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Keywords:

  • basal ganglia;
  • bipolar disorder;
  • brain;
  • energy metabolism;
  • intracellular pH;
  • phosphomonoester;
  • phosphorus magnetic resonance spectroscopy

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Abstract  The authors have previously reported that intracellular pH measured by phosphorus-31 magnetic resonance spectroscopy (31P-MRS) was decreased in the frontal lobes of patients with bipolar disorder. In the present study, phosphorus metabolism in the basal ganglia was examined in 13 patients with bipolar disorder and 10 matched controls by localized 31P-MRS. While no significant alteration of peak area ratios was found for all phosphorus metabolites, intracellular pH was significantly reduced in the basal ganglia in patients with bipolar disorder (7.014 ± 0.045) compared with control subjects (7.066 ± 0.047, P < 0.05). Unexpectedly, non-localized 31P-MR spectra also showed significantly lower levels of intracellular pH (6.970 ± 0.025) than controls (6.986 ± 0.024, P < 0.05). These results suggest that decreased intracellular pH in the brain of patients with bipolar disorder is not caused by dysfunction of the frontal lobes but reflect altered metabolism at the cellular level.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Bipolar disorder is a major mental disorder that affects approximately 1% of population and causes severe psychosocial impairment.1 Although recent studies revealed neuroprotective effects of lithium and valproate by increasing the expression of B-cell lymphoma/leukemia-2 (bcl-2) on mitochondrial outer membrane,2 molecular pathology corresponding to this effect of mood stabilizer has not been fully elucidated. Structural imaging studies suggested some alteration in the frontal and temporal lobes, basal ganglia, hippocampus, and amygdala but the results are not consistent.3 Therefore, it is still not clear as to which region of the brain has an important role in the pathology of bipolar disorder.

In vivo neurochemical examination using magnetic resonance spectroscopy (MRS), which can detect various important metabolites in the brain, is one of the most powerful tools in the study of the biochemical basis of bipolar disorder. Phosphorus-31 magnetic resonance spectroscopy (31P-MRS) is a unique tool that can examine energy metabolism, membrane phospholipid metabolism, and intracellular pH, in vivo. Using this method, several findings have been reported in euthymic patients with bipolar disorder: (i) decreased levels of phosphomonoester (PME) in the frontal4–6 and temporal lobes;7 (ii) decrease of intracellular pH in the frontal lobes;4,5 (iii) decrease of phosphocreatine (PCr) in the frontal lobes in patients with bipolar II disorder;8 and (iv) increase of the phosphodiester (PDE) peak in the frontal lobes.6

In contrast, 1H-MRS studies suggested that the choline-containing compounds (Cho)/Cr ratio in the basal ganglia was increased in patients with bipolar disorder in the euthymic9–11 and depressive states11 except for other studies suggesting no change of Cho.12,13 Reduced Cho signal14 and myo-inositol level15 were reported in the anterior cingulate cortex. We previously reported that creatine was decreased in the frontal cortex.16 Decreased N-acetylaspartate (NAA)/Cr ratio was recently reported in the hippocampus17–19 and frontal lobes,20,21 except for a study reporting that NAA did not differ from controls at baseline but increased after lithium treatment.22

As summarized here, various alterations of metabolite levels have been reported in many brain structures but the primary biochemical event underlying these findings has not been fully elucidated.

Among these findings, alteration of PME and PDE in the frontal lobe and Cho in the basal ganglia and cingulate could reflect the change of soluble choline containing compounds related to membrane phospholipid metabolism, phosphocholine (PC) and glycerophosphocholine (GPC), which contribute to both of these peaks. The PC is included in the PME and Cho peaks, while GPC is in the PDE and Cho resonances.23 However, there is no report on the difference of PME and PDE in the basal ganglia. The purpose of the present study was to characterize the neurochemical changes in the basal ganglia of patients with bipolar disorder using 31P-MRS.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Subjects

The subjects consisted of 13 patients with bipolar disorder (seven male, six female; mean age, 49.4 ± 12.1 years) hospitalized in the Shiga University of Medical Science Hospital. They were diagnosed by the consensus of at least two senior psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders (3rd edn, revised; DSM-III-R) criteria. They were in the euthymic state assessed by the DSM-III-R criteria. They were treated with various psychotropic drugs such as lithium carbonate (n = 8), carbamazepine (n = 5), sodium valproate (n = 1), antidepressants (n = 2), benzodiazepines (n = 12), and antipsychotics (n = 10). Healthy control subjects having no history of major mental disorders whose ages and sexes were comparable to the bipolar patients (six male, four female; mean age, 43.2 ± 13.7 years) were also examined. No subjects had major structural abnormalities in the brain examined by T1-weighted magnetic resonance images (MRI). They gave written informed consent to the study. The ethics committee of the Shiga University of Medical Science approved the present study.

Procedure

Magnetic resonance spectroscopy was done with a Signa 1.5 T MR system (GE Medical Systems, Milwaukee, USA) with a quadrature for phosphorus and linear for proton (QPLH) head coil double tuned for 1H and 31P (GE Medical Systems).

T1-weighted scout MR images were obtained to visualize the volume of interest (VOI). The VOI was 50 cm in the left–right axis and 35 cm in the posterior–anterior axis, selected by the outer volume suppression method, which was originally developed and used for the preselection of VOI within the head to suppress the lipid signal from the subcutaneous tissue and scalp.24 The 30 cm axial slice was selected by using the depth resolved surface coil spectroscopy (DRESS) sequence.25 As a result, the VOI was the 50 × 35 × 30 cm voxel, predominantly in the basal ganglia, but including other surrounding structures such as white matter and nearby cortexes (Fig. 1). Using a 1H-MR signal of water obtained by the DRESS sequence without outer volume supression, the magnetic field over the VOI was optimized. Non-localized 31P-MR spectra were obtained using one pulse (SPECREC) with a repetition time (TR) of 3 s, number of averaged spectra of 128, and a center frequency of 25.85 MHz. Subsequently, localized 31P-MR spectra were obtained with a TR of 3 s, the number of average, 400, and center frequency of 25.84 MHz. Total time of the measurement was 45 min.

image

Figure 1. T1-weighted magnetic resonance image showing the volume of interest examined by localized 31P-magnetic resonance spectroscopy (MRS), the 50 × 35 × 30 cm voxel, predominantly in the basal ganglia.

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The post-processing was performed using the omega csi software (GE medical systems) on the SUN workstation. Exponential filter with 15 Hz line broadening, Fourier transformation, first- and zero-order phase correction, and baseline correction were applied to the free induction decay (FID). The following seven peaks were resolved: PME, inorganic phosphate (Pi), PDE, PCr and three resonances from adenosine triphosphate (γ, α and β-ATP). The peak areas were shown as percent values of the total phosphorus signal. Intracellular pH was calculated from the chemical shift difference of PCr and Pi.

The peak areas of seven peaks were calculated by automatic curve fitting using home-built software according to the simplex method using Lorenzian curves. For statistical analysis, Mann–Whitney U-test was used. Except for the comparison testing of whether or not previously reported finding in the frontal lobes can be also found in the basal ganglia (i.e. pH, PME, and PDE), Bonferroni correction was applied to three peaks of ATP, PCr and Pi by setting the significance level at 0.01 (= 0.05/5)

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

The representative 31P-MR spectra are shown in Fig. 2 (non-localized spectra) and Fig. 3 (localized spectra in the basal ganglia). The results of the peak areas are shown in the Table 1. No significant difference of peak area ratios between patients with bipolar disorder and controls was found in the basal ganglia (Table 1).

image

Figure 2. Non-localized 31P-magnetic resonance spectra of the whole head in a patient with bipolar disorder and a healthy control subject. The dotted vertical line indicates the chemical shift of the inorganic phosphate (Pi) peak in the spectra of the control subject. p.p.m., parts per million; PME, phosphomonoester; PDE, phosphodiester; ATP, adenosine triphosphate.

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image

Figure 3. Localized 31P-magnetic resonance spectra of the basal ganglia in a patient with bipolar disorder and a healthy control subject.

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Table 1.  Peak area ratios and intracellular pH measured by 31p-MRS in the basal ganglia and whole brain in patients with bipolar disorder and control subjects
 ControlBipolar disorder
 nMeanSDnMeanSD
  1.  PME, phosphomonoester; Pi, inorganic phosphate; PDE, phosphodiester; PCr, phosphocreatine; ATP, adenosine triphosphate.

  2. *Mann–Whitney  U-test,  U = 30,  Z = −1.7,  P = 0.042  (one-tailed); ** Mann–Whitney  U-test,  U = 20,  Z = −2.1,  P = 0.031 (two-tailed).

Age (years)1043.213.71349.412.1
GenderF4/M6  F6/M7  
Basal ganglia (outer volume suppression)
pH 8 7.0660.04712 7.0140.045**
PME 8 8.3%1.6%12 9.6%4.4%
Pi 8 5.2%1.4%12 5.4%2.9%
PDE 827.7%5.7%1224.1%8.0%
PCr 819.0%3.8%1219.5%4.5%
γATP 810.7%2.5%1211.0%2.9%
αATP 817.2%2.9%1218.0%3.1%
βATP11.9%2.5%1212.4%2.8%
Non-localized
pH10 6.9860.02411 6.9700.025*
PME10 9.3%0.8%1110.1%1.6%
Pi10 5.8%0.9%11 5.6%0.8%
PDE1027.6%2.4%1128.8%1.9%
PCr1014.9%1.3%1114.5%2.6%
γATP10 8.6%1.5%11 7.8%0.8%
αATP1019.7%1.7%1118.4%2.3%
βATP1014.2%2.6%1114.8%1.8%

Intracellular pH in the basal ganglia was significantly lower in the patients with bipolar disorder compared with control subjects (Fig. 4, Table 1; P < 0.05, two-tailed Mann–Whitney U-test). Because decreased pH was commonly found in two brain regions, the frontal lobes4,5 and the basal ganglia (present study), whether or not this is a general finding throughout the brain was tested by applying the one-tailed Mann–Whitney test to the intracellular pH in the non-localized spectra. This finding of lower intracellular pH was also confirmed in the non-localized 31P-MR spectra obtained from the whole head (P < 0.05, one-tailed Mann–Whitney U-test). There was no significant difference of peak area ratios in the non-localized spectra between the two groups.

image

Figure 4. Intracellular pH in the basal ganglia measured by localized 31P-magnetic resonance spectroscopy in patients with bipolar disorder and controls. Each symbol represents the value of one subject. *P < 0.05 by two-tailed Mann–Whitney U-test.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

No significant difference of peak area ratios in the basal ganglia was found between the patients with bipolar disorder and control subjects. Increased peak ratio of choline containing compounds (Cho) has been reported in the basal ganglia of patients with bipolar disorder.9–11 The Cho peak could contain signals from PC, GPC, phosphatydylcholine, acetylcholine and free choline. Of those, contribution of phosphatydylcholine and acetylcholine is negligible.23 Among other choline-containing compounds contributing to the Cho peak in vivo, PC is included in the PME peak, while GPC is in the PDE peak. The lack of significant difference of PME and PDE in the basal ganglia in the present study suggests that increased choline peak in this region was due to alteration of free choline level, otherwise caused by alteration of relaxation times of the Cho signal.

Although these data suggest that altered membrane phospholipid metabolism may be limited to the frontal lobes in bipolar disorder, intracellular pH was significantly lower in the basal ganglia in patients with bipolar disorder, similarly to the frontal lobes. Unexpectedly, this finding of low intracellular pH was also observed in the 31P-MR spectra obtained from the whole head. As shown in Figs 2 and 3, the signal-to-noise ratio of the whole head spectra is much better than that of the localized spectra of the basal ganglia, and the pH measurement is more accurate. By the non-localized measurement using the head coil, not only the brain but also surrounding tissues such as muscles could contribute to the signal observed. However, because the non-localized 31P-MR spectra were also characterized by prominent PDE and moderate PCr peaks (Fig. 2), the contribution of signals from muscles seemed to be low.

These results suggest that decreased intracellular pH in the brain of patients with bipolar disorder is not caused by dysfunction of the frontal lobes but reflects altered metabolism at the cellular level. Although the biochemical process causing low intracellular pH in bipolar disorder has not yet been identified, this finding cannot be explained by the effects of drugs because this finding was confirmed in drug-free patients with bipolar disorder.26 Several kinds of cellular metabolic abnormalities, such as altered calcium concentration, have been reported in peripheral blood cells of patients with bipolar disorder.27

We proposed that patients with bipolar disorder have altered mitochondrial function27,28 due to accumulation of mtDNA deletions,29 mtDNA polymorphisms,30,31 and polymorphisms of nuclear encoded mitochondrial genes.32 Rango et al. examined the brain energy metabolism in patients with a mitochondrial disease, chronic external ophthalmoplegia (CPEO), without clinical central nervous system involvement during photic stimulation.33 They showed that healthy volunteers had a significant increase of intracellular pH during activation, while patients did not have a significant increase of pH during activation. Their finding may be relevant to the brain metabolism of patients with bipolar disorder because all three types of autosomal dominantly inherited CPEO, caused by ANT1,34Twinkle,35 and polymerase γ,36 were reported to be comorbid with bipolar disorder or depression. In addition, we previously reported that mtDNA genotype was associated with intracellular pH in the frontal lobes.30

These findings suggest that accumulation of deleted mtDNA or certain mtDNA haplotype causes mitochondrial dysfunction reflected by decreased intracellular pH, and increases the risk of bipolar disorder.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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