Heritability Heightens Brain Metabolite Differences in Schizophrenia

Considerable evidence has accumulated implicating genetic factors in the etiology of schizophrenia. Brain imaging has revealed subtle but significant abnormalities in schizophrenic patients. Several studies of schizophrenic patients have associated certain genotypes with specific morphologic or functional abnormalities. N-acetylaspartate (NAA) levels in the brain are measurable in vivo by proton magnetic resonance spectroscopy (¹H-MRS). A trend toward a decreased ratio of NAA to choline-containing compounds (Cho) in the anterior cingulate region has been observed in the offspring of schizophrenic patients in comparison to healthy subjects with no family history of schizophrenia.¹ Using ¹H-MRS, Callicott et al.² recently have demonstrated that both schizophrenic patients and their unaffected siblings showed significant reductions in hippocampal NAA/creatine-phosphocreatine (Cr) ratios compared with healthy subjects. These findings suggest that low NAA may be heritable.

Several methods have been used to investigate heritability in schizophrenia. Taking one of these approaches, Buckley et al.³ have examined brain metabolites measured by ¹H-MRS in schizophrenic patients in terms of presence or absence of a family history (FH) of schizophrenia, finding no significant association. However, these authors considered a patient whose first- or second-degree relatives were affected by manic-depressive psychosis to have a positive FH. The heterogeneity of “positive FH” in that study, then, could have affected the results. An epidemiologic study has found relatives of schizophrenic patients to have an increased risk for schizophrenia, schizoaffective disorder, paranoid disorder, and atypical psychosis.⁴ We therefore excluded patients with FH of a mood disorder within second-degree relatives when we examined whether FH of psychotic disorders affected metabolite levels in the left medial temporal lobe in schizophrenic patients.

METHODS

Sixty-four Japanese patients (34 men and 30 women, mean age 36.5±7.2 years; means±SD are reported) who met DSM-III-R⁵ criteria for schizophrenia were recruited from the psychiatric wards in Fujimoto and Kagoshima University hospitals. None of the patients had a history of neurologic disorders, metabolic disorders (e.g., diabetes mellitus, hypothyroidism), severe head trauma, or electroconvulsive therapy. No patients had been diagnosed with alcoholism, and none had access to alcohol or nonprescribed drugs for the more than 6 months that followed their admission. All patients were receiving typical neuroleptics such as haloperidol, perphenazine, propericiazine, nemonapride, and chlorpromazine at the time of MRS examination (mean chlorpromazine equivalent, 617±399 mg/day),⁶ and 33 patients also were receiving anticholinergic agents such as biperiden or trihexyphenidyl. They had been ill for 14.9±7.8 years. A comparison group of 51 healthy subjects included hospital staff and university students (25 men and 26 women, mean age 35.5±7.9 years). None had a history of psychiatric or neurologic illness. All subjects were right-handed, as determined by the Edinburgh Inventory;⁷ a laterality score greater than 80 qualified them as right-handed.⁸ The Edinburgh Inventory score did not differ significantly between the patient and control groups (patient vs. control: 91.8±6.5 vs. 93.1±7.5; t=1.027, df=113, P=0.307). Nutritional condition was not systematically matched between groups. Subjects with total cholesterol greater than 240 mg/dl or abnormal electrolyte concentrations in routine blood chemistry screening were excluded. The definition of a positive FH was determined in accordance with the Family History Research Diagnostic Criteria.⁹ Patients whose first- or second-degree relatives had histories of schizophrenia, delusional disorder, schizophreniform disorder, or schizoaffective disorder were defined as having a positive FH. Diagnoses in these relatives were made on the basis of information from their treating psychiatrists or physicians. Age and gender were not significantly different between patients with and without FH (with FH vs. without FH: mean age, 38.4±5.7 vs. 36.0±7.5 years; t=1.112, df=62, P=0.271; gender, 7 men and 6 women vs. 27 men and 24 women; χ²=0.003, df=1, P=0.999). The Edinburgh Inventory score was similar between the groups (with FH vs. without FH: mean score 91.6±6.3 vs. 91.9±6.6, t=0.159, df=62, P=0.874). All subjects gave written informed consent for their participation in the study.

The method of MRS data acquisition was similar to that in the previous study by Fukuzako and colleagues;¹⁰ however, some parameters were different and the method of peak area measurement was improved. Investigations were conducted by using an MR system (Siemens-Asahi Meditec; Erlangen, Germany) with a magnetic field strength of 2.0 tesla. A quadrature detection head coil with a diameter of 29 cm was used. The head coil was tuned to 84.5 MHz for proton imaging and MRS. The volume of interest (VOI) was localized on the basis of series of T₁-weighted images (fast low-angle shot sequence, TR 23 ms, TE 10 ms, flip angle 35 degrees, slice thickness 5 mm, matrix size 192×256). Voxel placement was performed by a single expert (T. Matsumoto) who located the anterior hippocampus in the center of the VOI. The VOI (2×2×2 cm³) included mainly the left medial temporal lobe (hippocampal formation, entorhinal cortex, and amygdala). Water-suppressed ¹H MR spectra were obtained by using stimulated echo acquisition mode (STEAM) pulse sequences. MR parameters were as follows: TR, 2,000 ms; TE, 60 ms; dwell time per point, 1,000 μs for 1,024 points; and filter band width, 500 Hz. After optimizing magnetic field homogeneity by shimming on the water signal, we obtained 256 measurements. Water-line widths below 4 Hz were achieved. Spectra were obtained through Fourier transformation of the raw data without line broadening. Chemical shifts were noted at NAA resonance (–CH₃) for 2.0 parts per million. Spectra of NAA (–CH₃), Cho, and Cr (–CH₃) were quantified by measurement of peak area. After automatic baseline correction (spline approximation), peak parameters such as height, position, and width were obtained by means of the standard software provided with the MAGNETOM Vision (Siemens, Germany; Lorentzian curve-fitting procedure). For each spectrum, integrated areas of the three metabolites were measured and ratios of NAA/Cr and Cho/Cr were calculated. ¹H-MRS was performed twice, 1 to 2 weeks apart, in 9 patients for the assessment of test-retest reliability. Coefficients of variation for the metabolite ratios were 9.2% and 10.4% for NAA/Cr and Cho/Cr, respectively.

Statistical analysis was performed by using StatView 4.5 software. Two-tailed t-tests were used to assess differences between groups. The relationships between the two kinds of values were tested by using Pearson's product-moment correlation coefficient (r).

RESULTS

Thirteen patients were found to have a positive FH. Ratios of NAA/Cr and Cho/Cr are presented in Table 1. A significantly lower NAA/Cr ratio was present in the medial temporal region in schizophrenic patients compared with healthy subjects (t=6.733, df=113, P<0.001). The Cho/Cr ratio did not differ significantly between patients and control subjects (t=1.465, df=113, P=0.146). The NAA/Cr reduction was more prominent for patients with an FH than for those without FH (t=2.860, df=62, P=0.006). No significant difference was observed for the Cho/Cr ratio with respect to FH (t=1.400, df=62, P=0.167). The NAA/Cr ratio was inversely correlated with age (r=–0.219, P=0.082) and duration of illness (r=–0.265, P=0.034). However, the duration of illness was similar in patients with and without FH (16.9±8.7 vs. 14.2±7.3 years; t=1.162, df=62, P=0.250). No significant correlations were seen between daily dose of neuroleptic and NAA/Cr ratio (r=–0.054, P=0.675) or Cho/Cr ratio (r=–0.071, P=0.577). Anticholinergic agents did not significantly affect metabolite ratios (NAA/Cr: 1.398±0.207 vs. 1.366±0.230; t=0.577, df=62, P=0.566; Cho/Cr: 1.188±0.187 vs. 1.172±0.242; t=0.282, df=62, P=0.779).

DISCUSSION

This study partly replicated earlier findings,¹⁰ confirming a decreased NAA/Cr ratio in schizophrenic patients, whereas a previously noted elevation of the Cho/Cr ratio was not seen at this time. The previously noted increase in Cho/Cr ratio may have reflected small sample size (n=15 in patient and control groups) and an incompletely developed technique for peak area measurement (a manually guided area measurement without curve fitting) in the previous study. The present findings are consistent with the majority of studies using ¹H-MRS.^2,10–13 Our finding of a greater reduction in NAA/Cr in the patients with positive FH may suggest segregation of lower NAA in pedigrees involving schizophrenia, and is in line with the results of Callicott's study.² Measurement of absolute concentration in each brain structure using more advanced in vivo MRS methods will determine if NAA concentration indicates a true phenotype of chemical intermediates representing genetic susceptibility.

The present study did not indicate significant effects of neuroleptics on the metabolites. Bertolino et al.¹⁴ found that a pattern of reduction of hippocampal and prefrontal NAA/Cr was similar in untreated schizophrenic patients and medicated patients. However, two recent reports suggest that the NAA/Cr ratio in the frontal lobe is influenced by administration of neuroleptics.^15,16 As opposed to findings for the hippocampus, Callicott et al.² did not find evidence for heritability of NAA/Cr ratio in the prefrontal region. Findings from untreated patients will be required in order to confirm heritability of brain metabolite phenotypes detected by ¹H-MRS, especially with respect to the frontal lobe.

ACKNOWLEDGMENTS

The author thanks Drs. T. Fukuzako, K. Yamada, K. Takeuchi, and T. Fujimoto for their assistance. This study was supported by grants from the National Center of Psychiatry and Neurology of the Ministry of Health and Welfare (3A-5) and from the Ministry of Education, Science, and Culture (No. 05770735, 06770764, 10672129) of Japan.

TABLE 1. Peak area ratios of metabolites

Enlarge table