- Email: [email protected]
Age-dependent alteration of metabolic response to photic stimulation in the human brain measured by 31P MR-spectroscopy
Brain Research 818 Ž1999. 72–76
Research report
Age-dependent alteration of metabolic response to photic stimulation in the human brain measured by 31 P MR-spectroscopy Jun Murashita a , Tadafumi Kato b
b, )
, Toshiki Shioiri a , Toshiro Inubushi c , Nobumasa Kato
a,b
a Department of Psychiatry, Shiga UniÕersity of Medical Science, Otsu, Shiga, 520-2121, Japan Department of Neuropsychiatry, Faculty of Medicine, UniÕersity of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo, 113-8655, Japan c Molecular Neurobiology Research Center, Shiga UniÕersity of Medical Science, Otsu, Shiga, 520-2121, Japan
Accepted 24 November 1998
Abstract Effects of photic stimulation ŽPS. on energy metabolism were examined in the occipital cortex of 25 healthy volunteers aged 23–69 years old using phosphorus-31 magnetic resonance spectroscopy Ž31 P-MRS.. A significant effect of photic stimulation was found only for intracellular pH Ž p - 0.05 by repeated measures analysis of variance. but not for any peak area ratios. An interaction between intracellular pH and age were statistically significant Ž p - 0.005., and the interaction between phosphocreatine and age was close to significance Ž p s 0.06.. In subjects aged more than 40 years old, phosphocreatine was significantly decreased during the photic stimulation Ž p - 0.05, multiple comparison by Dunnett’s method., and intracellular pH tended to be elevated just after the stimulation Ž p s 0.07.. There were no significant changes in these values in younger subjects. These results suggest that no significant effect of photic stimulation on brain energy metabolism was found in younger subjects, and that significant effects of photic stimulation on intracellular pH and phosphocreatine were found in middle-aged subjects. Metabolic response of the human brain to photic stimulation may be dependent on age. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus-31 magnetic resonance spectroscopy; Brain energy metabolism; Creatine phosphate; Intracellular pH; Membrane phospholipid; Photic stimulation
1. Introduction Since Chance et al. w1x have first applied nuclear magnetic resonance spectroscopy ŽMRS. to human skeletal muscles, this technique has been widely used for physiological studies of energy metabolism in living human organs. In human skeletal muscles, exercise induces reduction of phosphocreatine ŽPCr., increase of inorganic phosphate ŽPi., and decrease of intracellular pH w3,9,10x. Measurement of exercise-induced alteration of muscle energy metabolism by means of 31 P-MRS has been used for diagnosis of metabolic muscular disorders w10x. 31 P-MRS has also been used for the investigation of brain energy metabolism in many kinds of neurological or psychiatric disorders w11,16x. In these studies, 31 P-MRS
) Corresponding author. Fax: q81-3-5800-6894; E-mail: [email protected]
was examined at rest with no particular physiological stimulation. All the findings observed by 31 P-MRS, such as decrease of PCr, were not specific to a particular neuropsychiatric disease. If a physiological stimulation was applied, it might help us to distinguish different metabolic abnormalities in the human brain. There are only few reports of physiological alteration of energy metabolism detected by 31 P-MRS in the human brain. Van Rijen et al. w15x have reported that hyperventilation causes increase of intracellular pH detected by 31 PMRS. Sappey-Marinier et al. w13x reported that PCr in the occipital cortex was significantly decreased during photic stimulation ŽPS. in the brain in six normal control subjects. Intracellular pH was slightly increased during stimulation although it was not statistically significant. The authors w6x also reported that PCr was decreased and intracellular pH tended to be increased during PS in nine normal volunteers. They also reported that PCr returned to a normal level soon after the stimulation. In contrast to these two studies reporting effects of PS on energy metabolism in the
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 8 5 - 2
J. Murashita et al.r Brain Research 818 (1999) 72–76
occipital lobes, Chen et al. w2x have reported no significant effect of PS in five subjects. In this study, 25 normal volunteers aged 23–69 years old were examined by 31 P-MRS during 12 min of photic stimulation to confirm and extend our previous report on photic stimulation-induced alteration of brain phosphorus metabolism.
2. Subjects Subjects were 25 normal volunteers who were recruited from hospital staffs and students. They were 37.0 " 14.0 Žmean " S.D., 23–69. years old. Thirteen were female and 12 were male. The data in nine of these subjects were reported in the previous report w6x. Their past history and family history were examined by a questionnaire and an interview by a physician, and they did not have neuropsychiatric disorder, epilepsy, general medical disease, and opthalmologic disease. They did not have any major structural abnormalities in the brain examined by T1-weighted magnetic resonance imaging ŽMRI.. They did not have any medications at the time of the examination. 31 P-MRS was examined between 1800 and 2000 h before supper. They gave written informed consent to the study. This study was approved by the ethical committee of the Shiga University of Medical Science.
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3. Method 3.1.
31
P-magnetic resonance spectroscopy
MRS data were acquired with a SIGNA 1.5 Tesla MR system ŽGE Medical System, Milwaukee. with a standard spectroscopy package and surface coils for 1 H and 31 P supplied by the manufacturer. The surface coil for proton was horizontally set on the stretcher. The subjects lay so as to their occipital region is on the coil. Their heads were fixed by molded sponge. The position of the head was monitored by 1 H-magnetic resonance imaging ŽMRI. in a sagittal view. The head was repositioned when necessary, so that the occipital lobes were in the center of the coil. Slice selection was applied to make a volume of interest ŽVOI. of 5 cm thick axial slice including the occipital lobes to eliminate contamination from signals from neck muscles. The extent of VOI was visualized by the 1 H-MRI with the surface coil. The region examined was predominantly the occipital lobes. The magnetic field over the VOI was optimized using the water signal to establish the line width at less than 10 Hz. The surface coil for 1 H was then replaced with the coil for 31 P. The flip angle in the VOI was set to 908 in the phantom experiment. The 31 P-MR spectra were obtained with 1024 data points, spectral width of 4000 Hz, repetition time ŽTR. of 3 s, delay time of 1.5 ms, and center frequency of 25.84 MHz. One hundred
Fig. 1. Stacked plot of phosphorus-31 magnetic resonance spectra Ž31 P-MRS.. Six spectra serially recorded in one experiment are shown. Peaks are assigned to phosphomonoester Ž6.5 ppm., inorganic phosphate ŽPi, 4.8 ppm., phosphodiester Ž3 ppm., phosphocreatine ŽPCr, 0 ppm., and g-, a-, and b-adenosine triphosphate Žy3, y8, y16 ppm.. The bottom spectrum is obtained before photic stimulation, and other spectra obtained thereafter are stacked behind this spectrum. ppm s parts per million.
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J. Murashita et al.r Brain Research 818 (1999) 72–76
acquisitions were averaged. Each spectrum was acquired in 6 min. Six spectra were obtained in each session, during the course of photic stimulation. Free induction decay ŽFID. were transported to a SPARC2 work station ŽSUN micro system, California., and processed with OMEGA CSI software ŽGE Medical Systems.. FIDs were exponentially filtered with 15 Hz-line broadening. After Fourier transformation was applied, the first-order phase correction with fixed values, manual zero-order phase correction, and baseline correction with polynomial interpolation were applied. The same values for phase correction and the same polynomial function were used for processing of all six spectra in one session. The 31 P-MR spectra are shown in Fig. 1. The following seven peaks were resolved: phosphomonoester ŽPME., inorganic phosphate ŽPi., phosphodiester ŽPDE., PCr and three resonances from adenosine triphosphate Ž g, a and b-ATP.. The peak areas were calculated by automatic peak fitting with Lorentzian curves by SIMPLEX method using a software package which was programmed in our laboratory. The g-ATP peak was simulated by two peaks. The peak areas are shown as percent values of the total phosphorus signal. Intracellular pH was calculated from the chemical shift difference of PCr and Pi. 3.2. Photic stimulation The method of photic stimulation was minutely described in our previous article w6x. In brief, white light flashing at 10 Hz was used to stimulate the occipital cortex. A metal halide lamp ŽUshio Electric, Kobe, Japan. was used to emit white light for photic stimulation. The light was interrupted by an aluminum wheel with two holes which rounds at 5 Hz. Light was guided into the MR room using optical fibers. The optical fibers were divided into six segments, and each segment was attached to a titan dioxide coated glass rod with diameter of 3 cm and length of 15 cm ŽAsahi Glass, Tokyo, Japan., which scatters the light from optical fibers. These six glass rods set just before eyes of the subject to cover most of the visual field of the subject. Strength of the light at the position of eyes of the subjects was 5500 lx.
3.4. Statistics All data were reported as mean " S.D. For statistical analysis, repeated measures analysis of variance ŽANOVA. with factor of PS Žsix points in the course of the experiment. and a covariate of age was used. Multiple comparison by Dunnett’s method was used to examine changes of the MRS values during the course of experiments in comparison with the value before the photic stimulation. Pearson’s coefficient of correlation and Student’s t-test were also supplementary used.
4. Results 4.1. Effects of photic stimulation In 25 normal volunteers, repeated measures ANOVA revealed significant effects of PS on intracellular pH Ž F s 3.0, p - 0.05.. A significant interaction between PS and age was found for intracellular pH Ž F s 4.6, p - 0.005.. Interaction between PS and age was close to significance in PCr Ž F s 2.0, p s 0.07.. No significant effect of PS was found for any of other peak area ratios. No significant alteration of PCr and intracellular pH during the course of the experiment were found by multiple comparison in the 25 subjects. To delineate the significant interaction between PS and age, the subjects were divided into two groups at the middle point of age in this sample; older than and younger than 40 years old. The older group was 54.4 " 10.5 Žmean
3.3. Protocol of the experiment After the preparation of 31 P-MRS was finished, the first spectrum ŽPre. was obtained. Just after that, photic stimulation ŽPS. was started. During 12 min of PS, two spectra were obtained ŽPS1 and PS2.. Just after the PS was finished, three spectra ŽPost1, Post2, and Post3. were obtained during following 18 min. Total six spectra were obtained in 36 min for each subject. The room light in the MR scanning room turned off at the beginning of the experiment. Subject were directed to open their eyes throughout the experiment.
Fig. 2. Comparison of the photic stimulation-induced alteration of phosphocreatine ŽPCr. between younger subjects aged less than 40 years old Ž28.9"4.9 wmean"SDx years old, ns17. and middle-aged subjects aged more than 40 years old Ž54.4"10.5 years old, ns8.. Ordinate is PCrrtotal phosphorus signal peak area percentage. Pre: before PS; PS1: the first 6 min of PS; PS2: the latter 6 min of PS; Post1, Post2, Post3: 0–6, 6–12, and 12–18 min after PS. The PCr peak area during PS ŽPS1: 11.4"1.4. was significantly lower than that before PS ŽPre: 13.8"2.3, p- 0.05. in middle-aged subjects. The PCr level before stimulation was significantly higher in middle-aged subjects Ž ns8, 13.8"2.3. than that in younger subjects Ž ns 7, 11.5"1.8. Ž p- 0.05..
J. Murashita et al.r Brain Research 818 (1999) 72–76
Fig. 3. Comparison of the photic stimulation-induced alteration of intracellular pH between younger subjects and middle-aged subjects. Ordinate is intracellular pH. Intracellular pH after the PS ŽPost1: 7.22"0.32. tended to be lower than that before PS ŽPre: 7.00"0.12, ps 0.07. in middle-aged subjects. Intracellular pH in middle-aged subjects before PS was significantly lower than that in the younger subjects Ž7.11"0.10, p- 0.005..
" S.D.. years old Ž n s 8, 3 males and 5 females. and the younger group was 28.9 " 4.9 years old Ž n s 17, 9 males and 8 females.. In the older subjects, the PCr peak ratio was significantly decreased during the first 6 min during PS Ž11.4 " 1.4. compared to that before PS Ž13.8 " 2.3, p - 0.05.. No significant change of PCr was found in younger subjects during the course of the experiment ŽFig. 2.. Intracellular pH tended to be increased just after PS Ž7.22 " 0.32. compared with that before the stimulation Ž7.00 " 0.12, p s 0.07.. No significant change of intracellular pH was found in younger subjects during the course of the experiment ŽFig. 3.. 4.2. Effects of age on the MRS Õalues before PS In the older subjects, the PCr peak area before stimulation Ž13.8 " 2.3. was significantly higher and intracellular pH was significantly lower Ž7.00 " 0.12. than that in the younger subjects Ž11.6 " 1.9, p - 0.05 for PCr, and 7.11 " 0.06, p - 0.01 for pH.. Before the PS, intracellular pH Ž r s y0.47, p - 0.05. and Pi Ž r s y0.51, p - 0.01. were significantly negatively correlated with age. On the other hand, PCr Ž r s 0.41, p - 0.05. was significantly positively correlated with age.
5. Discussion In this study, our previous finding that PCr significantly decreased during PS in young volunteers Ž32.7 " 9.8 years old Ž n s 9. was not confirmed in a larger subject population Ž37.0 " 14.0 years old, n s 25.. This result suggests that decrease of PCr observed in our previous report, may be due to type I error because of small number of subjects in the previous study. These results are also contradictory to the report by Sappey-Marinier et al. w13x who also
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reported decrease of PCr in six volunteers whose ages are not reported. This discrepancy might have arose not only from the number of subjects Ž n s 6 vs. n s 25., but also from the difference of methodology, such as method of photic stimulation, pulse sequences, coils, and method of spectral processing. Our finding is in accordance with the data by Chen et al. w2x who reported no effect of PS on PCr in seven healthy volunteers whose ages were not reported. A statistically significant effect of PS was found for intracellular pH, however, when age of the subjects was considered. Moreover, statistically significant interaction of PS and age was found for intracellular pH and nearly significant interaction of PS and age was found for PCr. In middle-aged subjects Ž43–68, 54.4 " 10.5 years old, n s 8., PCr significantly decreased during PS and intracellular pH tended to be increased after PS. What causes this difference of response to PS between younger subjects and middle-aged subjects? It has been reported that regional cerebral blood flow ŽrCBF. and oxygen utilization ŽCMRO 2 . decrease with age in the frontal lobes but not in the occipital lobes w7,17x. Oxygen extraction ratio is also maintained relatively constant in aging w7x. These reports suggest that rCBF and CMRO 2 in the occipital lobe are not affected by age. Therefore, biological basis of age-dependent changes of PCr and intracellular pH in the occipital lobes is not known. In this study, the PCr level in the occipital lobe at rest was significantly higher in middle-aged subjects compared with younger subjects, while the PCr peak ratio in these subjects during the photic stimulation did not differ from that of younger subjects ŽFig. 2.. It has been reported that the PCr level in the frontal lobes also positively correlated with age w4,8,14x. This correlation between PCr and age may be caused by either a decrease of energy demand or increased efficiency or capacity of energy delivery with age. Intracellular pH increased just after the PS in middleaged subjects in this study. Although an effect of age on brain intracellular pH has never been reported so far, intracellular pH negatively correlated with age, and it was lower in middle-aged subjects compared to younger subjects before PS. In this subject population, intracellular pH was negatively correlated with PCr Ž r s y0.56, p - 0.005 by Pearson’s coefficient of correlation.. Therefore, these two correlations, PCr vs. age and intracellular pH vs. age, may reflect the same age-dependent biological phenomenon, although it is not yet characterized. It has been reported that intracellular pH is decreased in the subcortical hyperintensity lesions detected by T2-weighted MRI w12x. Because T2 hyperintensity lesions become more frequent with age, this might have affected the present results. In this study, definite conclusion cannot be made because T2-weighted MRI was not examined in these subjects. We should be cautious to interpret the finding that intracellular pH was increased after PS, considering the
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J. Murashita et al.r Brain Research 818 (1999) 72–76
large standard deviation of intracellular pH during and after the PS. Increased pH in these period might be due to inaccuracy of chemical shift of inorganic phosphate peak. Therefore, increase of intracellular pH in middle-aged subjects needs to be confirmed in a larger subject population using a more reliable method.
w3x
w4x
5.1. Technical limitations This study has several technical limitations. Because the repetition time was not long enough Ž3 s. in this study, the change of peak areas observed in this experiment might be caused by parameters besides concentrations, such as relaxation times. Absolute concentration determination could not be used in order to achieve sufficient time resolution in this experiment. Instead, we used the peak area percent of metabolites to the total phosphorus signal. Although other investigators may prefer other values such as the PCrrPi or PCrrb-ATP ratio, our experience suggests that the peak area percent to the total phosphorus signal is the most reliable indicator when the DRESS method is used. In the DRESS method, the Pi peak may be obscured by the large hump from phospholipid signals around the PDE peak, and the b-ATP peak is broadened due to T2 relaxation. We previously reported the inter-assay intra-individual coefficient of variations ŽCVs. of 31 P-MRS data acquisition by the DRESS method in 11 subjects examined twice. The inter-assay intra-individual CV of the PCr peak area percent to the total phosphorus signal was 4.2% w5x. Because the authors used a rough localization method in order to get a high time resolution, contamination of signals from muscles cannot be completely eliminated. However, it does not matter when the response to photic stimulation is investigated intra-individually. In spite of these limitations, this is the first study implicating age-dependent alteration of brain energy metabolism in the human brain detected by 31 P-MRS during photic stimulation.
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Acknowledgements This study was supported by a Research Grant for Nervous and Mental Disorders from the Japanese Ministry of Health and Welfare Ž8B-2., a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education ŽNo. 09770738., and a grant from Takeda Science Foundation.
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References w1x B. Chance, S. Eleff, J.S. Leigh, Noninvasive, nondestructive approaches to cell bioenergetics, Proc. Natl. Acad. Sci. USA 77 Ž1980. 7430–7434. w2x W. Chen, X.H. Zhu, G. Adriany, K. Ugurbil, Increase of creatine kinase activity in the visual cortex of human brain during visual
w17x
stimulation: a 31 P magnetization transfer study, Magn. Reson. Med. 38 Ž1997. 551–557. A.R. Coggan, A.M. Abduljalil, S.C. Swanson, M.S. Earle, J.W. Farris, L.A. Mendenhall, P.M. Robitaille, Muscle metabolism during exercise in young and older untrained and endurance-trained men, J. Appl. Physiol. 75 Ž1993. 2125–2133. T. Kato, S. Takahashi, T. Shioiri, J. Murashita, H. Hamakawa, T. Inubushi, Reduction of brain phosphocreatine in bipolar II disorder detected by phosphorus-31 magnetic resonance spectroscopy, J. Affect. Disord. 31 Ž1994. 125–133. T. Kato, T. Shioiri, J. Murashita, H. Hamakawa, T. Inubushi, S. Takahashi, Phosphorus-31 magnetic resonance spectroscopy and ventricular enlargement in bipolar disorder, Psychiatry Res.: Neuroimaging 55 Ž1994. 41–50. T. Kato, J. Murashita, T. Shioiri, H. Hamakawa, T. Inubushi, Effect of photic stimulation on energy metabolism in the human brain measured by 31 P-MR spectroscopy, J. Neuropsychiat. Clin. Neurosci. 8 Ž1996. 417–422. K.L. Leenders, D. Perani, A.A. Lammertsma, J.D. Heather, P. Buckingham, M.J.R. Healy, J.M. Gibbs, R.J.S. Wise, J. Hatazawa, S. Herold, R.P. Beaney, D.J. Brooks, T. Spinks, C. Rhodes, R.S.J. Frackowiak, T. Jones, Cerebral blood flow, blood volume and oxygen utilization: normal values and effect of age, Brain 113 Ž1990. 27–47. R. Longo, C. Ricci, L.D. Palma, R. Vidimari, A. Giorgini, J.A. den Holander, C.M. Segebarth, Quantitative 31 P MRS of the normal adult human brain. Assessment of interindividual differences and aging effects, NMR Biomed. 6 Ž1993. 53–57. K.K. McCully, H. Kakihira, K. Vandenborne, J.K. Braun, Noninvasive measurements of activity-induced changes in muscle metabolism, J. Biomechanics 24 Ž1991. 153–161, Suppl. 1. K.K. McCully, R.A. Fielding, W.J. Evans, J.S. Leigh Jr., J.D. Posner, Relationship between in vivo and in vitro measurements of metabolism in young and old human calf muscles, J. Appl. Physiol. 75 Ž1993. 813–819. B. Ross, T. Michaelis, Clinical applications of magnetic resonance spectroscopy, Magn. Reson. Q. 10 Ž1994. 191–247. D. Sappey-Marinier, R.F. Deicken, G. Fein, G. Calabrese, B. Hubesch, C.V. Dyke, W.P. Dillon, L. Davenport, D.J. Meyerhoff, M.W. Weiner, Alterations in brain phosphorus metabolite concentrations associated with areas of high signal intensity in white matter at MR imaging, Radiology 183 Ž1992. 247–256. D. Sappey-Marinier, G. Calabrese, G. Fein, J.W. Hugg, C. Biggins, M.W. Weiner, Effect of photic stimulation of human visual cortex lactate and phosphates using 1 H and 31 P magnetic resonance spectroscopy, J. Cereb. Blood Flow Metab. 12 Ž1992. 584–592. C.D. Smith, L.C. Pettigrew, M.J. Avison, J.E. Kirsch, A.J. Tinkhtman, F.A. Schmitt, D.P. Wermeling, D.R. Wekstein, W.R. Markesberry, Frontal lobe phosphorus metabolism and neuropsychological function in aging and in Alzheimer’s disease, Ann. Neurol. 38 Ž1995. 194–201. P.C. Van Rijen, P.R. Luyten, J.W. Berkelbach van der Sprenkel, V. Kraaier, A.C. van Huffelen, C.A. Tulleken, J.A. den Hollander, 1 H and 31 P NMR measurement of cerebral lactate, high-energy levels, and pH during voluntary hyperventilation: associated EEG, capnographic, and Doppler findings, Magn. Reson. Med. 10 Ž1989. 182– 193. J. Vion-Dury, D.J. Meyerhoff, P.J. Cozzone, M.W. Weiner, What might be the impact on neurobiology if the analysis of brain metabolism by in vivo magnetic resonance spectroscopy?, J. Neurol. 241 Ž1994. 354–371. T. Yamaguchi, I. Kanno, K. Uemura, F. Shishido, A. Inugami, T. Ogawa, M. Murakami, K. Suzuki, Reduction in regional cerebral metabolic rate of oxygen during human aging, Stroke 17 Ž1986. 1220–1228.
Research report
Age-dependent alteration of metabolic response to photic stimulation in the human brain measured by 31 P MR-spectroscopy Jun Murashita a , Tadafumi Kato b
b, )
, Toshiki Shioiri a , Toshiro Inubushi c , Nobumasa Kato
a,b
a Department of Psychiatry, Shiga UniÕersity of Medical Science, Otsu, Shiga, 520-2121, Japan Department of Neuropsychiatry, Faculty of Medicine, UniÕersity of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo, 113-8655, Japan c Molecular Neurobiology Research Center, Shiga UniÕersity of Medical Science, Otsu, Shiga, 520-2121, Japan
Accepted 24 November 1998
Abstract Effects of photic stimulation ŽPS. on energy metabolism were examined in the occipital cortex of 25 healthy volunteers aged 23–69 years old using phosphorus-31 magnetic resonance spectroscopy Ž31 P-MRS.. A significant effect of photic stimulation was found only for intracellular pH Ž p - 0.05 by repeated measures analysis of variance. but not for any peak area ratios. An interaction between intracellular pH and age were statistically significant Ž p - 0.005., and the interaction between phosphocreatine and age was close to significance Ž p s 0.06.. In subjects aged more than 40 years old, phosphocreatine was significantly decreased during the photic stimulation Ž p - 0.05, multiple comparison by Dunnett’s method., and intracellular pH tended to be elevated just after the stimulation Ž p s 0.07.. There were no significant changes in these values in younger subjects. These results suggest that no significant effect of photic stimulation on brain energy metabolism was found in younger subjects, and that significant effects of photic stimulation on intracellular pH and phosphocreatine were found in middle-aged subjects. Metabolic response of the human brain to photic stimulation may be dependent on age. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Phosphorus-31 magnetic resonance spectroscopy; Brain energy metabolism; Creatine phosphate; Intracellular pH; Membrane phospholipid; Photic stimulation
1. Introduction Since Chance et al. w1x have first applied nuclear magnetic resonance spectroscopy ŽMRS. to human skeletal muscles, this technique has been widely used for physiological studies of energy metabolism in living human organs. In human skeletal muscles, exercise induces reduction of phosphocreatine ŽPCr., increase of inorganic phosphate ŽPi., and decrease of intracellular pH w3,9,10x. Measurement of exercise-induced alteration of muscle energy metabolism by means of 31 P-MRS has been used for diagnosis of metabolic muscular disorders w10x. 31 P-MRS has also been used for the investigation of brain energy metabolism in many kinds of neurological or psychiatric disorders w11,16x. In these studies, 31 P-MRS
) Corresponding author. Fax: q81-3-5800-6894; E-mail: [email protected]
was examined at rest with no particular physiological stimulation. All the findings observed by 31 P-MRS, such as decrease of PCr, were not specific to a particular neuropsychiatric disease. If a physiological stimulation was applied, it might help us to distinguish different metabolic abnormalities in the human brain. There are only few reports of physiological alteration of energy metabolism detected by 31 P-MRS in the human brain. Van Rijen et al. w15x have reported that hyperventilation causes increase of intracellular pH detected by 31 PMRS. Sappey-Marinier et al. w13x reported that PCr in the occipital cortex was significantly decreased during photic stimulation ŽPS. in the brain in six normal control subjects. Intracellular pH was slightly increased during stimulation although it was not statistically significant. The authors w6x also reported that PCr was decreased and intracellular pH tended to be increased during PS in nine normal volunteers. They also reported that PCr returned to a normal level soon after the stimulation. In contrast to these two studies reporting effects of PS on energy metabolism in the
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 2 8 5 - 2
J. Murashita et al.r Brain Research 818 (1999) 72–76
occipital lobes, Chen et al. w2x have reported no significant effect of PS in five subjects. In this study, 25 normal volunteers aged 23–69 years old were examined by 31 P-MRS during 12 min of photic stimulation to confirm and extend our previous report on photic stimulation-induced alteration of brain phosphorus metabolism.
2. Subjects Subjects were 25 normal volunteers who were recruited from hospital staffs and students. They were 37.0 " 14.0 Žmean " S.D., 23–69. years old. Thirteen were female and 12 were male. The data in nine of these subjects were reported in the previous report w6x. Their past history and family history were examined by a questionnaire and an interview by a physician, and they did not have neuropsychiatric disorder, epilepsy, general medical disease, and opthalmologic disease. They did not have any major structural abnormalities in the brain examined by T1-weighted magnetic resonance imaging ŽMRI.. They did not have any medications at the time of the examination. 31 P-MRS was examined between 1800 and 2000 h before supper. They gave written informed consent to the study. This study was approved by the ethical committee of the Shiga University of Medical Science.
73
3. Method 3.1.
31
P-magnetic resonance spectroscopy
MRS data were acquired with a SIGNA 1.5 Tesla MR system ŽGE Medical System, Milwaukee. with a standard spectroscopy package and surface coils for 1 H and 31 P supplied by the manufacturer. The surface coil for proton was horizontally set on the stretcher. The subjects lay so as to their occipital region is on the coil. Their heads were fixed by molded sponge. The position of the head was monitored by 1 H-magnetic resonance imaging ŽMRI. in a sagittal view. The head was repositioned when necessary, so that the occipital lobes were in the center of the coil. Slice selection was applied to make a volume of interest ŽVOI. of 5 cm thick axial slice including the occipital lobes to eliminate contamination from signals from neck muscles. The extent of VOI was visualized by the 1 H-MRI with the surface coil. The region examined was predominantly the occipital lobes. The magnetic field over the VOI was optimized using the water signal to establish the line width at less than 10 Hz. The surface coil for 1 H was then replaced with the coil for 31 P. The flip angle in the VOI was set to 908 in the phantom experiment. The 31 P-MR spectra were obtained with 1024 data points, spectral width of 4000 Hz, repetition time ŽTR. of 3 s, delay time of 1.5 ms, and center frequency of 25.84 MHz. One hundred
Fig. 1. Stacked plot of phosphorus-31 magnetic resonance spectra Ž31 P-MRS.. Six spectra serially recorded in one experiment are shown. Peaks are assigned to phosphomonoester Ž6.5 ppm., inorganic phosphate ŽPi, 4.8 ppm., phosphodiester Ž3 ppm., phosphocreatine ŽPCr, 0 ppm., and g-, a-, and b-adenosine triphosphate Žy3, y8, y16 ppm.. The bottom spectrum is obtained before photic stimulation, and other spectra obtained thereafter are stacked behind this spectrum. ppm s parts per million.
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acquisitions were averaged. Each spectrum was acquired in 6 min. Six spectra were obtained in each session, during the course of photic stimulation. Free induction decay ŽFID. were transported to a SPARC2 work station ŽSUN micro system, California., and processed with OMEGA CSI software ŽGE Medical Systems.. FIDs were exponentially filtered with 15 Hz-line broadening. After Fourier transformation was applied, the first-order phase correction with fixed values, manual zero-order phase correction, and baseline correction with polynomial interpolation were applied. The same values for phase correction and the same polynomial function were used for processing of all six spectra in one session. The 31 P-MR spectra are shown in Fig. 1. The following seven peaks were resolved: phosphomonoester ŽPME., inorganic phosphate ŽPi., phosphodiester ŽPDE., PCr and three resonances from adenosine triphosphate Ž g, a and b-ATP.. The peak areas were calculated by automatic peak fitting with Lorentzian curves by SIMPLEX method using a software package which was programmed in our laboratory. The g-ATP peak was simulated by two peaks. The peak areas are shown as percent values of the total phosphorus signal. Intracellular pH was calculated from the chemical shift difference of PCr and Pi. 3.2. Photic stimulation The method of photic stimulation was minutely described in our previous article w6x. In brief, white light flashing at 10 Hz was used to stimulate the occipital cortex. A metal halide lamp ŽUshio Electric, Kobe, Japan. was used to emit white light for photic stimulation. The light was interrupted by an aluminum wheel with two holes which rounds at 5 Hz. Light was guided into the MR room using optical fibers. The optical fibers were divided into six segments, and each segment was attached to a titan dioxide coated glass rod with diameter of 3 cm and length of 15 cm ŽAsahi Glass, Tokyo, Japan., which scatters the light from optical fibers. These six glass rods set just before eyes of the subject to cover most of the visual field of the subject. Strength of the light at the position of eyes of the subjects was 5500 lx.
3.4. Statistics All data were reported as mean " S.D. For statistical analysis, repeated measures analysis of variance ŽANOVA. with factor of PS Žsix points in the course of the experiment. and a covariate of age was used. Multiple comparison by Dunnett’s method was used to examine changes of the MRS values during the course of experiments in comparison with the value before the photic stimulation. Pearson’s coefficient of correlation and Student’s t-test were also supplementary used.
4. Results 4.1. Effects of photic stimulation In 25 normal volunteers, repeated measures ANOVA revealed significant effects of PS on intracellular pH Ž F s 3.0, p - 0.05.. A significant interaction between PS and age was found for intracellular pH Ž F s 4.6, p - 0.005.. Interaction between PS and age was close to significance in PCr Ž F s 2.0, p s 0.07.. No significant effect of PS was found for any of other peak area ratios. No significant alteration of PCr and intracellular pH during the course of the experiment were found by multiple comparison in the 25 subjects. To delineate the significant interaction between PS and age, the subjects were divided into two groups at the middle point of age in this sample; older than and younger than 40 years old. The older group was 54.4 " 10.5 Žmean
3.3. Protocol of the experiment After the preparation of 31 P-MRS was finished, the first spectrum ŽPre. was obtained. Just after that, photic stimulation ŽPS. was started. During 12 min of PS, two spectra were obtained ŽPS1 and PS2.. Just after the PS was finished, three spectra ŽPost1, Post2, and Post3. were obtained during following 18 min. Total six spectra were obtained in 36 min for each subject. The room light in the MR scanning room turned off at the beginning of the experiment. Subject were directed to open their eyes throughout the experiment.
Fig. 2. Comparison of the photic stimulation-induced alteration of phosphocreatine ŽPCr. between younger subjects aged less than 40 years old Ž28.9"4.9 wmean"SDx years old, ns17. and middle-aged subjects aged more than 40 years old Ž54.4"10.5 years old, ns8.. Ordinate is PCrrtotal phosphorus signal peak area percentage. Pre: before PS; PS1: the first 6 min of PS; PS2: the latter 6 min of PS; Post1, Post2, Post3: 0–6, 6–12, and 12–18 min after PS. The PCr peak area during PS ŽPS1: 11.4"1.4. was significantly lower than that before PS ŽPre: 13.8"2.3, p- 0.05. in middle-aged subjects. The PCr level before stimulation was significantly higher in middle-aged subjects Ž ns8, 13.8"2.3. than that in younger subjects Ž ns 7, 11.5"1.8. Ž p- 0.05..
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Fig. 3. Comparison of the photic stimulation-induced alteration of intracellular pH between younger subjects and middle-aged subjects. Ordinate is intracellular pH. Intracellular pH after the PS ŽPost1: 7.22"0.32. tended to be lower than that before PS ŽPre: 7.00"0.12, ps 0.07. in middle-aged subjects. Intracellular pH in middle-aged subjects before PS was significantly lower than that in the younger subjects Ž7.11"0.10, p- 0.005..
" S.D.. years old Ž n s 8, 3 males and 5 females. and the younger group was 28.9 " 4.9 years old Ž n s 17, 9 males and 8 females.. In the older subjects, the PCr peak ratio was significantly decreased during the first 6 min during PS Ž11.4 " 1.4. compared to that before PS Ž13.8 " 2.3, p - 0.05.. No significant change of PCr was found in younger subjects during the course of the experiment ŽFig. 2.. Intracellular pH tended to be increased just after PS Ž7.22 " 0.32. compared with that before the stimulation Ž7.00 " 0.12, p s 0.07.. No significant change of intracellular pH was found in younger subjects during the course of the experiment ŽFig. 3.. 4.2. Effects of age on the MRS Õalues before PS In the older subjects, the PCr peak area before stimulation Ž13.8 " 2.3. was significantly higher and intracellular pH was significantly lower Ž7.00 " 0.12. than that in the younger subjects Ž11.6 " 1.9, p - 0.05 for PCr, and 7.11 " 0.06, p - 0.01 for pH.. Before the PS, intracellular pH Ž r s y0.47, p - 0.05. and Pi Ž r s y0.51, p - 0.01. were significantly negatively correlated with age. On the other hand, PCr Ž r s 0.41, p - 0.05. was significantly positively correlated with age.
5. Discussion In this study, our previous finding that PCr significantly decreased during PS in young volunteers Ž32.7 " 9.8 years old Ž n s 9. was not confirmed in a larger subject population Ž37.0 " 14.0 years old, n s 25.. This result suggests that decrease of PCr observed in our previous report, may be due to type I error because of small number of subjects in the previous study. These results are also contradictory to the report by Sappey-Marinier et al. w13x who also
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reported decrease of PCr in six volunteers whose ages are not reported. This discrepancy might have arose not only from the number of subjects Ž n s 6 vs. n s 25., but also from the difference of methodology, such as method of photic stimulation, pulse sequences, coils, and method of spectral processing. Our finding is in accordance with the data by Chen et al. w2x who reported no effect of PS on PCr in seven healthy volunteers whose ages were not reported. A statistically significant effect of PS was found for intracellular pH, however, when age of the subjects was considered. Moreover, statistically significant interaction of PS and age was found for intracellular pH and nearly significant interaction of PS and age was found for PCr. In middle-aged subjects Ž43–68, 54.4 " 10.5 years old, n s 8., PCr significantly decreased during PS and intracellular pH tended to be increased after PS. What causes this difference of response to PS between younger subjects and middle-aged subjects? It has been reported that regional cerebral blood flow ŽrCBF. and oxygen utilization ŽCMRO 2 . decrease with age in the frontal lobes but not in the occipital lobes w7,17x. Oxygen extraction ratio is also maintained relatively constant in aging w7x. These reports suggest that rCBF and CMRO 2 in the occipital lobe are not affected by age. Therefore, biological basis of age-dependent changes of PCr and intracellular pH in the occipital lobes is not known. In this study, the PCr level in the occipital lobe at rest was significantly higher in middle-aged subjects compared with younger subjects, while the PCr peak ratio in these subjects during the photic stimulation did not differ from that of younger subjects ŽFig. 2.. It has been reported that the PCr level in the frontal lobes also positively correlated with age w4,8,14x. This correlation between PCr and age may be caused by either a decrease of energy demand or increased efficiency or capacity of energy delivery with age. Intracellular pH increased just after the PS in middleaged subjects in this study. Although an effect of age on brain intracellular pH has never been reported so far, intracellular pH negatively correlated with age, and it was lower in middle-aged subjects compared to younger subjects before PS. In this subject population, intracellular pH was negatively correlated with PCr Ž r s y0.56, p - 0.005 by Pearson’s coefficient of correlation.. Therefore, these two correlations, PCr vs. age and intracellular pH vs. age, may reflect the same age-dependent biological phenomenon, although it is not yet characterized. It has been reported that intracellular pH is decreased in the subcortical hyperintensity lesions detected by T2-weighted MRI w12x. Because T2 hyperintensity lesions become more frequent with age, this might have affected the present results. In this study, definite conclusion cannot be made because T2-weighted MRI was not examined in these subjects. We should be cautious to interpret the finding that intracellular pH was increased after PS, considering the
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large standard deviation of intracellular pH during and after the PS. Increased pH in these period might be due to inaccuracy of chemical shift of inorganic phosphate peak. Therefore, increase of intracellular pH in middle-aged subjects needs to be confirmed in a larger subject population using a more reliable method.
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5.1. Technical limitations This study has several technical limitations. Because the repetition time was not long enough Ž3 s. in this study, the change of peak areas observed in this experiment might be caused by parameters besides concentrations, such as relaxation times. Absolute concentration determination could not be used in order to achieve sufficient time resolution in this experiment. Instead, we used the peak area percent of metabolites to the total phosphorus signal. Although other investigators may prefer other values such as the PCrrPi or PCrrb-ATP ratio, our experience suggests that the peak area percent to the total phosphorus signal is the most reliable indicator when the DRESS method is used. In the DRESS method, the Pi peak may be obscured by the large hump from phospholipid signals around the PDE peak, and the b-ATP peak is broadened due to T2 relaxation. We previously reported the inter-assay intra-individual coefficient of variations ŽCVs. of 31 P-MRS data acquisition by the DRESS method in 11 subjects examined twice. The inter-assay intra-individual CV of the PCr peak area percent to the total phosphorus signal was 4.2% w5x. Because the authors used a rough localization method in order to get a high time resolution, contamination of signals from muscles cannot be completely eliminated. However, it does not matter when the response to photic stimulation is investigated intra-individually. In spite of these limitations, this is the first study implicating age-dependent alteration of brain energy metabolism in the human brain detected by 31 P-MRS during photic stimulation.
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Acknowledgements This study was supported by a Research Grant for Nervous and Mental Disorders from the Japanese Ministry of Health and Welfare Ž8B-2., a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education ŽNo. 09770738., and a grant from Takeda Science Foundation.
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