Alterations in brain high-energy phosphate and membrane phospholipid metabolism in first-episode, drug-naive schizophrenics. A pilot study of the dorsal prefrontal cortex by in vivo phosphorus 31 nuclear magnetic resonance spectroscopy.

In this pilot study, membrane phospholipid and high-energy phosphate metabolism were studied in the dorsal prefrontal cortex of 11 drug-naive, first-episode schizophrenic patients and compared with those of 10 healthy control volunteers comparable in age, education, and parental education. The schizophrenic patients had significantly reduced levels of phosphomonoesters and inorganic orthophosphate and significantly increased levels of phosphodiesters and adenosine triphosphate compared with the controls. The levels of phosphocreatine and adenosine diphosphate did not differ in the two subject groups. The adenosine triphosphate and inorganic orthophosphate findings suggest functional hypoactivity of the dorsal prefrontal cortex. The phosphomonoester and phosphodiester findings are compatible with either premature aging or an exaggeration of normal programmed regressive events occurring in the neural systems sampled.

31P nuclear magnetic resonance study of the brain in Alzheimer's disease.

The histopathological hallmarks of Alzheimer's disease have long been considered to be neurofibrillary tangles (NFT) and neuritic (senile) plaques (SP). Neither of these structures, however, are unique to Alzheimer's disease, and both probably represent end-stage markers of the disorder. NFT have been demonstrated in many disorders; SP occur in small numbers with normal aging. Evidence is presented for elevation of phosphomonoesters (PME) in Alzheimer's brain compared to non-Alzheimer's diseased controls and normal controls. The PME detected by 31P nuclear magnetic resonance (NMR) spectroscopy of autopsy brain are predominantly anabolic precursors of membrane phospholipids. Elevated PME could be secondary to a metabolic block at the rate-limiting enzyme in membrane phospholipid synthesis, which is cytidine triphosphate (CTP): phosphocholine (or phosphoethanolamine) cytidyltransferase (EC 2.7.7.15). Elevated PME could also be secondary to decreased breakdown of PME by phospholipase D activity. Since CTP: phosphocholine cytidyltransferase is inactivated by phosphorylation and since there is independent evidence for hyperphosphorylation of tau and MAP-2 proteins in AD brain, enhanced protein kinase activity could be a common factor. Preliminary evidence suggests that PME could interact with N-methyl-D-aspartate receptors and potentially act as false neurotransmitters. Further studies will be needed to investigate these possibilities.

Changes in brain energy and phospholipid metabolism during development and aging in the Fischer 344 rat.

The effects of brain development and aging on high-energy phosphate and membrane phospholipid metabolism were studied from birth to senescence in the Fischer 344 rat using 31P nuclear magnetic resonance spectroscopy. Marked developmental and smaller aging-related changes were observed in brain high-energy phosphates, phospholipid precursors and phospholipid breakdown products. The biochemical changes correlate with known histological and electrophysiological changes occurring in the brain during development (neuritic sprouting and onset of brain electrical activity) and aging (loss of dendritic processes). These findings provide a framework for interpreting the effects of physiological insults during different developmental and aging periods.

31P nuclear magnetic resonance studies of phosphoglyceride metabolism in developing and degenerating brain: preliminary observations.

31P nuclear magnetic resonance (NMR) studies were conducted on perchloric acid extracts of the brain of one control, two Huntington's disease (HD), one probable Alzheimer's disease (AD), and one AD patient. These studies demonstrated significant elevations (over control) in the levels of phosphomonoesters in all brain areas of the patients with HD and AD even in areas devoid of neuropathological findings. Elevations of phosphodiesters were also observed, but they tended to reflect the degree of neuropathological change. We postulate that the 31P NMR findings represent molecular alterations with corresponding metabolic correlates which either antedate or occur in the absence of changes in cellular morphology or structure. As such the 31P NMR findings may reflect a subcellular "molecular neuropathology."

Magnetic resonance spectroscopy in schizophrenia: methodological issues and findings--part I.

Our knowledge of the biological basis of schizophrenia has significantly increased with the contribution of in vivo proton and phosphorus magnetic resonance spectroscopy (MRS), a noninvasive tool that can assess the biochemistry from a localized region in the human body. Studies thus far suggest altered membrane phospholipid metabolism at the early stage of illness and reduced N-acetylaspartate, a measure of neuronal volume/viability in chronic schizophrenia. Inconsistencies remain in the literature, in part due to the complexities in the MRS methodology. These complexities of in vivo spectroscopy make it important to understand the issues surrounding the design of spectroscopy protocols to best address hypotheses of interest. This review addresses these issues, including 1) understanding biochemistry and the physiologic significance of metabolites; 2) the influence of acquisition parameters combined with spin-spin and spin-lattice relaxation effects on the MRS signal; 3) the composition of spectral peaks and the degree of overlapping peaks, including the broader underlying peaks; 4) factors affecting the signal-to-noise ratio; 5) the various types of localization schemes; and 6) the objectives to produce accurate and reproducible quantification results. The ability to fully exploit the potentials of in vivo spectroscopy should lead to a protocol best optimized to address the hypotheses of interest.

Magnetic resonance spectroscopy in schizophrenia: methodological issues and findings--part II.

Magnetic resonance spectroscopy allows investigation of in vivo neurochemical pathology of schizophrenia. "First generation" studies, focusing on phosphorus and proton magnetic resonance spectroscopy, have suggested alterations in membrane phospholipid metabolism and reductions in N-acetyl aspartate in the frontal and temporal lobes. Some discrepancies remain in the literature, perhaps related to the variations in medication status and phase of illness in the patients examined, as well as in magnetic resonance spectroscopy methodology; the pathophysiologic significance of the findings also remains unclear. Technologic advances in magnetic resonance spectroscopy in recent years have expanded the potential to measure several other metabolites of interest such as the neurotransmitters glutamate and gamma-aminobutyric acid and macromolecules such as membrane phospholipids and synaptic proteins. Issues of sensitivity, specificity, measurement reliability, and functional significance of the magnetic resonance spectroscopy findings need to be further clarified. The noninvasive nature of magnetic resonance spectroscopy allows longitudinal studies of schizophrenia both in its different phases and among individuals at genetic risk for this illness. Future studies also need to address confounds of prior treatment and illness chronicity, take advantage of current pathophysiologic models of schizophrenia, and be hypothesis driven.

A preliminary 31P MRS study of autism: evidence for undersynthesis and increased degradation of brain membranes.

In this pilot study, brain high energy phosphate and membrane phospholipid metabolism were investigated in the dorsal prefrontal cortex of 11 high-functioning autistic adolescent and young adult men (the age range is 12-36 years) and 11 age-, gender-, IQ, race- and socioeconomic status-matched normal controls using in vivo 31P nuclear magnetic resonance spectroscopy (MRS). The autistic group had decreased levels of phosphocreatine and esterified ends (alpha ATP + alpha ADP + dinucleotides + diphosphosugars) compared to the controls. When the metabolite levels were compared within each subject group with neuropsychologic and language test scores, a common pattern of correlations was observed across measures in the autistic group, but not in the control group. As test performance declined in the autistic subjects, levels of the most labile high energy phosphate compound and of membrane building blocks decreased, and levels of membrane breakdown products increased. No significant correlations were present with age in either group or with IQ in the control group, suggesting that these findings were not the consequence of age or IQ effects. This pilot study provides tentative evidence of alterations in brain energy and phospholipid metabolism in autism that correlate with the neuropsychologic and language deficits. The findings are consistent with a hypermetabolic energy state and undersynthesis of brain membranes and may relate to the neurophysiologic and neuropathologic abnormalities in autism.

Increased cerebrospinal fluid glutamine levels in depressed patients.

BACKGROUND: There is increasing evidence for an association between alterations of brain glutamatergic neurotransmission and the pathophysiology of affective disorders. METHODS: We studied the association between cerebrospinal fluid (CSF) metabolites, including glutamine, in unipolar and bipolar depressed patients versus control subjects using a proton magnetic resonance spectroscopy technique. Cerebrospinal fluid samples were obtained from 18 hospitalized patients with acute unmedicated severe depression without medical problems and compared with those of 22 control subjects. RESULTS: Compared with the control group, the depressed patient group had significantly higher CSF glutamine concentrations, which correlated positively with CSF magnesium levels. CONCLUSIONS: These findings suggest an abnormality of the brain glial-neuronal glutamine/glutamate cycle associated with N-methyl-D-aspartate receptor systems in patients with depression.

Chronic myo-inositol increases rat brain phosphatidylethanolamine plasmalogen.

BACKGROUND: Oral myo-inositol (12--18 g/day) has shown beneficial effect in placebo-controlled studies of major depression, panic disorder, and obsessive compulsive disorder, and preliminary data suggest it also may be effective in bipolar depression. Evidence linking antidepressant activity to membrane phospholipid alterations suggested the examination of acute and chronic myo-inositol effects on rat brain membrane phospholipid metabolism. METHODS: With both (31)P nuclear magnetic resonance (NMR) and quantitative high-performance thin-layer chromatography (HPTLC; hydrolysis) methods, rat brain phospholipid levels were measured after acute (n = 20, each group) and chronic myo-inositol administration (n = 10, each group). With (31)P NMR, we measured myo-inositol rat brain levels after acute and chronic myo-inositol administration. RESULTS: Brain myo-inositol increased by 17% after acute myo-inositol administration and by 5% after chronic administration, as compared with the control groups. Chronic myo-inositol administration increased brain phosphatidylethanolamine (PtdEtn) plasmalogen by 10% and decreased brain PtdEtn by 5%, thus increasing the ratio PtdEtn plasmalogen (PtdEtn-Plas)/PtdEtn by 15%. Phosphatidylethanolamine plasmalogen levels quantified by (31)P NMR and HPTLC were highly correlated. The validity and reliability of the (31)P NMR method for phospholipid analysis were demonstrated with phospholipid standards. CONCLUSIONS: The observed alteration in the PtdEtn-Plas/PtdEtn ratio could provide insights into the therapeutic effect of myo-inositol in affective disorders.

The effect of lithium on the membrane molecular dynamics of normal human erythrocytes.

Erythrocytes from normal adults with no personal or family history of bipolar affective disorder were analyzed by fluorescence spectroscopy to determine what effect, if any, acute in vitro incubation with lithium had on erythrocyte membrane dynamics. The effects on erythrocyte membrane molecular dynamics of varying concentrations of Li2CO3 (0.25-2.0 meq/liter), varying incubation temperatures (25-40 degrees C), and varying incubation times (5-185 min) were investigated. Following incubation with Li2CO3, the erythrocytes were labeled with either 4-phenylspiro-[furan-2(3H),--1'phthalan]--3,3'-dione (fluorescamine), which binds to membrane surface primary amines, or 12(9)anthroyl stearate [12(9)AS], which inserts deep in the membrane hydrocarbon core. The membrane molecular dynamics were then determined by fluorescence anisotropy measurements. These studies demonstrate that clinically relevant concentrations of Li+ incubated with intact normal human erythrocytes significantly alters molecular dynamics on the erythrocyte membrane surface, with less striking changes in the hydrocarbon core. A possible interpretation of these findings is that hydrated Li+ alters the electrostatic interaction of membrane surface molecules, as well as the surrounding solvent (water) structure, with a resultant increase in the molecular motion of these molecules. Alterations in membrane receptor motion could potentially alter receptor functional activity. If similar motional alterations were to occur in the interior of a membrane channel, such as an ionophore, the functional activity of the channel could also be potentially altered. These findings provide additional insight into possible biological actions of Li+, as well as potential molecular alterations in bipolar affective disorder erythrocytes.

Landmark-based morphometric analysis of first-episode schizophrenia.

BACKGROUND: The goal of this investigation was to utilize landmark-based shape analysis and image averaging to determine the sites and extent of specific structural changes in first-episode schizophrenia. METHODS: Neuroanatomic structures identified on midsagittal magnetic resonance imaging (MRI) scans were compared between 20 patients with schizophrenia and 22 normal control subjects. The difference between averaged landmark configurations in the two groups was visualized as a shape deformation by a thin-plate spline and through averaged MRI images for both groups. RESULTS: A shape difference was found to be statistically significant; by inspection, it is contrast between differences in two closely abutting regions, involving primarily the posterior corpus callosum and upper brain stem--the "focus" is the relation between them. CONCLUSIONS: The findings are consistent with prior studies suggesting involvement in schizophrenia of the corpus callosum and the limbic structures contributing to the corpus callosum; the possibility of local pathology primarily involving the brain stem cannot be excluded. The methods of landmark-based shape analysis and image averaging utilized in this study can complement the "region-of-interest" method of investigating morphometric abnormalities by characterizing the spatial relationships among structural brain abnormalities in schizophrenia.

Endogenous inhibitors of human choline acetyltransferase present in Alzheimer's brain: preliminary observation.

We have previously demonstrated the presence in Alzheimer's disease brain of an endogenous inhibitor of choline acetyltransferase activity. Selected properties of these compounds were investigated. There appear to be two distinct classes of inhibitor present, both phosphomonoesters and nonphosphorylated substances. They are not proteins, pass through 500 mm dialyses membranes and are not lipoidal. There are both different sensitivities of individual control cytosotic activity to inhibition and differences in intrinsic inhibitory activity present in individual Alzheimer's disease brain samples. There is a competitive type of inhibition with respect to acetyl CoA as substrate and a noncompetitive type with respect to choline as substrate.

Membrane interactions of a phosphomonoester elevated early in Alzheimer's disease.

Phosphoserine (L-PS) is among several phosphomonoesters found to be elevated in autopsied Alzheimer's disease (AD) brain tissue. To investigate the molecular interactions of L-PS with membrane lipid bilayers, small angle X-ray diffraction and high resolution differential scanning calorimetry (DSC) approaches were used with liposomes composed of lecithin and cholesterol. A one-dimensional electron density profile of a control dimyristoyl phosphatidylcholine (DMPC)/cholesterol lipid bilayer with a unit cell dimension of 52 A at 37 degrees C was generated from the X-ray diffraction data. Following incubation with 2.0 mM L-PS, a broad decrease in electron density +/- 4-12 A from the lipid bilayer center was observed concomitant with an increase in the width of the phospholipid headgroup electron density and a 3 A reduction in lipid bilayer width. The interactions of L-PS with DMPC lipid bilayers were concentration-dependent, highly affected by cholesterol content and reproduced in egg phosphatidylcholine/cholesterol liposomes. DSC analysis showed that millimolar (1.0-5.0 mM) L-PS levels decrease the phase transition cooperative unit size of DMPC liposomes in a highly concentration-dependent manner which was significantly greater in preparations containing cholesterol. The endotherm width at half-maximum doubled at 5.0 mM and 1.25 mM L-PS, respectively, for DMPC and DMPC/cholesterol liposomes. These data provide direct evidence that elevated phosphomonoester levels modulate the biophysical properties of the membrane lipid bilayer which may, in turn, lead to altered structure/function relationships in membranes during AD.

Chrysamine-G binding to Alzheimer and control brain: autopsy study of a new amyloid probe.

Chrysamine-G (CG) is a carboxylic acid analogue of Congo red, a histologic dye which stains amyloid. CG binds to the beta-amyloid protein of Alzheimer's disease (AD) in vitro and partitions into the brain of normal mice. In this study, we demonstrate increased binding of [14C]CG to homogenates of several regions of AD brain as compared to control. The total binding of CG to AD brain was approximately two- to three-fold that of control brain. The cerebellum could be used as an internal standard for each brain as CG binding to cerebellum did not differ between AD and control. The binding of [14C]CG correlated with numbers of senile plaques and neurofibrillary tangles. In addition, CG could be used to stain cerebrovascular amyloid in tissue sections. These results suggest that CG may prove useful as an in vivo probe of amyloid deposition in AD.

Development of small molecule probes for the beta-amyloid protein of Alzheimer's disease.

This study describes the synthesis and in vitro testing of small molecule probes that may eventually prove useful as markers of amyloid deposition in living patients. The prototype agent, Chrysamine G (CG), is a derivative of Congo red. CG binds synthetic beta-amyloid well in vitro, as does a fluorinated derivative. The mechanism of binding appears to be the same as Congo red--through a bidentate attachment spanning several amyloid peptide chains. CG is much more lipophilic than Congo red and crosses the blood-brain barrier in normal mice, achieving a brain/blood ratio over 10/1. There was no acute toxicity in mice at doses 10 times those used in the distribution studies. CG appears to be a relatively high affinity probe for beta-amyloid that appears to have low toxicity and can cross the blood-brain barrier. These characteristics are promising for development of in vivo amyloid probes similar to CG.

Elevated phosphocholine and phosphatidylcholine following rat entorhinal cortex lesions.

At early stages of Alzheimer's disease, phosphomonoesters (PMEs) including phosphocholine (P-choline) are present at elevated levels. PMEs also are elevated in the developing brain during the period of neurite extension. To determine if the elevation of PMEs in AD could reflect neuritic sprouting, 31P-NMR was used to examine phospholipid metabolites and membrane phospholipids at various times following unilateral lesions of the entorhinal cortex, a well-defined model of neuritic sprouting. Two to 7 days postlesion, P-choline levels were elevated 48% in the hippocampus ipsilateral to the entorhinal cortex lesion, but not in the contralateral hippocampus or cerebral cortex. P-choline levels declined by day 15, and reached control levels 45 days following the lesion. The lesion-induced elevation in P-choline could result from increased P-choline synthesis via choline kinase, decreased activity of CTP:phosphocholine cytidylyltransferase, or breakdown of phosphatidylcholine (PC). To distinguish between these possibilities, the membrane phospholipids PC and phosphatidylethanolamine (PE) were measured. Both phospholipids were maintained at or above control levels at each of the postlesion time points, arguing against membrane breakdown or decreased PC synthesis contributing to the elevation of P-choline levels. Other alterations included a widespread elevation in inositol phosphate 2 days postlesion, but not at later time points. The alterations in phospholipid metabolites observed in the rat hippocampus following entorhinal cortex lesions closely resemble those observed in the human brain in the early stages of AD.

Clinical and neurochemical effects of acetyl-L-carnitine in Alzheimer's disease.

In a double-blind, placebo study, acetyl-L-carnitine was administered to 7 probable Alzheimer's disease patients who were then compared by clinical and 31P magnetic resonance spectroscopic measures to 5 placebo-treated probable AD patients and 21 age-matched healthy controls over the course of 1 year. Compared to AD patients on placebo, acetyl-L-carnitine-treated patients showed significantly less deterioration in their Mini-Mental Status and Alzheimer's Disease Assessment Scale test scores. Furthermore, the decrease in phosphomonoester levels observed in both the acetyl-L-carnitine and placebo AD groups at entry was normalized in the acetyl-L-carnitine-treated but not in the placebo-treated patients. Similar normalization of high-energy phosphate levels was observed in the acetyl-L-carnitine-treated but not in the placebo-treated patients. This is the first direct in vivo demonstration of a beneficial effect of a drug on both clinical and CNS neurochemical parameters in AD.

Changes in brain membrane phospholipid and high-energy phosphate metabolism precede dementia.

A 52-year-old Caucasian male was followed with Mattis and 31P MRS examinations every 6 months for 33 months. At entry into the study, the subject had a normal clinical examination and normal Mattis scores but had alterations in MRS measures of membrane phospholipid and high-energy phosphate metabolism indistinguishable from those previously reported in mildly demented AD patients. After 33 months of follow-up, the subject had clinical and Mattis findings suggestive of possible incipient dementia and after 46 months of follow-up there was sufficient cognitive decline to make the diagnosis of dementia with a frontal lobe preponderance. The findings in this subject support the contention that alterations in brain membrane phospholipid and high-energy metabolism can be noninvasively detected by 31P MRS years before any clinical manifestations of the disease.

Alterations of cerebral metabolism in probable Alzheimer's disease: a preliminary study.

Previous in vitro and in vivo 31P MRS studies of Alzheimer's disease patients have revealed alterations in membrane phospholipid metabolism and PET studies have shown alterations in glucose and oxidative metabolism. This study of probable Alzheimer's disease patients demonstrates severity dependent alterations in measures of both high-energy phosphate and membrane phospholipid metabolism. Mildly demented Alzheimer's patients compared to the controls, have increases in the levels of phosphomonoesters, decreases in the levels of phosphocreatine and probably adenosine diphosphate, and an increased oxidative metabolic rate. As the dementia worsens, the levels of phosphocreatine and adenosine diphosphate increase, the levels of phosphomonoesters decrease, and the oxidative metabolic rate decreases. The phosphomonoester findings replicate previous findings and provide a new dimension to the molecular pathology of Alzheimer's disease, implicating basic defects in membrane metabolism. The changes in oxidative metabolic rate suggest the AD brain is under energetic stress. The changes in energy metabolites with increasing dementia could be a consequence of nerve terminal degeneration and are consistent with previous PET findings. 31P MRS provides new diagnostic and metabolic insights into this disease and would be a noninvasive method to follow the progression of the disease and the metabolic response to therapeutic interventions.

Analysis of magnetic resonance spectra by mole percent: comparison to absolute units.

A variety of metabolites present in perchloric acid extracts of brain tissue were measured by 1H and 31P magnetic resonance spectroscopy (MRS) and HPLC in the same tissue sample and the MRS results were expressed both in terms of mole % and mumole/g based on an internal standard. The levels of 16 metabolites were compared by linear regression analysis and the mole % results were found to correlate very well with the results expressed as mumole/g. To compare the two units under typical experimental conditions, the percent change in metabolites in a group of Alzheimer's disease brains was compared to a control group using both units. The results were essentially identical for the mole % and mumole/g methods. We conclude that the use of the mole % method of expressing MRS data yields results which are equivalent to those expressed in absolute units and suggest that, for in vivo MRS studies, use of the mole % method is preferable because fewer artifacts, such as partial volume effects, are introduced.