J Appl Physiol 98: 282–287, 2005.
First published September 24, 2004; doi:10.1152/japplphysiol.00437.2004.
Noninvasive measurement of temperature and fractional dissociation of
imidazole in human lower leg muscles using 1H-nuclear magnetic
resonance spectroscopy
Yoshichika Yoshioka,1 Hiroshi Oikawa,2 Shigeru Ehara,2 Takashi Inoue,3
Akira Ogawa,3 Yoshiyuki Kanbara,4 and Manabu Kubokawa1
Departments of 1Physiology II, 2Radiology, and 3Neurosurgery, School of Medicine, Iwate Medical University, Morioka;
and 4High Field Magnetic Resonance Imaging Research Institute, Iwate Medical University, Takizawa, Japan
Submitted 26 April 2004; accepted in final form 15 September 2004
alphastat; pH; carnosine; gastrocnemius
imidazole would preserve protein structure and function as
temperature varies (18, 25, 28). Reeves (28) proposed the idea
that arterial and intracellular pH are not the primary regulated
variables but that the ratio of unprotonated to total protein
histidine imidazole (fractional dissociation of imidazole or
␣-imidazole) is primarily regulated to maintain the several
protein functions. The idea is called the “alphastat hypothesis.”
The alphastat hypothesis has been evaluated indirectly with the
help of the data on pH with changing temperature (16, 28, 34).
Direct measurements of ␣-imidazole with changing temperature, however, are obscure (5, 13), even for poikilothermic
animals. Although humans are homeothermic animals, a temperature gradient exists in the body, and the temperature near
the surface may be influenced by surrounding conditions. The
optimal pH strategy during hypothermic cardiopulmonary bypass and hypothermic treatments of humans remains controversial (29). Therefore, the applicability of the alphastat hypothesis to the human body, which suffers changes in tissue
temperature, is an interesting issue (18, 24).
An expression for the fractional dissociation of imidazole
can be derived from the following equations:
Im H ⫹7Im ⫹ H⫹ chemical equilibrium
K Im ⫽ [Im][H⫹]/[Im H⫹]
equilibrium constant
(1)
⫹
␣-imidazole ⫽ [Im]/([Im] ⫹ [Im H ])
where Im is the imidazole group and brackets denote concentration. ␣-Imidazole could also be calculated using the nuclear
magnetic resonance (NMR) chemical shift of the imidazole
proton (13)
␦ o ⫽ ␦acid 关Im H⫹]/([Im] ⫹ [Im H⫹])
ACID-BASE HOMEOSTASIS IS IMPORTANT
for normal body functions.
The imidazole group of histidine can reversibly bind a proton
within the physiological pH range and plays a key role in
maintaining the physiological pH (5, 25, 28). However, the pH
of fluids is known to vary with temperature even in the
presence of imidazole (5). It has been reported that the change
of pH with temperature is nearly equal to that of the equilibrium constant of imidazole (pKIm), suggesting that the ratio of
protonated to unprotonated imidazole is not altered by temperature (5, 25, 28). Because histidine is found at the functional
sites of many cellular proteins, a constant charge state for
where ␦acid is the chemical shift of the peak representing
completely protonated imidazole, ␦base is the peak position of
completely unprotonated imidazole, and ␦o is the peak under
ambient conditions.
Using in vivo and in vitro 1H-NMR spectroscopy, metabolites in the muscles such as creatines (creatine, phosphocre-
Address for reprint requests and other correspondence: Y. Yoshioka, Dept.
of Physiology II, School of Medicine, Iwate Medical Univ., Morioka 0208505, Japan (E-mail: [email protected]).
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. Section 1734 solely to indicate this fact.
282
⫹ ␦base [Im]/([Im] ⫹ [Im H⫹])
⫽ ␦acid ⫺ 共␦acid ⫺ ␦base兲关Im]/([Im] ⫹ [Im H⫹])
(2)
⬖ ␣-imidazole ⫽ 共␦acid ⫺ ␦o兲/共␦acid ⫺ ␦base兲
8750-7587/05 $8.00 Copyright © 2005 the American Physiological Society
http://www. jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on June 17, 2017
Yoshioka, Yoshichika, Hiroshi Oikawa, Shigeru Ehara,
Takashi Inoue, Akira Ogawa, Yoshiyuki Kanbara, and Manabu
Kubokawa. Noninvasive measurement of temperature and fractional
dissociation of imidazole in human lower leg muscles using 1H-nuclear
magnetic resonance spectroscopy. J Appl Physiol 98: 282–287, 2005.
First published September 24, 2004; doi:10.1152/japplphysiol.
00437.2004.—The temperature change of the fractional dissociation of imidazole (␣-imidazole) in resting human lower leg muscles was measured noninvasively using 1H-nuclear magnetic resonance spectroscopy at 3.0 and 1.5 T on five normal male volunteers aged 30.6 ⫾ 10.4 yr (mean ⫾ SD). Using 1H-nuclear
magnetic resonance spectroscopy, water, carnosine, and creatine in
the muscles could be simultaneously analyzed. Carnosine contains
imidazole protons. The chemical shifts of water and carnosine
imidazole protons relative to creatine could be used for estimating
temperatures and ␣-imidazole, respectively. Using the chemical
shift, the values of temperature in gastrocnemius (Gast) and soleus
muscles at ambient temperature (21–25°C) were estimated to be
35.5 ⫾ 0.5 and 37.4 ⫾ 0.6°C (means ⫾ SE), respectively (significantly different; P ⬍ 0.01). The estimated values of ␣-imidazole
in these muscles were 0.620 ⫾ 0.007 and 0.630 ⫾ 0.013 (means ⫾
SE), respectively (not significant). Alternation of the surface temperature of the lower leg from 40 to 10°C significantly changed the
temperature in Gast (P ⬍ 0.0001) from 38.1 ⫾ 0.5 to 28.0 ⫾ 1.2°C,
and the ␣-imidazole in Gast decreased from 0.631 ⫾ 0.003 to
0.580 ⫾ 0.011 (P ⬍ 0.05). However, the values of ␣-imidazole and
the temperature in soleus muscles were not significantly affected
by this maneuver. These results indicate that the ␣-imidazole in
Gast changed significantly with alternation in muscle temperature
(r ⫽ 0.877, P ⬍ 0.00001), and its change was estimated to be
0.0058/°C.
TEMPERATURE AND ␣-IMIDAZOLE IN HUMAN LOWER LEG MUSCLES
Fig. 1. An axial MRI in the lower leg muscles. NMR spectra were observed
at boxes A and B. The voxel sizes were 25 (1 ⫻ 5 ⫻ 5 cm) to 36 (1 ⫻ 6 ⫻ 6
cm) ml. Boxes A and B are gastrocnemius and soleus muscles, respectively.
J Appl Physiol • VOL
Fig. 2. Typical 1H-NMR spectra of human lower leg muscles (gastrocnemius)
at 3.0 T. The top spectrum was obtained under water suppression, and the
bottom without water suppression. The chemical shift (␦) was calculated based
on the shift of creatine [3.03 parts/million (ppm) from the dimethylsilapentane
sodium sulfonate (DSS) methyl signal]. Acquisition parameters were as follows: 3,000-ms repetition time, 144-ms echo time, 2,500-Hz spectral width,
and 256 aqusitions for the top spectrum and 8 for the bottom spectrum.
taneously, and the correlation between the temperature and
␣-imidazole could be assessed.
A part of this study has been reported at the First International Conference on Biomedical Spectroscopy: From Molecules to Men (2002, Cardiff, UK) (38).
MATERIALS AND METHODS
Materials and subjects. Five volunteers aged 30.6 ⫾ 10.4 yr
(mean ⫾ SD) were enrolled in this magnetic resonance spectroscopy
study. All of these volunteers were healthy men, and NMR spectra
were obtained from their legs at resting supine position. Phantom
solutions and excised pig skeletal muscles were also used in this study
for calibration of ␣-imidazole and temperature with NMR spectroscopy. The data for these calibrations are described in RESULTS in detail.
The Ethics Committee of Iwate Medical University has approved the
experimental protocols. NMR measurements for volunteers were
performed after obtaining appropriate consent forms signed by the
volunteers.
Spectroscopy and data processing in humans. 1H-NMR spectroscopy was performed at 3.0 and 1.5 T (General Electric, SIGNA
Horizon LX 3T and LX 1.5 T). The spectra of volunteer’s lower leg
were obtained from gastrocnemius (Gast) and soleus muscles (Sol),
which were identified by MRI. Figure 1 shows an example of lower
leg MRI. The magnetic resonance spectra were obtained from blocked
A and B regions. These regions indicate Gast and Sol, respectively.
Typical 1H-NMR spectra of a human Gast at 3.0 T are represented
in Fig. 2.
The upper spectrum shown in Fig. 2 was obtained under water
suppression for estimation of the ␣-imidazole and the lower one
without water suppression for estimation of the temperature. The
signal position of the imidazole C-2 proton and that of creatine methyl
protons, as shown in the upper spectrum of Fig. 2, were used for the
estimation of ␣-imidazole. The signal position of water and that of
creatine methyl protons, as shown in the lower spectrum of Fig. 2,
were used for the estimation of temperature. There were no differ-
98 • JANUARY 2005 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on June 17, 2017
atine, creatinine), cholines (choline, phosphoryl choline, glycerophosphocholine) and carnosine could be simultaneously
analyzed (1, 3, 36, 37, 39). Carnosine is abundant in human
muscles and contains imidazole protons. The imidazole NMR
signal of carnosine is detectable in human muscles in vivo (14,
26, 38). Therefore, we could estimate the ␣-imidazole of
carnosine using 1H-NMR spectroscopy. Hitzig et al. (13)
applied this method for intact amphibian skeletal muscle and
showed that ␣-imidazole remained constant with alterations in
body temperature. Binzoni et al. (4) examined the change in pH
in human muscles with changes in temperature (⫺0.016 pH
units/°C) and suggested the applicability of the alphastat hypothesis. However, direct measurement of ␣-imidazole in human tissues with changing temperature had hardly been
achieved. Thus we tried to measure ␣-imidazole directly with
changing muscle temperature using the idea provided by Hitzig
et al. (13).
Several noninvasive thermometers employing NMR techniques have been proposed based on the temperature dependence of NMR parameters, such as relaxation time (30),
diffusion coefficient (10), chemical shift (2, 6, 8, 20), or the
proton frequency method (11, 15, 19). Reliability with an error
range of less than ⫾1°C is required for application of these
techniques to the estimation of the temperature in the human
body under physiological conditions (21, 23, 32, 33). Cady et
al. (6) and Corbett et al. (8) have reported novel techniques for
estimating the absolute temperature of the human brain under
physiological conditions. They used the chemical shift difference between the water and N-acethylaspartate as the temperature probe. N-acethylaspartate is one of the endogenous metabolites, and, therefore, noninvasive temperature estimation
could be done. We used a similar approach to estimate temperature in human lower leg muscles in which the chemicalshift difference between water and creatine was used as the
probe. The temperature change of 1°C causes a shift of water
signal of ⬃0.01 part per million (ppm) (see RESULTS).
The temperature and ␣-imidazole, therefore, could be estimated by 1H-NMR spectroscopy in the human muscles simul-
283
284
TEMPERATURE AND ␣-IMIDAZOLE IN HUMAN LOWER LEG MUSCLES
RESULTS
Calibration for estimation of temperature. Calibration lines
for estimation of the muscle temperature were obtained using
16 excised pig skeletal muscles. The temperature of the muscles was monitored by a copper-constantan thermocouple (absolute accuracy: ⫾0.1°C). The temperature drift of the pig
muscles during measurements was ⬍1.0°C. Although the excised pig muscles had been stored in a refrigerator until use, the
energy statuses of the muscles were not controlled. The pH of
the pig muscles came to near 6. The change of pH, however,
has little effect on the chemical shift difference between water
and creatine. Relationships between actual temperature measured by copper-constantan thermocouple and NMR spectra
are shown in Fig. 3.
The difference between the chemical shifts for choline or
creatine and water showed a linear function of increasing
temperature from 20 to 40°C (slope: ⫺0.00971 and ⫺0.00999
ppm/°C; r ⫽ ⫺0.996 and ⫺0.996 for choline and creatine,
respectively). The slopes of these lines were comparable to
those of the temperature dependence of the water signal (12, 27).
J Appl Physiol • VOL
Fig. 3. Calibration lines for temperature estimation. These lines were obtained
using excised pig skeletal muscles at different temperatures. The temperature
of the muscle was monitored by a copper-constantan thermocouple. The drift
in muscle temperature during measurements was ⬍1.0°C. The ordinate is the
chemical shift difference between water and choline or creatine signals (␦).
These chemical shift differences showed a linear function of increasing
temperature from 20 to 40°C (slope: ⫺0.00971 and ⫺0.00999 ppm/°C; r ⫽
⫺0.996 and ⫺0.996 for choline and creatine, respectively).
Estimation of ␣-imidazole. The NMR signals of creatine and
carnosine were observed using standard buffer solutions
(Wako Pure Chemical Industries) at several temperatures. A
small amount (⬃1 mM) of dimethylsilapentane sodium sulfonate, creatine, and carnosine was dissolved into buffer solutions. The pH of each buffer solution was 1.68 (oxalate buffer),
4.01 (phthalate buffer), 6.86 (phosphate buffer), 7.41 (phosphate buffer), 9.18 (tetraborate buffer), and 10.01 (carbonate
buffer) at 25°C. To confirm the signal position of imidazole,
dimethylsilapentane sodium sulfonate, creatine, and carnosine
were added to the buffer solutions. The change in pH of the
buffer solutions induced by adding these chemicals was ⬍0.03
pH units. The buffer solutions were cooled or warmed to
several temperatures, and the spectra were observed. The
temperature drifts of the buffers during NMR measurements
were ⬍0.5°C. In this experiment, the accuracy and stability of
the pH were most important. Therefore, we used the standard
buffer solutions, sold to calibrate pH meters, and only a little
amount of metabolites was added to the buffers. Although the
ionic strengths of the buffer we used and the intracellular fluid
of human muscles were not equivalent, the effect of ions to the
chemical shift difference between carnosine and creatine is
little (26). Therefore, we could estimate ␣-imidazole of human
muscles by using the chemical shift data of carnosine and
creatine obtained with buffer solutions. The chemical shift of
the imidazole C-2 proton in carnosine, relative to the methyl
protons of creatine, changed with the temperature at pH 6.8 and
7.4, as shown in Fig. 4A.
The chemical shift values of the imidazole C-2 proton in
carnosine at 20, 30, and 40°C were calculated using the linear
regression lines shown in Fig. 4A and were fitted by Eq. 3 (1)
pH ⫽ pKIm ⫹ log[(␦acid ⫺ ␦o)/(␦o ⫺ ␦base)]
(3)
where ␦o equals the chemical shift of the imidazole (C-2
proton) signal relative to creatine (⫺CH3 signal), and ␦acid and
98 • JANUARY 2005 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on June 17, 2017
ences in measured chemical shift or calculated temperatures when the
two different field strengths were used. The standard deviation of the
chemical shift difference was less than ⫾0.002 ppm at both field
strengths.
The total sampling time for obtaining the spectra shown in Fig. 2
was ⬃14 min. After the NMR spectra at ambient temperature (21–
25°C) were obtained from the lower leg muscles, these muscles were
cooled or warmed from the exterior using cooling or warming elements. These elements were commercial cold- and heat-reserving
materials and had been cooled to ⫺10°C or warmed to 45°C before
use. A towel was inserted between the element and the leg. The NMR
spectra were obtained 10 – 40 min later from the beginning of cooling
or warming. The skin surface temperatures during cooling and warming were ⬃10 and 40°C, respectively. The changes of skin temperature during measurement were ⬍2°C. Acquisition parameters were as
follows: at 3.0 T, 3,000-ms repetition time, 30- to 144-ms echo time,
2-K data size, 2,500-Hz spectral width, 8 –256 acquisitions; at 1.5 T,
1,500-ms repetition time, 30- to 288-ms echo time, 2-K data size,
2,500-Hz spectral width, 16 –512 acquisitions. The voxel sizes were
25–36 ml for human lower leg muscles and 8 ml for pig skeletal
muscles and phantom solutions. The raw data were transferred from a
magnetic resonance scanner to a personal computer and processed on
the personal computer (zero filling; 2 K, apodization; 1 Hz, fast
Fourier transform, and peak fitting). The phase of the spectrum was
determined manually. NMR relaxation times (T1 and T2) are generally
shorter at 1.5 T than at 3 T, although we did not determine those
accurately. Therefore we adopted repetition time ⫽ 1,500 and 3,000
ms at 1.5 and 3 T, respectively. Then the accumulation number at 1.5
T was two times that at 3 T. The signal-to-noise ratio of the spectrum
obtained at 1.5 T was comparable to that at 3 T.
The NMR spectra were analyzed by an automatic curve-fitting
procedure and decomposed into Lorentzian peak components
(GRAMS/AI, Galactic Industries, Salem, NH). The fittings were done
for three separate regions of 1) imidazole protons of carnosine, 2)
water protons, and 3) methyl protons of choline and creatine. In the
fitting procedures of regions 1 and 3, a baseline correction was
performed. The origin of this baseline is the tail parts of water and
lipid signals. As a result of this fitting procedure, the height, full width
at half-maximum, position, and area of each peak were determined.
Statistical analyses. Scheffé’s test was used for post hoc multiple
comparisons between different pairs of means after one-way ANOVA
indicated significant differences. The correlations between the temperature and ␣-imidazole were examined by Pearson’s test.
TEMPERATURE AND ␣-IMIDAZOLE IN HUMAN LOWER LEG MUSCLES
285
Fig. 4. Chemical shift of the imidazole C-2 proton in
carnosine relative to creatine methyl protons. A: chemical shift as a function of temperature at several pH
values. Measured data are shown as points, and the line
results from linear regression of the experimental data.
B: chemical shift as a function of pH at 20, 30, 40°C.
Calculated data with the regression line in A are shown
as points, and the curve results from a fit of the calculated data to Eq. 2.
warmed conditions (40°C). Temperatures of Gast and Sol at
ambient conditions were 35.5 ⫾ 0.5 and 37.4 ⫾ 0.6°C
(means ⫾ SE), respectively (significantly different; P ⬍ 0.01).
These values are comparable to the previously reported values
(4, 32, 35). Alternation of the surface temperature of the lower
leg from 40 to 10°C significantly changed the temperature in
Gast (P ⬍ 0.0001) from 38.1 ⫾ 0.5 to 28.0 ⫾ 1.2°C but
induced no significant change in Sol (Table 1).
Estimated ␣-imidazole of volunteers. The values of ␣-imidazole in Gast and Sol at ambient temperature were estimated
to be 0.620 ⫾ 0.007 and 0.630 ⫾ 0.013 (means ⫾ SE),
respectively, using Eq. 2. On lowering the surface temperature
of the lower leg from 40 to 10°C, the ␣-imidazole decreased
significantly in Gast (P ⬍ 0.05) from 0.631 ⫾ 0.002 to 0.580 ⫾
0.011, but not in Sol (Table 1). Because the acid and basic
chemical shifts of imidazole are unaffected by temperature, we
could estimate the dissociation of imidazole (␣-imidazole)
independently of the temperature.
Correlation between ␣-imidazole and temperature. The
present study demonstrated that the temperatures and ␣-imidazole in the lower leg muscles could be estimated simultaneously. Finally, we examined the correlation between the
Table 1. Estimated values of the temperature and ␣-imidazole
in human lower leg muscles during cooling and
warming and at ambient temperature
Temperature, °C
Cooling
Ambient
Warming
␣-Imidazole
Cooling
Ambient
Warming
Gastrocnemius
Soleus
28.0⫾1.2
35.5⫾0.5‡
38.1⫾0.5‡
36.6⫾0.5
37.4⫾0.6
37.6⫾0.9
0.580⫾0.011
0.620⫾0.007*
0.631⫾0.003†
0.601⫾0.017
0.630⫾0.013
0.611⫾0.009
Values are means ⫾ SE. Significant difference between means of cooling
and ambient and of cooling and warming: *P ⬍ 0.05; †P ⬍ 0.01; ‡P ⬍
0.0001.
J Appl Physiol • VOL
Fig. 5. Correlation plots of the temperature and ␣-imidazole in the gastrocnemius examined with Pearson’s test. The slope is 0.0058/°C (r ⫽ 0.877, P ⬍
0.00001).
98 • JANUARY 2005 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on June 17, 2017
␦base are the limiting acidic and basic chemical shifts, respectively. The fitting curves are shown in Fig. 4B. The pH of the
buffer at each temperature was corrected according to the
technical data for the buffer (Wako Pure Chemical Industries).
In agreement with the previous reports (1, 9, 26), the limiting
chemical shifts were unaffected by temperature. The ␦acid and
␦base were estimated to be 5.533 and 4.625 relative to the
creatine signal, respectively. Therefore, if imidazole and creatine signals can be observed by 1H-NMR spectroscopy, ␣-imidazole can be estimated by Eq. 2 independently of the temperature. The value of pKIm was also estimated by using the
fitting curves shown in Fig. 4B to be 7.348 – 0.0154 T, where T
is temperature in degrees Celsius. The values of pKIm and
⌬pKIm/⌬T are comparable to those in previous reports (1, 9,
13, 26).
Estimated temperature of volunteers. Using the calibration
line shown in Fig. 3, the temperatures of human muscles could
be measured noninvasively. We used the creatine signal as an
internal chemical shift reference, since the signal-to-noise ratio
of this signal is higher than that for choline and the distortion
of the signal caused by the large water signal is minimal.
Linear regression analysis yielded ␦ ⫽ 2.0505– 0.009992 T,
where ␦ is the water proton signal chemical shift relative to the
methyl signal of creatine. The estimated values of temperature
in the Gast and Sol are shown in Table 1.
Cooling, ambient, and warming in Table 1 represent the
muscle temperature at cooled (10°C), ambient (21–25°C), and
286
TEMPERATURE AND ␣-IMIDAZOLE IN HUMAN LOWER LEG MUSCLES
␣-imidazole and the temperature in Gast. The ␣-imidazole in
Gast significantly correlated with the temperature (r ⫽ 0.877,
P ⬍ 0.00001) as shown in Fig. 5. The slope calculated was
0.0058/°C.
DISCUSSION
1
J Appl Physiol • VOL
GRANTS
This work was partly supported by Grants-in-Aid for Advanced Medical
Science Research from the Ministry of Education, Culture, Sports, Science and
Technology of Japan, and Grants-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan.
REFERENCES
1. Arús C, Bárány M, Westler WM, and Markley JL. 1H NMR of intact
muscle at 11 T. FEBS Lett 165: 231–237, 1984.
2. Arús C, Chang YC, and Bárány M. N-acetylaspartate as an intrinsic
thermometer for 1H NMR of brain slices. J Magn Reson 63: 376 –379,
1985.
3. Beech JS, Nolan KM, Iles RA, Cohen RD, Williams SCR, and Evans
SJW. The effects of sodium bicarbonate and a mixture of sodium bicarbonate and carbonate (“carbicarb”) on skeletal muscle pH and hemodynamic status in rats with hypovolemic shock. Metabolism 43: 518 –522,
1994.
4. Binzoni T, Hiltbrand E, Terrier F, Cerretelli P, and Delpy D. Temperature dependence of human gastrocnemius pH and high-energy phosphate concentration by noninvasive techniques. Magn Reson Med 43:
611– 614, 2000.
5. Burton RF. Temperature and acid-base balance in ectothermic vertebrates: the imidazole alphastat hypotheses and beyond. J Exp Biol 205:
3587–3600, 2002.
6. Cady EB, D’Souza PC, Penrice J, and Lorek A. The estimation of local
brain temperature by in vivo 1H magnetic resonance spectroscopy. Magn
Reson Med 33: 862– 867, 1995.
7. Cameron JN. Acid-base status of fish at different temperatures. Am J
Physiol Regul Integr Comp Physiol 246: R452–R459, 1984.
8. Corbett RJT, Laptook AR, Tollefsbol G, and Kim B. Validation of a
noninvasive method to measure brain temperature in vivo using 1H NMR
spectroscopy. J Neurochem 64: 1224 –1230, 1995.
9. Damon BM, Hsu AC, Stark HJ, and Dawson MJ. The carnosine C-2
proton’s chemical shift reports intracellular pH in oxidative and glycolytic
muscle fibers. Magn Reson Med 49: 233–240, 2003.
10. De Poorter J, De Wagter C, De Deene Y, Thomsen C, Ståhlberg F, and
Achten E. The proton-resonance-frequency-shift method compared with
molecular diffusion for quantitative measurement of two-dimensional
time-dependent temperature distribution in a phantom. J Magn Reson
B103: 234 –241, 1994.
11. De Poorter J, De Wagter C, De Deene Y, Thomsen C, Ståhlberg F, and
Achten E. Noninvasive MRI thermometry with the proton resonance
98 • JANUARY 2005 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on June 17, 2017
H-NMR spectroscopy provided a noninvasive method of
estimating the temperature and ␣-imidazole in human lower
leg muscles simultaneously. The 1H-NMR chemical shift differences between the water and creatine of pig skeletal muscles
vary in a linear fashion with temperature, and these differences
could be used to determine muscle temperature. The temperature-dependent change in chemical shift obtained from pig
skeletal muscles was nearly identical to that obtained from
water (12, 27). Because we applied the results of pig skeletal
muscles to those of human muscles, the measured temperature
of human muscle with 1H-NMR might not be absolutely
accurate. However, large merit of 1H-NMR spectroscopy is its
ability to estimate temperatures at various regions of interest
noninvasively. Our results showed that the temperature in Sol
was 1.9°C higher than that in Gast at ambient temperature and
indicated that the temperature in Gast was significantly affected by surrounding conditions but not in Sol.
Hitzig et al. (13) developed a method for direct measurement
of ␣-imidazole in intact, unanesthetized amphibian skeletal
muscles using 1H-NMR spectroscopy and reported that ␣-imidazole remained constant with alterations in body temperature. Their results support the alphastat hypothesis. We applied
their direct method on human lower leg muscles. Our results,
however, showed that ␣-imidazole in human Gast decreased
gradually on cooling from the surface of the lower leg and also
that the values correlated with the muscle temperature significantly. One of the reasons for this discrepancy may be as
follows. Hitzig et al. (13) used water signal as the internal
reference standard for the chemical shift of the carnosine
peaks. The chemical shift of the water signal changes with a
temperature change of ⫺0.01 ppm/°C (12, 27). Therefore, the
chemical shift of carnosine in amphibian muscles should
change with alterations in body temperature. The change of
⫺0.01 ppm/°C in the carnosine signal corresponds to the
change of 0.01/°C in the ␣-imidazole. This value is about two
times larger than that of our results (0.0058/°C). It could be
considered that the ␣-imidazole estimated by us and also by
Hitzig et al. changes with alternating muscle temperature. In
fact, if the chemical shift is calculated in reference to the water
signal, the change in the imidazole chemical shift with temperature is very small, and it may be hard to pick up the change
of the imidazole signal in vivo.
The temperature dependence of ␣-imidazole estimated by us
corresponds to ⫺0.005 pH units/°C. This value is one-third of
that proposed in the alphastat hypothesis (⫺0.015 pH units/
°C). Therefore, this result may indicate that some buffering
systems with small ⌬pK/⌬T values contribute in human muscles with changes in muscle temperature (7, 22). Inorganic
phosphate is one of the candidates with a small ⌬pK/⌬T value,
i.e., ⫺0.006 to ⫺0.001 (17, 31). Although concentrations of
inorganic phosphate are often very low, inorganic phosphate
can sometimes contribute significantly to intracellular buffering. It is considered that the concentration of inorganic phos-
phate is raised to some extent by hydrolysis of ATP and
phosphocreatine (16).
Binzoni et al. (4) reported on the pH in the human Gast over
a temperature range from 30 to 36°C using 31P-NMR spectroscopy. They showed that the pH varies, as was suggested by the
alphastat hypothesis (⫺0.016 pH units/°C). Our results were
obtained with a wider temperature range, between 23 and 40°C
(⫺0.005 pH units/°C). Our data on ␣-imidazle on cooling was
significantly smaller than those of the other two protocols
(Table 1). The values at ambient temperature and on warming
were not significantly different (Table 1). Therefore, our results
at high temperatures may be consistent with the data of Binzoni
et al. The decrease of ␣-imidazole at lower temperatures may
be significant (Fig. 5).
Carnosine is distributed in the cytosol but not in mitochondria or myofibrils (39). Therefore, our results may indicate that
the ␣-imidazole of human muscle cytosol changes with alternating muscle temperature, and, therefore, some buffering
systems with small ⌬pK/⌬T values other than imidazole could
contribute in muscle cytosol on changes in temperature.
In summary, we have here described a method using 1HNMR spectroscopy for measuring the temperature and ␣-imidazole in resting human lower leg muscles noninvasively and
indicated that ␣-imidazole in the muscles correlates with muscle temperature.
TEMPERATURE AND ␣-IMIDAZOLE IN HUMAN LOWER LEG MUSCLES
12.
13.
14.
15.
16.
17.
18.
20.
21.
22.
23.
24.
25.
J Appl Physiol • VOL
26. Pan JW, Hamm JR, Rothman DL, and Shulman RG. Intracellular pH
in human skeletal muscle by 1H-NMR. Proc Natl Acad Sci USA 85:
7836 –7839, 1988.
27. Peters RD, Hinks RS, and Henkelman RM. Ex vivo tissue-type independence in proton-resonance frequency shift MR thermometry. Magn
Reson Med 40: 454 – 459, 1998.
28. Reeves RB. An imidazole alphastat hypotesis for vertebrate acid-base
regulation: tissue carbon dioxide content and body temperature in bullfrogs. Respir Physiol 14: 219 –236, 1972.
29. Sakamoto T, Kurosawa H, Shin’oka T, Aoki M, and Isomatsu Y. The
influence of pH strategy on cerebral and collateral circulation during
hypothermic cardiopulmonary bypass in cyanotic patients with heart
disease: results of a randomized trial and real-time monitoring. J Thorac
Cardiovasc Surg 127: 12–19, 2004.
30. Schwarzbauer C, Zange J, Adolf H, Deichmann R, Nöth U, and Haase
A. Fast measurement of temperature distributions by rapid T1 mapping. J
Magn Reson 106: 178 –180, 1995.
31. Seo Y, Murakami M, Watari H, Imai Y, Yoshizaki K, Nishikawa H,
and Morimoto T. Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological
system. J Biochem (Tokyo) 94: 729 –734, 1983.
32. Shellock FG, Swan HJC, and Rubin SA. Muscle and femoral vein
temperatures during short-term maximal exercise in hear failure. J Appl
Physiol 58: 400 – 408, 1985.
33. Shiraki K, Sagawa S, Tajima F, Yokota A, Hashimoto M, and
Brengelmann GL. Independence of brain and tympanic temperatures in
an unanesthetized human. J Appl Physiol 65: 482– 486, 1988.
34. Swain JA, McDonald TJ Jr, Robbins RC, and Balaban RS. Relationship of cerebral and myocardial intracellular pH to blood pH during
hypothermia. Am J Physiol Heart Circ Physiol 260: H1640 –H1644, 1991.
35. Williams DB and Karl RC. Measurement of deep muscle temperature in
ischemic limbs. Am J Surg 139: 503–507, 1980.
36. Williams SR, Gadian DG, Proctor E, Sprague DB, Talbot DF, Young
IR, and Brown FF. Proton NMR studies of muscle metabolites in vivo.
J Magn Reson 63: 406 – 412, 1985.
37. Yoshioka Y, Masuda T, Nakano H, Miura H, Nakaya S, Itazawa SI,
and Kubokawa M. In vitro 1H-NMR spectroscopic analysis of metabolites in fast- and slow-twitch muscles of young rats. Magn Reson Med Sci
1: 7–13, 2002.
38. Yoshioka Y, Oikawa H, Ehara S, Inoue T, Ogawa A, Kambara Y,
Itazawa SI, and Kubokawa M. Noninvasive estimation of temperature
and pH in human lower leg muscles using 1H nuclear magnetic resonance
spectroscopy. Spectroscopy 16: 183–190, 2002.
39. Yoshizaki K, Seo Y, and Nishikawa H. High-resolution proton magnetic
resonance spectra of muscle. Biochim Biophys Acta 678: 283–291, 1981.
98 • JANUARY 2005 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.33.6 on June 17, 2017
19.
frequency (PRF) method: in vivo results in human muscle. Magn Reson
Med 33: 74 – 81, 1995.
Hindman JC. Proton resonance shift of water in the gas and liquid states.
J Chem Phys 44: 4582– 4592, 1966.
Hitzig BM, Perng WC, Burt T, Okunieff P, and Johnson DC. 1H-NMR
measurement of fractional dissociation of imidazole in intact animals.
Am J Physiol Regul Integr Comp Physiol 266: R1008 –R1015, 1994.
Hu J, Willcott MR, and Moore GJ. Two-dimensional proton chemicalshift imaging of human muscle metabolites. J Magn Reson 126: 187–192,
1997.
Ishihara Y, Calderon A, Watanabe H, Okamoto K, Suzuki Y, Kuroda
K, and Suzuki Y. A precise and fast temperature mapping using water
proton chemical shift. Magn Reson Med 34: 814 – 823, 1995.
Johnson DC, Burt CT, Perng WC, and Hitzig BM. Effects of temperature on muscle pHi and phosphate metabolites in newts and lungless
salamanders. Am J Physiol Regul Integr Comp Physiol 265: R1162–
R1167, 1993.
Kost GJ. pH standardization for phosphorus-31 magnetic resonance heart
spectroscopy at different temperatures. Magn Reson Med 14: 496 –506,
1990.
Krause WL, Kazemi H, and Burton MD. Potential role for imidazole in
the rhythmic respiratory activity of the in vitro neonatal rat brainstem.
Neurosci Lett 251: 153–156, 1998.
Kuroda K, Suzuki Y, Ishihara Y, Okamoto K, and Suzuki Y. Temperature mapping using water proton chemical shift obtained with 3DMRSI: feasibility in vivo. Magn Reson Med 35: 20 –29, 1996.
Lutz NW, Kuesel AC, and Hull WE. A 1H-NMR method for determining temperature in cell culture perfusion systems. Magn Reson Med 29:
113–118, 1993.
Mariak Z, Lewko J, Luczaj J, Polocki B, and White MD. The
relationship between directly measured human cerebral and tympanic
temperatures during changes in brain temperatures. Eur J Appl Physiol 69:
545–549, 1994.
Mark FC, Bock C, and Pörtner HO. Oxygen-limited thermal tolerance
in antarctic fish investigated by MRI and 31P-MRS. Am J Physiol Regul
Integr Comp Physiol 283: R1254 –R1262, 2002.
Mellergård P. Intracerebral temperature in neurosurgical patients: intracerebral temperature gradients and relationships to consciousness level.
Surg Neurol 43: 91–95, 1995.
Mortola JP and Frappell PB. Ventilatory responses to changes in
temperature in mammals and other vertebrates. Annu Rev Physiol 62:
847– 874, 2000.
Nattie EE. The alphastat hypothesis in respiratory control and acid-base
balance. J Appl Physiol 69: 1201–1207, 1990.
287