Psychiatry Research: Neuroimaging, 55:41-50 Elsevier

Phosphorus-31 Magnetic Resonance Spectroscopy Ventricular Enlargement in Bipolar Disorder

Tadafumi Kato, Toshiki Shioiri, Jun Murashita, Hiroshi Hamakawa, Toshiro Inubushi, and Saburo Takahashi

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and

Received Jul_v 23, 1993; revised version received November 8, 1993; accepted February 18. 1994.

Abstract. Phosphorus-31 magnetic resonance spectroscopy (JiP-MRS) was used to examine whether reduced levels of phosphomonoesters (PME) were correlated with ventricular enlargement in 40 patients with bipolar disorder and 60 age- matched normal control subjects. Ventricular enlargement was assessed by magnetic resonance imaging (iH-MRI) using the following three methods: Evans ratio (ER), Huckman number (HN), and minimum distance of caudate nuclei (MDCN). Although MDCN and ER were significantly larger in the patient group, no significant correlations were found between 3iP-MRS and IH-MRI. PME was negatively correlated with age in bipolar disorder. Decreased levels of PME were found only in bipolar I disorder. Intracellular pH was positively correlated with duration of lithium treatment. These results suggest that the observed PME reduction was not related to ventricular enlargement, but the issue should be further studied with volumetric MRI analysis.

Key Words. Affective disorder, magnetic resonance imaging, brain, lithium, age, phosphomonoesters.

Phosphorus-31 magnetic resonance spectroscopy (3rP-MRS) demonstrated de- creased levels of phosphomonoester (PME) and intracellularpH in the brains of 17

euthymic patients with a diagnosis of bipolar affective disorder (Kato et al., 1992, 1993). It was speculated that an alteration in membrane phospholipid metabolism might be responsible for the reduction of PME observed in bipolar disorder. The reported reduction of PME is, however, not specific to bipolar disorder; a similar reduction has been found in schizophrenia (Pettegrew et al., 1991; Williamson et al.,

1991; Shioiri et al., in press). The PME peak detected by JiP-MRS in the brain includes phosphoethanolamine

(PEt), phosphocholine (PC), and various sugar phosphates (Glonek et al., 1982), with PEt being the greatest contributor to this peak (Gyulai et al., 1984). PMEs appear mainly in the anabolic pathway of membrane phospholipid metabolism

(Pettegrew et al., 1987).

Tadafumi Kato, M.D., Toshiki Shioiri, M.D., PhD., Jun Murashita, M.D., and Hiroshi Hamakawa, M.D., are Assistants, Department of Psychiatry; Saburo Takahashi, M.D., Ph.D., is Professor, Department of Psychiatry; and Toshiro Inubushi, Ph.D., is Professor, Molecular Neurobiology Research Center, Shiga University of Medical Science, Otsu, Shiga, Japan. (Reprint requests to Dr. T. Kato, Department of Psychiatry, Shiga University of Medical Science, Seta-tsukinowa-cho, Otsu, Shiga, 520- 21, Japan.)

0165-1781/94/$07.00 @ 1994 Elsevier Science Ireland Ltd.

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Elevated levels of PME have been reported under conditions of increased mem- brane phospholipid synthesis such as brain tumors (Segebarth et al., 1987) and

neonatal brain (van der Knaap et al., 1990). A decrease in PME, on the other hand. has been associated with chronic cerebral infarction (Sappey-Marinier et al.. 1992); severe demyelinating disorders (van der Knaap et al., 1992); schizophrenia, especially cases characterized by negative symptoms (Pettegrew et al., 1991; Williamson et al.. 1991; Shioiri et al., in press); and anorexia nervosa (Kato et al., 1994). To date. the biochemical basis of reduced PME in these disorders has been difficult to examine because of the poor spectral resolution of in vivo MRS and the lack of post-mortem

in vitro 3IP-MRS studies that can be compared with in vivo MRS studies. It should be noted that most of the disorders in which reductions of PME have been reported

are also characterized to varying degrees by brain atrophy. The reported PME reductions could reflect decreased membrane phospholipid metabolism, and such a

metabolic change could be related to brain atrophy in these disorders. In other words, the PME reduction might be a nonspecific finding observed in brain

disorders accompanied with brain atrophy. Ventricular enlargement in bipolar disorder has been found in computed

tomographic studies (for review, see Andreasen et al., 1990), an observation that was

partly replicated in a recent volumetric study by proton magnetic resonance imaging (‘H-MRI) (Swayze et al., 1990). Therefore. we hypothesized that a decrease in PME might be correlated with ventricular enlargement in bipolar disorder. In addition to examining whether PME measured by j’P-MRS were linked to ventricular

enlargement indices measured by ‘H-MRI, it was a goal of this study to examine background factors underlying reduced PME and intracellularpH in lithium-treated euthymic patients with a diagnosis of bipolar disorder.

Methods

Subjects. The sample comprised 40 patients with bipolar disorder, of whom 3 1 had a subtype diagnosis of bipolar disorder I (BP-I) and 9 of bipolar disorder not otherwise specified (NOS). Diagnoses were made on the basis of DSM-III-R criteria (American Psychiatric Association, 1987) through two interview sessions by senior psychiatrists for 1 hour each. All the patients with bipolar disorder NOS had had major depressive and hypomanic episodes, so that they were subclassified as suffering from bipolar II disorder (BP-II). Ten of 40 patients (6 women and 4 men) had psychotic features. They were all inpatients hospitalized in the Shiga University of Medical Science Hospital and provided written informed consent to participate in the study. They had been treated with lithium carbonate for more than 2 weeks before the MRS examination. Duration of lithium treatment was 14-2865 days (mean = 3 13.7 days, SD = 556.8, median = 84.5). Seventeen of the patients were given additional treatment with antipsychotics (n = II) or antidepressants (n = 6). They were examined in the euthymic state. The 71P-MRS data in 17 of these patients were previously reported elsewhere (Kato et al.. 1993).

Sixty normal control subjects who were mentally and physically healthy and recruited from among hospital workers or their family members were also examined. Their ages and sexes were comparable to those of the patients. The normal control subjects had significantly more years of education (mean = 15.4, SD = 2.2, t = 5.9, p < 0.01) than did the patients with bipolar disorder (mean = 12.4, SD = 2.6) (Table 1).

Procedure. The 3lP-MRS protocol was basically the same as that described in our previous

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Table 1. Characteristics of the subiects

Control Bipolar disorder (n = 60) (n = 401

Females

Males

Age (yr)

Education (yr)

Duration of lithium treatment

(days)

Duration of illness fvrl

33 24

27 16

39.6 f 13.9 42.0 f 12.4

15.4 zt 2.2’ 12.4 f 2.6

- 3131t556

6.8 f 6.9

Note. Values are presented as mean f SD.

1. p < 0.01 to bipolar disorder

report (Kato et al., 1992, 1993). A General Electric (GE) Signa IS-Tesla MR system with a standard spectroscopy package was used. Surface coils for proton (‘H) and phosphorus (3lP) were supplied by the manufacturer. The diameter of the receiver coils was 7.5 cm. Subjects were lying in the magnet with their heads positioned (fixed with Velcro straps and molded foam head supports) so that the orbitomeatal line was vertical to the axis of the magnet. A surface coil for iH was placed on the forehead. The T,-weighted images in the axial planes with spin-echo pulse sequences, repetition time (TR) of 500 msec and echo time (TE) of 20 msec, were used for measurements of ventricular enlargement. Typical parameters of ‘H-MRI used were as follows: 256 X 128 matrix, 16-cm field of view, 0.5 excitations, 5-mm thick, and 1.5mm interscan gap. The position of the coil was monitored in iH-MRI and the coil was about 45 mm apart from the center of the volume of interest (VOI).

After the iH-MRI procedure, the magnetic field over the VOI was optimized by the signal from water. After the surface coil for proton was replaced by that for phosphorus, 3iP-MRS data were obtained with depth-resolved surface coil spectroscopy (DRESS) (Bottomley et al., 1984). VOI was the center 30-mm slice between the front pole and the front edge of the corpus callosum, parallel to the coil, predominantly of the frontal lobe (Fig. 1). One hundred twenty- eight scans were averaged with repetition time (3 seconds), delay time (1.5 msec), number of data points (1024) and center frequency (25.85 MHz). A GE 1280 data station with GEN software was used to process free induction decays (FIDs). The spectra were zero-filled twice, exponentially filtered (line broadening, 15 Hz), and had baseline distortion canceled by the convolution difference method (150 Hz, k = -0.85). Zero order and first order phase correction was manually performed, and a baseline correction with linear tilt was applied after deciding five points known to have no signal manually.

Fig. 2 shows 31P-MR spectra. Seven peaks were resolved: phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr), and three resonances from adenosine triphosphate (y-, a-, P-ATP). Intracellular pH was calculated from the chemical shift difference of PCr and Pi (Petroff et al., 1985). Peak areas were calculated by manual curve fitting with the mixture of 80% Gaussian and 20% Lorentzian curves. Because of peak splitting due to spin-spin coupling, y-ATP was simulated by synthesis of two similar peaks. Each peak area was shown as percent value versus the total phosphorus signal. Peak fitting was not performed without knowledge of diagnosis. Interrater reliability of peak area calculation, which was measured in 12 data sets by three raters, was < 6.5% except for Pi, 8.4%. Inter-assay coefficients of variation (CVs) calculated in 11 patients examined more than twice were as follows: PME 8.7%, Pi 20.9%, PDE 7.3%, PCr 4.2%, P-ATP 9.0%, and intracellular pH 0.80%. Signal-to-noise ratio in this experiment was about 8.

Linear measurement was used to evaluate degrees of ventricular enlargement. The Evans ratio (ER), Huckman number (HN), and the minimum distance of caudate nuclei (MDCN) (Huckman et al., 1975; Reveley, 1985) were used to measure ventricular enlargement with a translucent ruler from MRI film. ER is the measure of the maximum distance between tips of

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Fig. 1. Tl-weighted proton magnetic resonance image in a control subject with spin-echo pulse sequence

Repetition trme was 500 msececho trme, 20 msec; 512 n 256 matrix; number of excrtatrons, 0.5; and slrce thrckness. 5 mm Volume of interest in phosphorus-31 magnetic resonance spectroscopic examination is denoted by a rectangular box.

Fig. 2. Phosphorus-31 magnetic resonance spectra in a patient with bipolar disorder (64 years old, female) by depth-resolved surface coil spectroscopy (DRESS) in the frontal lobe

4

3

6

Repetition time, 3 seconds; delay time, 1.5 msec; 126 averages. Peak assignment is as follows: 1. PME (phosphomonoester); 2. Pi (inorganic phosphate): 3. PDE (phosphodiester); 4. PCr (creatine phosphate); 5. y-ATP (y-nucleotide triphosphate, mainly adenosine triphosphate); 6. a-ATP; 7. I).ATP.

frontal horns (FH) divided by the maximum transverse outer diameter of the skull along this

line. MDCN is the min~um distance between the heads of caudate nuclei just anterior to the foramen of Monroe. HN represents the sum of FH and MDCN. These measurements were performed by raters without knowledge of diagnoses and “P-MRS data. interrater reliability was assessed through measurements of 10 films by four raters; the average interrater CVs were 3.1% for ER, 3.1% for HN, and 6.3% for MDCN. Intermeasurement variability was assessed in 28 subjects examined more than twice, and average intermeasurement CVs were 4.6% for ER, 4.6% for HN, and 8.4% for MDCN.

For statistical analysis, two-sample t test, Pearson’s correlation analysis, and Spearman’s rank-correlation test were used. To’ test the ~producibility of previously reported findings such as low PME and low intracellularpH, one-tailed I tests were used.

Results

Correlation of 3lP-MRS and 1H-MRI. Table 2 summarizes the 3*P-MRS and ventricular enlargement indices. Values of PME (%) in the 40 bipolar patients (mean = 10.6, SD = 1.7) were lower than those of normal control subjects (mean = 11.2, SD = I .S, t = 1.95, p < 0.05).

Ventricular size, as measured by ER (mean = 0.315, SD = 0.027) and MDCN (mean = 13.8, SD = 3.4) with iH-MRI, was greater in 39 bipolar patients (‘H-MRI data were missing in one BP-I patient) than in the normal control subjects (ER: mean =I 0.301, SD = 0.034, c = 2.28, p < 0.05; MDCN: mean = 12.0, SD = 3.3, t = 2.32, p < 0.05). There were no significant differences in HN measures between the two groups (bipolar patients: mean = 39.9, SD = 11.5; control subjects: mean Z 38.4, SD = 11.7, t = 0.70). The patients with psychotic features (n = 10) had significantly greater venticular enlargement as measured by the ER (mean = 0.33, SD = 0.02) compared with those without psychotic features (mean = 0.30, SD = 0.02, t = 2.5, p < O.OS), while other values did not differ between these two groups.

Table 2. Results in 31P-MRS and lH-MRI

Control Bipolar disorder

Mean SD - Mean SD

PME% 11.2 1.5 10.6 1.7

PH 7.05 0.04 7.01 2 0.04

MDCN (mm) 12.0 3.3 1 3.83 3.4

Huckman number (mm) 38.4 11.7 39.9 11.5

Evans ratio 0.301 0.034 0.3154 0.027

Note. MDCN = minimum distance of caudate nuclei. PME = phosp~omon~ste~. MRS = magnetic resonance spectroscopy. MRl= magnetic resonance imaging.

1. p < 0.05, t = 1.9. 2.p<O.Ol,t=2.9. 3. p < 0.05, t = 2.3. 4. p < 0.05, t = 2.2.

There were no correlations between educational level and ER or MDCN measures of ventricular size, while a weak but significant correlation was found between educational level and ventricular size as measured by HN (r = -0.24, p < 0.05) when all the data obtained from normal control subjects and patients were combined.

In 39 patients with bipolar disorder, there were no significant correlations between

PME and three measures of ventricular enlargement: ER (r = 0.02, t = 0.17). MDCN (r = -0.1 1, I = 0.68), and HN (r = 0.23, I = 1.44) (Table 3). No correlation was found between these values after patients with psychotic features were excluded.

Clinical Factors Related to Reduced PME and Intracellular pH. PME values were negatively correlated with age in patients with bipolar disorder (r = -0.4 I. t = 3.02, p < 0.01) but not in normal control subjects (r = -0.07, t = 0.81). There was no significant correlation between PME and duration of illness (r = -0.23, I = I .5), age of onset (r = -0.25, I = I .65), or duration of lithium treatment (r = 0.07.

t = 0.43). IntracellularpH in 40 patients with bipolar disorder (mean = 7.0 I, SD = 0.04) wab

lower than that in the normal control subjects (mean = 7.05, SD = - 0.04, t = 2.9, p < 0.0 I). There were no significant correlations between intracellular pH and any of the following factors: age (r = 0.03, t = 0.19), duration of illness (r = 0.0 I, t = 0.10).

or age of onset (r = 0.0 1, t == 0.07). Intracellular pH showed a positive correlation

with duration of lithium treatment (r = 0.33, p < 0.05, t = 2.13) (Table 3). The J’P-MRS measures, including PME and intracellular pH, showed no

significant correlation with years of education when all the data obtained from normal control subjects and patients with bipolar disorder were combined. There were no differences in PME and intracellularpH between those patients treated with antipsychotics and those without antipsychotics. The use of antidepressants also did not affect PME and pH. There were no significant differences of PME and pH between the patients with psychotic features and those without psychotic features.

Table 3. Coefficients of correlation of 31P-MRS with lH-MRI and clinical factors in bipolar disorder

PME DH

MDCN -0 11

Huckman number 0.23 -

Evans ratio 0.02 -

Age -0.43’ 0.03

Age of onset -0.25 0.01

Duration of illness -0.23 0.01

Duration of lithium treatment 0.07 0.332

Note. MDCN = minimum distance of caudate nuclei. PME = phosphomonoesters. MRS = magnetic resonance spectroscopy. MRI = magnetic resonance imaging.

1.p < 0.01, t = 3.0. 2.p < 0.05, t = 2.1.

Differences Between BP-I and BP-II Subtypes. Because all the patients with BP-II were female, only female subjects in the BP-I (n = 15), BP-II (n = 9), and control (n = 33) groups were compared. Age, education, and duration of illness did not differ significantly among the three groups.

PME was lower in BP-I females (mean = 10.2, SD = 1.8) than in the normal females (mean = 11.2, SD = 1.5, t = 1.8, p < 0.05). PME in BP-II females (mean = I 1.4, SD = 1.3) did not differ from that in normal control females, and tended to

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be higher than that in BP-I females (C = 1.5, p 3 &IO), although the difference was not statistically significant. Intracellular pH in the BP-I females (mean = 7.01, SD = 0.04) was lower than that in the normal control females (mean = 7.04, SD = 0.04, p < 0.05, t = 2.1). The values of intracellular pH in BP-II females (mean = 7.02, SD = 0.03) were intermediate between those in the BP-I and normal control females.

Discussion

In a replication study with a larger sample size, we confirmed our previous finding (Rato et al., 1992, 1993) of lower PME and intracelluIar pH in lithium-treated euthymic patients with bipolar disorder, although the difference of PME observed between bipolar patients and control subjects was not large.

PME showed a negative correlation with age in bipolar disorder. Such age dependence of PME could not be found in the normal control subjects. There are two possible explanations for this finding: (1) PME decreases with age in bipolar disorder or (2) late-onset bipolar disorder differs in its pathophysiology from early onset bipolar disorder. We cannot determine which of these possible explanations is likely to be correct because there was no correlation between PME and duration of illness or age of onset. A longitudinal followup study will be needed to resolve this question. Although the educational level of the normal control subjects differed significantly from that in the patients with bipolar disorder, this factor did not seem to be related to J’P-MRS measures. It is difficult to determine from the present data whether lithium treatment contributed to the PME decrease. Reduced PME was found only in BP-I patients, which may indicate that the reduction of PME is specific to the BP-I subtype, suggesting that the BP-I and BP-II dichotomy that has been suggested by clinical studies (Endicott et al., 1985) may be an important distinction in biological studies of affective disorders.

On the other hand, it is difficult to conclude from the present results whether intracellular pH is specific to BP-I disorder. The positive correlation between intracellular pH and duration of lithium treatment does not seem to support the hypothesis that low intracellular pH in bipolar disorder is due to lithium treatment, On the contrary, we can hypothesize that long-term lithium treatment restores abnormally reduced intracellular pH in bipolar disorder. Future study in drug-free bipolar patients may clarify this problem.

Of three methods of measuring ventricular enlargement, both ER and MDCN were significantly increased in bipolar disorder. No difference was found in these values between BP-I and BP-II patients. Although ventricular enlargement has been reported in bipolar disorder (for review, see Andreasen et al., 1990), this finding is still controversial (Swayze et al., 1990). The present results are consistent with previous results with linear measurement (Schlegel and Kretzschmar, 1987). We cannot exclude the possibility that a difference in the educational level between the patients and the normal control subjects influenced the findings of ventricular enlargement.

Contrary to our hypothesis, no correlation was found between PME and ventricular enlargement scores in 39 patients with bipolar disorder. From the present

4x

results, ventricular enlargement found in bipolar disorder did not seem to be the consequence of altered membrane phospholipid metabolism. Swayze et al. (1990) speculated that ventricular enlargement observed in bipolar disorder may be a predisposing factor rather than a causative factor. because it showed no correlation with duration of illness and it was seen only in male patients who have a higher rate of birth abnormalities. Nasrallah (1991) also speculated that ventricular enlargement

in bipolar disorder may be a neurodevelopmental abnormality, because the hippocampus amygdala complex and the cerebellum were small in bipolar disorder and there was not a progressive increase in ventricular size in a followup study (Woods et al.. 1990).

From the present results, together with these hypotheses, we cannot say that the reduction of PME observed in bipolar disorder is a nonspecific finding associated

with ventricular enlargement. However, we should take many other confounding factors into account before

coming to any conclusion: I. Temporal dissociation between reduction of PME and ventricular enlargement

may confound the results. In other words, it would take many years for an alteration of membrane phospholipid metabolism to cause ventricular enlargement.

2. The linear measurements that we used to assess ventricular enlargement may

have confound the results. Although linear measurement is a reliable method when used properly (Reveley. 1985). other forms of measurement offer more precision in the quantificaiton of brain atrophy. Because we used a surface coil to acquire IH- MRI in this study, we could not use other indexes such as the ventricle-brain ratio or

volumetric measurement. A study with “‘P-MRS and volumetric MRI will be

required to rule out the effects of atrophy on “P-MRS measures. 3. Reduction of the PME peak area observed does not always reflect reduced

concentration of PMEs in the brain. Reduction of the PME peak area might be due to changes in T, and or T, relaxation times. Because the T, relaxation time of phosphoethanolamine in the human brain. 2.9 seconds (Gruetter et al.. 1993). is

comparable to the TR we used in this study, prolongation of T, may decrease the PME peak area. Moreover, signals with very short Tz may be lost because of the relatively long delay time in this experiment, 1.5 msec. Because the DRESS method that we used examines heterogeneous tissues such as the brain, cerebrospinal flood (CSF), bones, skin, and muscles, the PME peak area obtained may not exactly reflect PME in the brain. Because the VOI was close to the ventricular cavity. the VOI might be contaminated by ventricles. However, it would not affect peak ratios.

4. Gray matter: white matter ratios in the VOI may differ between subject>. Because “P-MR spectra differ between gray matter and white matter (Kilby et al., 1991), heterogeneity of their ratios in the VOI among subjects may confound the results.

5. A number of factors can influence the reliability of “P-MRS examinations: the position of the coil, the loading factor, the size of the head, movement artifacts, and resonance offset artifacts. The exact shape of the VOI in a DRESS experiment cannot be known. It is difficult to measure intracellular pH if the Pi peak arises from the broad shoulder of the PDE peak. There are also many confounding factors in data processing: phase correction, baseline correction, and peak area calculation b!,

49

manual curve fitting. These factors might confound the present results. Nevertheless, the significant age-dependent increase of PCr in the normal control subjects (r = 0.35, f = 2.92, p < O.Ol), which was found in the present data and which was reported by other authors (Long0 et al., 1993), supports the reliability of our method.

Although there are still many shortcomings in 3iP-MRS studies such as the one reported here, the method offers a valuable means of detecting in vivo metabolism in the human brain, This is the first study in a sizable number of patients with bipolar disorder to reveal an age-dependent decrease of PME, an absence of correlation of PME values with ventricular enlargement, and a difference in PME values between BP-I and BP-II subgroups.

Today, advanced MRS techniques are available that provide higher sensitivity and better localization of the signal. Such methods may have potential impact on the reliability of MRS investigations in psychiatry.

Acknowledgments. This study was supported in part by a Research Grant (5B-2) for Nervous and Mental Disorders from the Ministry of Health and Welfare and by a Grant-in- Aid from the Japanese Ministry of Education, N05770723.

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