarly, no HbO2and/or
Mb concentration as
Table 1 HbCO absorbance property
kb
eb
Est. e538
ka
ea
Est. e568
Species Source
540 13.413.3 56913.4 13.3Human (Antonini and Brunori 1971)
540 14.013.9 568
568.5b
14.1 14.1 Human(de Duve 1948)
53914.3 14.3
14.4 14.3Human(van Kampen and Zijlstra 1965)
53914.414.3 568.514.314.3Human (van Assendelft 1970)
537.5 14.8 14.856814.8 14.8Human(Horecker 1943)
537.515.0 15.0568 15.015.0Calf(Horecker 1943)
aScaled to expected extinction coefficients (using 16.8 g dry pigment per liter) from de Duve’s measured extinction coefficients using 1 g dry
pigment per liter: 0.833 at 540 nm, 0.826 at 538 nm, and 0.840 at 568 nm (de Duve 1948)
bAn average value derived from the report’s table entry, ‘‘569–568’’
Table 2 MbCO optical absorbance property
kb
eb
Est. e538
ka
ea
Est. e568
SpeciesSource
54214.2 13.558012.210.9 Human(de Duve 1948)a
540 15.415.3 57913.912.0Horse(Antonini and Brunori 1971)
54014.9 14.857713.011.5Horse (Bowen 1949)
54215.114.758013.211.3Guinea pig(Helwig and Greenberg 1952)
542 15.615.2 57813.611.9 Guinea pig(Helwig and Greenberg 1952)
542 14.013.657912.210.6 Sperm whale(Antonini and Brunori 1971)
aScaled to expected extinction coefficients (using 16.8 g dry pigment per liter) from de Duve’s measured extinction coefficients using 1 g dry
pigment per liter: 0.844 at 542 nm, 0.806 at 538 nm, 0.727 at 580 nm, and 0.649 at 568 nm (de Duve 1948)
490630610590570 550
Wavelength, nm
530510
A
B
C
Fig. 4 Deconvolution of Mb and Hb in the optical spectra: a observed
opticalspectrumfromunperfusedmouseheartcontainingbothMbandHb;
b Computer modeling of Mb and Hb contribution; c spectral difference
(a - b). The difference spectrum shows no significant residual error
Eur J Appl Physiol (2008) 104:41–48 45
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metHb signals appear (Ho and Russu 1981). Moreover, the
spectra show no detectable signals of cytochrome C (Feng
et al. 1990). The NMR analysis yields tissue Mb of
0.28 ± 0.04 and 0.26 ± 0.03 from perfused and unperfused
heart homogenate, respectively. These values agree with the
optical determination of Mb from buffer-perfused heart.
Mb concentration and equipoise PO2
The Mb concentration influences the intracellular oxygen
delivery by establishing an equipoise PO2, the PO2where
the contribution from free O2flux equals the Mb O2flux as
expressed in the equation
FMb
O2
FO2
O2
¼
DMbCMb
K0ðPO2þ P50Þ
where FMb
free O2, DMbis Mb diffusion coefficient, CMbis Mb con-
centration, K0is Krogh’s diffusion constant for free O2,
PO2is the partial pressure of O2at the cell surface, and
P50 is PO2that will half saturate Mb (Lin et al. 2007a, b).
Given the experimentally determined DMbof 7.85 9 10-7
cm2s-1in heart at 35?C and a literature-reported Mb
concentration of 0.19 mM, previously reported analysis has
determined an equipoise PO2of 1.7 mm Hg (Lin et al.
2007a, b). With the newly determined value of 0.26 mM in
this report, the equipoise PO2rises only to 1.8 mm Hg.
With a resting intracellular PO2well above 10 mm Hg and
a fully saturated Mb signal even at 29 the basal work load
in the heart, a 37% increase in Mb concentration from 0.19
to 0.26 mM alters insignificantly the equipoise PO2
(Kreutzer et al. 2001; Zhang et al. 1999). Such a viewpoint
raises questions about any simplistic interpretation of O2
delivery enhancement following Mb increase with exercise
training (Hickson 1981; Masuda et al. 1998).
O2is the O2flux from Mb, FO2
O2is the O2flux from
Maxima and extinction coefficient error
The standard Reynafarje method deconvolutes the optical
spectra of Mb and Hb predicated on the assumption that the
HbCO peak at 538 nm exhibits the same intensity as the
peak at 568 nm. In contrast, the corresponding Mb peaks
show different extinction coefficients at these wavelengths,
14.7 versus 11.8 9 103cm-1M-1. With a mixture of
MbCO and HbCO found in blood-perfused tissue, the
optical density difference at 538 and 568 nm should yield
only the Mb contribution. Dividing the intensity difference
spectra by MbCO extinction coefficients at 538 and
568 nm leads to the determination of Mb in the presence of
Hb (de Duve 1948; Reynafarje 1963). In a similar
approach, Nakatani has used the Soret instead of the a and
b bands (Nakatani 1988).
Some of the underlying assumptions of the standard
method to deconvolute the Mb from Hb do not always hold.
The Hb absorbance maxima of different species do not
always appear at the same wavelength or with the same
intensities. Consequently, the peaks at 538 and 568 nm can
exhibit quite different intensity values. In mammalian Hb,
themaximapositionscanvarybyatleast±1 nm,andthetwo
extinctioncoefficientscanvaryfrom11.4–15.0 9 103cm-1
M-1. As a consequence, the specified absorbance values at
538 and 568 nm will also vary. In particular, mouse Hb has
extinction coefficients of 15.1 and 14.9 9 103cm-1M-1.
Similarly, the Mb absorbance maxima and extinction
coefficients also vary widely. The literature shows the
MbCO b band appearing from 532 to 542 nm and having
extinction coefficients ranging from 11.9–14.8 9 103cm-1
M-1. The corresponding a band can appear from 562 to
580 nm and can have an extinction coefficient between
10.6–12.3 9 103cm-1M-1. Specifically, mouse Mb has
the b and a bands at 540 and 577 nm, respectively. The
Reynafarje method, however, sets MbCO extinction coef-
ficients as 14.7 and 11.8 9 103cm-1M-1and HbCO
extinction coefficients as 14.7 and 14.7 9 103cm-1M-1
at 538 and 568 nm for all species.
Modified deconvolution algorithm
Simply assuming a constant extinction coefficient of
11.8 9 103cm-1M-1for the MbCO peak intensity at
Table 3 Mb concentration in mouse myocardium
HeartWeight (g)
n
Reynafarje method (mM)
1H-NMR spectroscopy (mM)Deconvolution algorithm (mM)
[MbCO]
Perfused0.13 ± 0.0280.26 ± 0.060.28 ± 0.040.22 ± 0.05
Unperfused0.14 ± 0.0280.36 ± 0.07 0.26 ± 0.030.23 ± 0.07
[HbCO]
Perfused0.13 ± 0.028000
Unperfused0.14 ± 0.0280.61 ± 0.20 0.57 ± 0.33 0.76 ± 0.18
Extinction
568 nm = 14.7 9 103cm-1M-1andHbCOat538 nm = HbCOat568 nm = 14.7 9 103cm-1M-1(Reynafarje1963).Values = mean ± SD
coefficientsfor theopticalmeasurement areasfollows: MbCO at538 nm = 11.8 9 103cm-1M-1, MbCO at
46 Eur J Appl Physiol (2008) 104:41–48
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568 nm already starts the Mb determination with an input
error. For example, horse and sperm whale MbCO have
estimated extinction coefficients of 12.0 and 10.6 9
103cm-1M-1at 568 nm. Using 11.8 9 103cm-1M-1
instead of the actual extinction coefficients introduces an
error of 1.6 and 11.3%, respectively.
Using a constant 14.7 9 103cm-1M-1at 538 nm also
introduces a similar error. Human and horse MbCO spectra
haveestimatedextinction
14.8 9 103cm-1M-1at 538 nm, leading to an error of
8.2 and 0.6%.
These errors can then propagate. In sperm whale, the
MbCO exhibits absorbance maxima at 542 and 579 nm
with respective extinction coefficients of 14.0 and
12.2 9 103cm-1M-1. Assuming the mouse and sperm
whale MbCO spectra share similar features in the b and a
bands leads to an extrapolation of the extinction coeffi-
cients as 13.6 and 10.6 9 103cm-1M-1at the required
Reynafarje wavelengths of 538 and 568 nm. For human
MbCO, the corresponding extinction coefficients differ,
being 13.5 and10.9 9 103cm-1M-1,
Based on the Reynafarje method, the Mb concentra-
tion,
CMbCO= (OD538- OD568) 9 (14.7 - 11.8)-1=
(OD538- OD568) 9 3.45 9 10-1. Using the estimated
extinction coefficients at 538 and 568 nm from the
sperm whale spectra yields, CMbCO= (OD538- OD568) 9
(13.6 - 10.6)-1(OD538- OD568) 9 3.33 9 10-1. Using
the values from human MbCO leads to a CMbCO=
(OD538- OD568) 9 (13.5 - 10.9)-1= (OD538- OD568) 9
3.85 9 10-1. Using the Reynafarje extinction coefficients
at 538 and 568 leads to a 3.6% error in sperm whale Mb
determination but a 10% error in human Mb determination.
Different MbCO will exhibit a different degree of error
propagation. Given the small propagated error range,
however, the variation in extinction coefficients cannot
account for all the observed error in the optical difference
approach, which overestimates the Mb concentration by
38%. Another significant error source must exist.
One significant contribution arises from baseline errors
during spectral subtraction, which reflects individual
spectrophotometer’s performance characteristics. Relying
simply on constant extinction coefficients at fixed wave-
lengths, 538 and 568 nm, would overlook these errors.
Instead, using a baseline corrected visible spectra of pure
1 mM Mb and Hb as the reference basis set and applying a
nonlinear least squares routine to approximate the baseline
corrected spectra from blood-perfused tissue, yields a Mb
value of 0.22 mM, in excellent agreement with the optical
and NMR analysis of the buffer-perfused heart homogenate
(Press et al. 2007). Such a deconvolution approach presents
a simple modification of the Reynafarje method and
appears to dramatically improve the accuracy of the tissue
Mb determination. It serves as a simple alternative to the
coefficientsof13.5and
respectively.
more sophisticated technique employing partial least
squares fit of second derivative spectra but requires further
study to clarify the underlying chemometrics (Marcinek
et al. 2007).
Mb in marine mammals
The error in the Reynafarje method might appear to apply
only to terrestrial mammalian tissue, where blood Hb can
interfere significantly with the determination of the low
tissue Mb concentration. In marine mammals, the high Mb
concentration and the very low Hb interference would
seem to rise above the error concerns in the present study.
An investigator, who overlooks the negligible Hb contri-
bution and uses only the prescribed Mb extinction
coefficient of 11.8 9 103cm-1M-1at 568 nm to deter-
mine the Mb concentration of sperm whale, would
introduce immediately a 10% error. For sperm whale, the
Reynafarje spectral differencing method appears to pro-
duce only a 3.6% error based on the extinction coefficient
variation. For other marine mammals, however, the error
may deviate significantly and depends upon the measured
extinction coefficients for Mb and Hb. However, a large
error contribution does not even relate to the issue of
extinction coefficients. It arises from baseline errors in
spectral differencing methodology, which contributes a
significant fraction of the observed 38% error observed in
this study (Truscott et al., unpublished observation). In
essence, the study results argue for a reassessment of the
tissue Mb concentration in marine mammals to clarify the
basis for interpreting the role of Mb in regulating metab-
olism during a dive.
Conclusion
The study shows that the unmindful use of the standard
Reynafarje method to determine Mb concentration in the
presence of Hb can lead to significant errors. Variations in
extinction coefficients and baseline fluctuation contribute
to these errors. Comparative analysis of homogenate from
unperfused and perfused heart reveals that a modified
algorithm that relies on a basis set of pure Mb and Hb
spectra will improve the Mb assay. The newly determined
Mb concentration of 0.26 mM reflects a 37% increase in
Mb concentration relative to the previously determined
value of 0.19 mM. Such a rise in Mb concentration, how-
ever, does not significantly alter the equipoise PO2
appreciably and, therefore, the O2transport function of Mb,
at least in the mammalian myocardium. The study suggests
that a reassessment of the tissue Mb concentration in dif-
ferent species would help clarify the role of Mb in
regulating metabolism.
Eur J Appl Physiol (2008) 104:41–48 47
123

Page 8

Acknowledgments
from the Japan Ministry of Education, Culture, Sports, Science and
Technology 15700410 (KM), NIH GM 58688 (TJ), Philip Morris
005510 (TJ), and the American Heart Association Western States
Affiliate 0265319Y (UK).
We gratefully acknowledge funding support
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