Clozapine but not lithium reverses aberrant tyrosine uptake in patients with bipolar disorder.

RATIONALE: Availability of the dopamine and noradrenaline precursor tyrosine is critical for normal functioning, and deficit in tyrosine transport across cell membrane and the blood-brain barrier has been reported in bipolar disorder and schizophrenia. Clozapine and lithium are two psychoactive agents used to treat psychosis, mood disorders and suicidal behavior, but their mechanism of action remains largely unknown. OBJECTIVE: To characterize immediate and delayed differences in tyrosine uptake between healthy controls (HC) and bipolar patients (BP) and see if these differences could be normalized by either clozapine, lithium or both. A second objective was to see if clozapine and lithium have additive, antagonistic or synergistic effects in this. METHOD: Fibroblasts from five HC and five BP were incubated for 5 min or 6 h with clozapine, lithium, or combination of both. Radioactive labelled tyrosine was used to quantify tyrosine membrane transport. RESULTS: There was significantly reduced tyrosine uptake at baseline in BP compared to HC, a deficit that grew with increasing incubation time. Clozapine selectively increased tyrosine uptake in BP and abolished the deficit seen under baseline conditions, while lithium had no such effect. Combination treatment with clozapine and lithium was less effective than when clozapine was used alone. CONCLUSIONS: There was significant deficit in tyrosine transport in BP compared to HC that was reversed by clozapine but not lithium. Clozapine was more effective when used alone than when added together with lithium. Potential clinical implications of this will be discussed.

Aberrant amino acid transport in fibroblasts from patients with bipolar disorder.

Aberrant tyrosine transport is a repeated finding in fibroblasts from schizophrenic patients. The transport aberration could lead to disturbances in the dopaminergic and noradrenergic neurotransmitter systems. Tyrosine and tryptophan are the precursors of the neurotransmitters dopamine and serotonin. Disturbed dopaminergic, noradrenergic and serotoninergic systems are implicated as causes of bipolar disorder. Hence, the aim of this study was to explore whether patients with bipolar disorder have an aberrant transport of tyrosine and/or tryptophan. Fibroblast cell lines from patients with bipolar type-1 disorder (n=10) and healthy controls (n=10) were included in this study. All patients fulfilled the DSM-IV diagnostic criteria. The transport of amino acids across the cell membranes was measured by the cluster tray method. The kinetic parameters, maximal transport velocity (V(max)) and affinity constant (K(m)) were determined. A significantly lower V(max) for tyrosine (p=0.027) was found in patients with bipolar type-1 disorder in comparison to healthy controls. No significant differences in K(m) for tyrosine and in the kinetic parameters of tryptophan between patients with bipolar type-1 disorder and healthy controls were observed. The decreased tyrosine transport (low V(max)) found in this study may indicate less access of dopamine in the brain, resulting in disturbed dopaminergic and/or noradrenergic neurotransmission, that secondarily could lead to disturbances in other central neurotransmitter systems, such as the serotoninergic system. However, as sample size was small in this study and an age difference between patients and controls existed, the present findings should be considered as pilot data. Further studies with larger sample number are needed to elucidate the transport aberration and the significance of these findings.

Detection of small implanted tumors growing during repeated magnetic resonance imaging of the rabbit liver: application of an interpretation model.

PURPOSE: To apply experimentally and further develop a new image interpretation model based on repeated imaging and aimed at improving assessments of technical efficacy and diagnostic accuracy in the detection of small lesions. MATERIAL AND METHODS: VX2 carcinoma was implanted in the liver of 14 rabbits as two 1.1-1.7 mm3 cores. Magnetic resonance imaging was performed before and 4 days after implantation and then every second day up to the 14th to 20th day. One T2-weighted sequence (TSE T2) and three T1-weighted sequences (SE T1, GE T1, and TFL T1) were used. Interpretation was performed stepwise: three readers independently interpreted image sequences chronologically (step 1). Tumors were included at the last examination (step 2). By concurrent interpretation of repeated examinations, the earliest day at which tumors became visible and tumor size were recorded (step 3). Records were corrected (step 4) and autopsy was performed (step 5). Two procedures for use in calculating repeated detection rates of tumors with different magnetic resonance imaging sequences are presented and discussed. RESULTS: Of 40 macroscopic tumors, 34 were included. They were mainly small (size range SE T1: 1-3mm, TSE T2: 1.5-5 mm) when they became visible as determined at step 3, which was consistently earlier than observed at step 1. TSE T2, SE T1, and GE T1 did not differ significantly regarding earliest day of detection (step 3), while TFL T1 revealed the tumors later. The initial repeated detection rates were higher with TSE T2 than with the other sequences. Frequency of false positives varied over time, indicating fluctuating criteria for reporting tumors. CONCLUSION: A theoretical image interpretation model previously described proved to be applicable for detection of experimental liver tumors. The model was improved by introducing calculations of repeated detection rates for initial image interpretation using an imaging reference standard.

Aberrant tyrosine transport across the cell membrane in patients with schizophrenia.

BACKGROUND: There is evidence that patients with schizophrenia exhibit abnormalities, not only in the brain but also in peripheral organs. An abnormal cell membrane composition has been suggested to be a common denominator, supported by findings of alterations in membrane phospholipid levels. In a previous study, the transport of amino acids across the plasma membrane was investigated with fibroblasts from patients with schizophrenia and controls. An isolated decrease in the maximal transport capacity (V(max)) of tyrosine was observed in the cells from patients. In this context, tyrosine transport across the fibroblast membrane was investigated in patients with schizophrenia and healthy control subjects. METHODS: Skin fibroblasts were obtained from 36 patients with schizophrenia (15 first episode and 21 chronic) and 10 healthy controls. Tyrosine transport across the cell membrane was studied in cultivated fibroblasts. The V(max) and the affinity of the tyrosine binding sites (K(m)) were determined. RESULTS: Significantly lower V(max) (F(1,41) = 12.80; P =.001; effect size = 1.36) and K(m) (F(1,41) = 24.85; P<.001; effect size = 1.00) were observed in fibroblasts from the patients. The findings were present in both neuroleptic-naive patients with their first episode and patients with chronic schizophrenia. CONCLUSIONS: The lower V(max) and K(m) are compatible with a cell membrane disturbance and support the view of schizophrenia as a systemic disorder. The decreased V(max) and K(m) observed in cells from schizophrenic patients probably reflect a genetic trait, as the changes were transmitted through several cell generations of cultured fibroblast.

Synthesis of mevalonate pathway lipids in fibroblasts from Zellweger and X-linked ALD patients.

Fibroblasts were cultured to determine the involvement of peroxisomes in cholesterol and dolichol synthesis. For this purpose, the behavior of cells from patients with Zellweger syndrome, with X-linked adrenoleukodystrophy, and from nondiseased control subjects was studied. Cells both after pretreatment with mevinolin and without pretreatment were incubated in a medium containing [3H]-mevalonate. In fibroblasts from patients with peroxisomal defects, the cholesterol content and mevalonate incorporation into cholesterol were decreased by 10-20% in comparison with control cells. Mevinolin pretreatment decreased the incorporation rate of [3H]-mevalonate into cholesterol but increased the labeling of ubiquinone and dolichol both in diseased and control cells. Squalene synthase activity was unchanged, whereas the activity of farnesyl-pyrophosphate synthase was increased in the diseased states. The results show that in patients with peroxisomal deficiency neither the amount nor the rate of synthesis of cholesterol and dolichol is reduced to any greater extent.

Fatty acid oxidation in fibroblasts from patients with defects in beta-oxidation and in the respiratory chain.

Fatty acid oxidation has been studied with the tritium release assay in cultured fibroblasts from patients with defects in beta-oxidation and in the mitochondrial respiratory chain. Cells from all patients with beta-oxidation defects and cells from 10 of 16 patients with respiratory chain defects showed an impairment of fatty acid oxidation. The result of the tritium release assay is not only dependent on the proper function of the beta-oxidation cycle but is also influenced by the reoxidation of reduced cofactors. The assay can thus be used to study the expression of respiratory chain defects in cultured fibroblasts.

MnDPDP enhancement in rabbit liver after intravenous bolus injection and slow infusion.

PURPOSE: To investigate the MR-enhancing effect of mangafodipir trisodium (MnDPDP, Teslascan) in the rabbit liver in relation to dose, mode of administration and imaging window. MATERIAL AND METHODS: MnDPDP was administered to 18 rabbits at a dose of 10 mumol/kg or 20 mumol/kg, as a bolus injection or infusion. MR imaging of the liver was performed at different time intervals. RESULTS: Peak liver enhancement was typically observed 10-30 min after administration and the enhancement declined with a half-time of about 5 h. This pattern was observed in all sequences (SE 400/15, FLASH, and SE 132/10), with both doses and with both rates of administration. The peak enhancement was greater, though not significantly so after 20 mumol/kg than after 10 mumol/kg. A higher relative peak signal was observed with SE 132/10 than with FLASH or SE 400/15. CONCLUSION: A good liver imaging result was obtained after a dose of 10 mumol/kg, either bolus or infusion, 10-30 min post-contrast with heavily T1-weighted sequences.

Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency with the G1528C mutation: clinical presentation of thirteen patients.

Long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase is one of three enzyme activities of the mitochondrial trifunctional protein. We report the clinical findings of 13 patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. At presentation the patients had had hypoglycemia, cardiomyopathy, muscle hypotonia, and hepatomegaly during the first 2 years of life. Seven patients had recurrent metabolic crises, and six patients had a steadily progressive course. Two patients had cholestatic liver disease, which is uncommon in beta-oxidation defects. One patient had peripheral neuropathy, and six patients had retinopathy with focal pigmentary aggregations or retinal hypopigmentation. All patients were homozygous for the common mutation G1528C. However, the enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities of the mitochondrial trifunctional protein were variably decreased in skin fibroblasts. Dicarboxylic aciduria was detected in 9 of 10 patients, and most patients had lactic acidosis, increased serum creatine kinase activities, and low serum carnitine concentration. Neuroradiologically there was bilateral periventricular or focal cortical lesions in three patients, and brain atrophy in one. Only one patient, who has had dietary treatment for 9 years, is alive at the age of 14 years; all others died before they were 2 years of age. Recognition of the clinical features of long-chain 3-hydroxyacyl-CoA deficiency is important for the early institution of dietary management, which may alter the otherwise invariably poor prognosis.

Detection of defective 3 beta-hydroxysterol delta 7-reductase activity in cultured human fibroblasts: a method for the diagnosis of Smith-Lemli-Opitz syndrome.

Patients with the autosomal recessive disorder Smith-Lemli-Optiz syndrome (SLO) have recently been shown to have markedly increased tissue levels of certain cholesterol biosynthesis intermediates, most notably 7-dehydrocholesterol. The findings strongly suggest a block in the step that catalyses reduction of 7-dehydrocholesterol to cholesterol. The accumulation of 7-dehydrocholesterol can generally easily be detected in serum by gas chromatography-mass spectrometry. However, it could not be totally ruled out that SLO patients with less severe enzyme defects could escape detection by this method. A more direct way of diagnosing a defect in 7-dehydrocholesterol reduction would be to assay the conversion of 7-dehydrocholesterol to cholesterol in cultured fibroblasts from patients with suspected SLO. In the present work, an assay for the conversion of [3H]lathosterol to [3H]cholesterol in cultured human fibroblasts is described. Lathosterol is the immediate precursor of 7-dehydrocholesterol in the cholesterol biosynthetic pathway and was chosen for the assay instead of 7-dehydrocholesterol owing to the difficulty in preparation and handling of the latter compound. Fibroblasts from control subjects converted [3H]lathosterol to [3H]cholesterol efficiently, whereas in fibroblasts from SLO patients the conversion did not go beyond 7-dehydrocholesterol. It is concluded that the present method is useful for the diagnosis of SLO.

Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: effects of the Swedish Alzheimer's disease APP 670/671 mutation on binding levels.

We have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimer's disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [3H] quinuclidinyl benzilate ([3H]QNB) and the M2/M4 selective antagonist [3H] (+/-)-5,11-dihydro-11-([(2-[(di-propylamino)methyl]-1- piperidinyl]ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6- one ([3H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [3H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine > himbacine > methoctramine > (+/-)-p-fluoro-hexahydro-sila-difenidol hydrochloride > pirenzepine > muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25-35% lower levels of muscarinic receptors labelled with [3H]QNB, [3H]N-methyl scopolamine and [3H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing.

Excessive production of amyloid beta-protein by peripheral cells of symptomatic and presymptomatic patients carrying the Swedish familial Alzheimer disease mutation.

The 39- to 43-amino acid amyloid beta-protein (A beta), which is progressively deposited in cerebral plaques and blood vessels in Alzheimer disease (AD), is secreted by cultured human cells during normal metabolism. In studies of cell lines transfected with beta-amyloid precursor protein (beta APP) cDNAs, the beta APP mutation K670N/M671L found in a Swedish familial AD (FAD) pedigree has previously been shown to cause a marked augmentation of A beta secretion. Here, we have conducted blinded analyses of beta APP metabolism in primary skin fibroblasts from affected members of the Swedish FAD pedigree and their unaffected siblings or spouses. These fibroblasts continuously secrete a homogenous population of A beta molecules starting at Asp-1 (D672 of beta APP). We found a consistent and significant approximately 3-fold elevation of A beta release from all biopsied skin fibroblasts bearing the FAD mutation. No significant alterations of other metabolic derivatives of beta APP were detected. The elevated A beta levels were found in cells from both patients with clinical AD and presymptomatic subjects. Thus, A beta overproduction in this FAD pedigree is not a secondary event but is consistent with a causal role in the development of the disease. Increased A beta secretion can begin many years prior to onset of symptoms, even in peripheral tissues, indicating that it does not require preexisting neural abnormalities.

Increased beta-amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation.

Cell lines transfected with the Swedish Alzheimer's disease amyloid precursor protein APP670/671 mutation release significantly more beta-amyloid than wild-type cells. Citron et al. [Proc. Natl. Acad. Sci. USA (1994) in press] have recently shown that fibroblasts carrying the APP670/671 mutation also release more beta-amyloid than control cells [1]. The present study confirms a ca. threefold increase in beta-amyloid release from mutation-bearing fibroblasts. APP mRNA levels did not differ between mutation-bearing and control cells, although mutation-bearing fibroblasts contained significantly more APP751/770 than controls. Mild stress decreased beta-amyloid secretion and increased APP751/770 levels in all cell lines. In conclusion, the proportion of APP committed to amyloidogenic processing is increased in fibroblasts from family members with the APP670/671 mutation, and this mutation may also compromise the APP stress response.

Tyrosine transport in schizophrenia.

Tyrosine transport was examined in cultured skin fibroblasts from patients with schizophrenia (DSM-III-R) and normal subjects. The transport capacity (Vmax) was lower in the patients. The results confirm previous findings of decreased tyrosine transport in schizophrenia. In cells incubated with psychotropic drugs at different concentrations, tyrosine transport was not differentially influenced across patients and normal subjects. Dopaminergic and beta-adrenergic receptor mechanisms did not seem to influence tyrosine uptake. There seems to be a primary disturbance of tyrosine transport in schizophrenia which indicates a generalized cell membrane dysfunction.

beta-Oxidation enzymes in fibroblasts from patients with 3-hydroxydicarboxylic aciduria.

The activities of 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase were measured in fibroblasts from eight patients with 3-hydroxydicarboxylic aciduria. Measurement of 3-hydroxyacyl-CoA dehydrogenase with 3-ketopalmitoyl-CoA as substrate provided conclusive evidence for a deficiency of the long-chain 3-hydroxyacyl-CoA dehydrogenase in seven of the patients. Measurement of the enzyme in the normal direction cannot be recommended because this gives a higher residual activity. A trifunctional enzyme protein is responsible for the 3-hydroxyacyl-CoA dehydrogenase as well as for the hydratase and thiolase activities. A slight decrease in one or both of the other two activities was observed in four of the seven deficient patients, indicating that a defect in the trifunctional enzyme protein may affect the three enzyme activities to different degrees.

Experimental tissue transplantation using a biopsy instrument and radiologic methods.

RATIONALE AND OBJECTIVES: The liver is the most common site for metastases from gastrointestinal tumors, malignant melanoma, and primary liver tumors. Early detection or exclusion of a neoplasm is important for appropriate treatment. The authors introduce a method for tumor transplantation into the rabbit liver for experimental purposes. METHODS: VX2 tumor cells initially were grown intraperitoneally in a New Zealand white rabbit. Using an automated biopsy instrument with an ultrathin-wall biopsy needle, standardized tumor samples were taken from the peritoneal tumor. Using the same technique, a tumor sample was transplanted into the left liver lobe in a series of seven rabbits. RESULTS: Tumor growth was achieved in all cases at the implantation site. The tumors were well delineated from the surrounding liver parenchyma. Metastases occurred only at later stages. CONCLUSION: The method is almost nontraumatic to the animals, and the technical procedure is simple, time-saving, and provides well-localized tumor growth.

Long-chain 3-hydroxyacyl-CoA dehydrogenase in chorionic villi, fetal liver and fibroblasts and prenatal diagnosis of 3-hydroxyacyl-CoA dehydrogenase deficiency.

Prenatal diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency was performed by analysis of the enzyme activity in a chorionic villus biopsy obtained in the 10th week of pregnancy. The diagnosis was confirmed in liver tissue and cultured fibroblasts from the aborted fetus.

The transport of tyrosine into the human brain as determined with L-[1-11C]tyrosine and PET.

An alteration of dopaminergic transmission in the brain has been proposed for schizophrenia. To explore this, the rate constant for the intransport of L-tyrosine across the blood-brain barrier in healthy controls and in patients with schizophrenia (DSM-III-R) was determined with PET and L-[1-11C] tyrosine as the tracer. Kinetics for tyrosine transport were determined according to a two-compartment model using radioactivity data of arterial blood and brain tissue sampled between 1 and 3.5 min after a bolus injection of L-[1-11C] tyrosine. Radioactivity was measured every second in the blood and in 10-sec intervals in the brain tissue. In the normal controls the brain intransport rate constant for tyrosine was 0.052 ml/g/min with an influx rate of 2.97 nmol/g/min. The patients had a similar intransport rate constant (0.045 ml/g/min) but a lower influx rate of tyrosine 1.95 nmol/g/min (p less than 0.05). The patients' tyrosine concentrations in the blood were lower. For data sampled between 5 and 25 min, the net accumulation rate of tyrosine into the brain was 0.015 ml/g/min in the controls which did not differ to the patients' rate. However, the net utilization of tyrosine was lower in the patients (0.672 nmol/g/min) than in the controls (0.883 nmol/g/min) despite similar tissue concentrations of tyrosine.

Retrospective diagnosis of 3-hydroxydicarboxylic aciduria by analysis of filter paper blood samples.

Five patients with 3-hydroxydicarboxylic aciduria have been investigated. Two of them had elder siblings who had died unexpectedly in early infancy. Stored filter paper blood samples obtained from the patients and their siblings for neonatal screening were retrieved. Elevated levels of 3-hydroxy fatty acids were observed in the samples from three of the five patients with 3-hydroxydicarboxylic aciduria and in the samples from both siblings.

Decreased tyrosine transport in fibroblasts from schizophrenic patients.

Amino acid transport was studied in vitro in cultured fibroblasts from schizophrenic patients and controls. An isolated decrease in the transport capacity (Vmax) for tyrosine was observed in cells from the patients. The Km for tyrosine transport was unaffected. The kinetic parameters for phenylalanine, tryptophan, leucine and glycine transport did not differ between patients and controls. Competitive inhibition among the amino acids transported by the L-system and its exchange properties were normal in cells from the patients. No differences in intracellular levels of amino acids between patients and controls were observed. The decreased tyrosine transport in the cells from schizophrenic patients appears not to be related to any known amino acid transport system and may reflect a more general defect in plasma membrane function in schizophrenia.