Proteomic analysis of nuclei dissected from fixed rat brain tissue using expression microdissection.

Expression microdissection (xMD) is a high-throughput, operator-independent technology that enables the procurement of specific cell populations from tissue specimens. In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate (EVA) film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p < 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.

Analysis of melatonin in human plasma by gas chromatography negative chemical ionization mass spectrometry.

Three techniques have been used to measure human plasma melatonin: bioassay, radioimmunoassay, and gas chromatography--mass spectrometry (GC-MS). GC-MS is theoretically capable of the greatest specificity, but in general suffers from insufficient sensitivity. Negative chemical ionization, a new technique, provides a 150-fold increase in GC-MS sensitivity for electron-capturing compounds. Negative chemical ionization GC-MS permits routine measurement in human plasma of melatonin at a concentration as low as 1 picogram per milliliter.

Galactonic Acid in galactosemia: identification in the urine.

Galactose is converted to galactonic acid in vivo in man. Galactonate was isolated from the urine of galactosemia patients who had been given galactose orally. The identity of the galactonate was established by gas-liquid chromatography and by the preparation of derivatives.

Acidic components of green river shale identified by a gas chromatography-mass spectrometry-computer system.

A system consisting of a gas chromatograph coupled to a mass spectrometer and computer has been used to characterize the extractable acidic components of Green River shale. This system accumulates mass spectra at every point of the gas chromatogram in a permanent form which permits one to observe mass spectra of minor as well as major constituents. One minor component, whose identification was confirmed by synthesis, was 6,10,14-trimethyl-pentadecanoic acid.

Light and propranolol suppress the nocturnal elevation of serotonin in the cerebrospinal fluid of rhesus monkeys.

Markedly elevated nighttime concentrations of serotonin in rhesus monkey cerebrospinal fluid were reduced to daytime levels by exposing the monkeys to continuous light or to the beta-adrenergic antagonist propranolol. Nighttime elevations of melatonin in cerebrospinal fluid were also suppressed by propranolol and light. Serotonin released in large quantities at night appears to be regulated like melatonin, and may act as a cerebroventricular hormone to influence brain and pituitary function at night.

Light suppresses melatonin secretion in humans.

Bright artificial light suppressed nocturnal secretion of melatonin in six normal human subjects. Room light of less intensity, which is sufficient to suppress melatonin secretion in other mammals, failed to do so in humans. In contrast to the results of previous experiments in which ordinary room light was used, these findings establish that the human response to light is qualitatively similar to that of other mammals.

Facile oxidative decarboxylation of 3,4-dihydroxyphenylacetic acid catalyzed by copper and manganese ions.

Under physiological conditions, we observed the rapid, pH- and temperature-dependent, oxidative decarboxylation and hydration of 3,4-dihydroxyphenylacetic acid (DOPAC) to form 3,4-dihydroxybenzyl alcohol (DBAlc). This product was oxidized and underwent tautomerization to form 3,4-dihydroxybenzaldehyde (DBAld). This reaction did not occur in the presence of EDTA, was catalyzed by copper (CuI, CuII) and manganese (MnII) and was oxygen dependent. A variety of mono- and dihydroxyphenyl carboxylic acids were tested and the reaction producing DBAlc as an intermediate was observed to be unique to DOPAC. 3.4-Dihydroxymandelic acid (DOMA) was rapidly oxidatively decarboxylated to form DBAld directly. The substrate and catalyst selectivity of this reaction suggest that this may have physiological relevance in the neurotoxic consequences of manganese and copper to the dopaminergic system in man.

Conversion of MHPG to vanillylmandelic acid. Implications for the importance of urinary MHPG.

Deuterium-labelled 4-hydroxy-3-methoxyphenylglycol (MHPG), when administered intravenously, is rapidly converted to 4-hydroxy-3-methoxymandelic acid (or vanillylmandelic acid [VMA]) or conjugates of MHPG. Since over half of either racemic D,L-MHPG or the natural D-MHPG is converted to VMA and since about half of urinary VMA is derived from MHPG, estimates of the proportion of urinary MHPG derived from the brain must be revised. The results indicate that only about one fifth of urinary MHPG is derived from the brain, and clearly urinary MHPG cannot be used as a valid index of brain norepinephrine metabolism. While these observations do not alter the value of urinary MHPG as a predictor of therapeutic response or in subclassifying affective disorders, it is clear that new research questions must be formulated and appropriate investigations completed before the relationship of urinary MHPG to affective disorders is understood.

Imipramine and desipramine in plasma and spinal fluid: relationship to clinical response and serotonin metabolism.

In a double-blind study of depressed patients treated with imipramine hydrochloride, levels of imipramine and desipramine were measured in plasma and in CSF. Levels of both drugs in CSF were approximately 10% of plasma levels, but the levels in the two body fluids were highly correlated. The levels of both drugs were approximately equal in plasma, but desipramine predominated in CSF (imipramine/desipramine ratio of 0.8). The imipramine-induced alteration in CSF levels of the serotonin metabolite (5-hydroxy-indoleacetic acid [5HIAA]) correlated with imipramine levels but not with desipramine. For the group of patients showing a clear antidepressant response, the mean drug levels were nearly double those of the nonresponder group, a difference that did not quite reach statistical significance in this relatively small sample. The desipramine levels showed no responder-nonresponder difference, while the ratio of imipramine/desipramine was significantly higher among the responders. On the average this particular patient group had relatively low pretreatment levels of 5HIAA in CSF, an observation that may partially account for the relatively low overall response rate to imipramine.

Antidepressants reduce whole-body norepinephrine turnover while enhancing 6-hydroxymelatonin output.

The effects of antidepressant treatment on noradrenergic function were studied in 27 patients with a major affective disorder. Twenty-four-hour urinary excretion of 6-hydroxymelatonin and "whole-body norepinephrine (NE) turnover," ie, 24-hour urinary output of NE and its major metabolites 3-methoxy-4-hydroxyphenylglycol, vanillylmandelic acid, and normetanephrine, were measured before and after treatment with the tricyclic desipramine hydrochloride, the aminoketone bupropion hydrochloride, the nonselective monoamine oxidase (MAO) inhibitor tranylcypromine sulfate, and the specific MAO type A inhibitor clorgiline. 6-Hydroxymelatonin excretion increased following antidepressant treatment, while at the same time whole-body NE turnover was reduced. These findings support the hypothesis that antidepressant therapy increases noradrenergic "efficiency," in that functional output, as measured by 6-hydroxymelatonin, is maintained while total NE production is decreased.

Light exposure reduces and pinealectomy virtually stops urinary excretion of 6-hydroxymelatonin by rhesus monkeys.

The major metabolite of the pineal hormone melatonin, conjugated 6-hydroxymelatonin, was hydrolyzed, separated from the urine of rhesus monkeys, and assayed mass spectrometrically. The daily excretion pattern reflected pineal melatonin synthetic activity, being 5- to 16-fold higher at night than during the day. Progressive lengthening of the daily photoperiod beyond 12 h decreased the daily excretion of 6-hydroxymelatonin proportional to the increase duration of light exposure. Constant light reduced daily excretion by 90%, and pinealectomy reduced daily excretion by 96%. These results demonstrate the absence of significant extrapineal contributions to the urinary melatonin metabolite and confirm the use of 6-hydroxymelatonin excretion rates as a valid index of pineal gland melatonin synthesis.

Melatonin metabolite excretion during childhood and puberty.

Daily urinary excretion of conjugated 6-hydroxymelatonin, the major metabolite of the pineal hormone melatonin, has been determined in 54 boys and 47 girls (aged 3-16 yr) and 20 normal adults to determine whether a change in melatonin production is seen during the maturation of reproductive function in humans. There was no correlation between daily excretion rates and age in children, and the excretion rates were similar to those in adults. In addition, children of all ages had normal circadian patterns of 6-hydroxymelatonin excretion from the earliest age tested. A significant increase in 6-hydroxymelatonin excretion was observed at the time of the onset of breast development (Tanner stage II) in girls. No similar difference was seen during puberty in males. The significance of this difference in Tanner II girls is not known.

Urinary 6-hydroxymelatonin excretion in patients with orthostatic hypotension.

The rates of melatonin formation and its diurnal fluctuations have been examined in patients with three types of orthostatic hypotension by measuring the urinary excretion rates of 6-hydroxymelatonin, the major metabolite of the pineal gland hormone. Deficiencies in the peripheral autonomic nervous system resulted in markedly diminished daily excretion (2.5 +/- 1.3 micrograms) relative to control (12.2 +/- 1.2). Patients with impaired central nervous system function exhibited low and/or abnormal excretion patterns. Two patients with sympathotonic orthostatic hypotension excreted greater amounts of 6-hydroxymelatonin than any of the control subjects. Melatonin secretion by the pineal gland can be used as an index of sympathetic nerve function. The study of patients with altered function may reveal the role of the pineal gland in human physiology.

Pinealectomy abolishes plasma melatonin in the rat.

Using gas chromatography-negative ionization mass spectrometry, plasma melatonin levels in pinealectomized and sham-operated rats were assessed. The pinealectomized rats consistently demonstrated an absence of plasma melatonin while the intact animals showed detectable amounts. This suggests that although melatonin may be formed in tissues other than a pineal gland, the contribution to plasma is of pineal origin. Thus, plasma melatonin levels can be used as a marker of circadian melatonin secretion by the pineal gland and of its beta-adrenergic regulation.

2-Amino-3-(methylamino)propanoic acid (BMAA) bioavailability in the primate.

2-Amino-3-(methylamino)-propanoic acid (BMAA) is a low potency excitatory amino acid present in the cycad plant that has been proposed as a factor in the high incidence of amyotrophic lateral sclerosis-parkinsonism dementia (ALS-PD) in the western Pacific region. We employed stable isotopic forms of BMAA to assess the oral bioavailability of this compound in cynomolgous monkeys (n = 3). The stable isotope labeled BMAA ([15N]-BMAA) was injected i.v. at the same time that the unlabeled compound was administered orally. Both forms of BMAA were then quantified in a 48h urine sample by gas chromatography-mass spectrometry (GC/MS). Following oral dosing, 80% of the administered BMAA was absorbed into the systemic circulation; thus, oral bioavailability was high and other routes of administration could not result in significantly higher circulating levels of BMAA for a given administered dose.

Neuroactive metabolites of L-tryptophan, serotonin and quinolinic acid, in striatal extracellular fluid. Effect of tryptophan loading.

Extracellular fluid levels of the neurotoxin quinolinic acid in the corpus striatum of rats, measured by in vivo microdialysis, were increased in a dose-dependent manner following the intraperitoneal administration of tryptophan. The lowest dose of tryptophan (12.5 mg/kg), equivalent to about 5% of the normal daily intake, increased peak quinolinic acid levels nearly 3-fold. At higher doses of tryptophan (up to 250 mg/kg), concentrations of quinolinic acid increased over 200-fold and exceeded potentially neurotoxic levels (10 microM). In contrast, the increase in extracellular serotonin following even the highest tryptophan dose was small (less than 2-fold). These data indicate that quinolinic acid is present in the extracellular fluid where it may function as a neuromodulator and that it is very responsive to physiological changes in precursor availability.

Decreased 6-hydroxymelatonin excretion in Korsakoff's psychosis.

Mean (+/- SEM) urinary excretion rate of the major melatonin metabolite 6-hydroxymelatonin (micrograms/day) was lower in 7 (2.8 +/- 1.0) of 8 men with Korsakoff's psychosis (KP) than in 15 healthy men (11.4 +/- 1.4). Treatment with the alpha 2-noradrenergic agonist clonidine decreased daily 6-hydroxymelatonin excretion (p less than 0.02). Reduced daily excretion of 6-hydroxymelatonin in KP reflects decreased melatonin synthesis in the pineal gland, perhaps as a residual effect of lesions due to past thiamine deficiency (Wernicke's encephalopathy).

2-Amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour: an unlikely cause of amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.

We conducted an investigation of the levels of the neurotoxin 2-amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour. Analysis of 30 flour samples processed from the endosperm of Cycas circinalis seeds collected on Guam indicated that more than 87% of the total BMAA content was removed during processing. Furthermore, in 1/2 the samples almost all (greater than 99%) of the total BMAA was removed. We found no significant regional differences in the BMAA content of flour prepared from cycad seeds collected from several villages on Guam. Testing of different samples prepared by the same Chamorro woman over 2 years suggests that the washing procedure probably varies in thoroughness from preparation to preparation but is routinely efficient in removing at least 85% of the total BMAA from all batches. Analysis of a flour sample that had undergone only 24 hours of soaking indicated that this single wash removed 90% of the total BMAA. We conclude that processed cycad flour as prepared by the Chamorros of Guam and Rota contains extremely low levels of BMAA, which are in the order of only 0.005% by weight (mean values for all samples). Thus, even when cycad flour is a dietary staple and eaten regularly, it seems unlikely that these low levels could cause the delayed and widespread neurofibrillary degeneration of nerve cells observed in amyotrophic lateral sclerosis and the parkinsonism-dementia complex of Guam (ALS-PD).

Inter-relationships between quinolinic acid, neuroactive kynurenines, neopterin and beta 2-microglobulin in cerebrospinal fluid and serum of HIV-1-infected patients.

Quinolinic acid (QUIN) is an neurotoxic N-methyl-D-aspartate receptor agonist and an L-tryptophan metabolite of the kynurenine pathway. Increased concentrations of QUIN occur in both cerebrospinal fluid (CSF) and blood of patients infected with human immunodeficiency virus (HIV)-1, particularly those with neurologic disturbances. In the present study of HIV-1 infected patients in Walter Reed stages 4, 5 and 6, reductions in L-tryptophan accompanied proportional increases in L-kynurenine and QUIN in both serum and CSF. Further, close inter-correlations exist between QUIN kynurenic acid and L-kynurenine with both beta 2-microglobulin and neopterin in CSF and serum. These correlations support the hypotheses that the kynurenine pathway is activated in association with inflammation and induction of indoleamine-2,3-dioxygenase. There were no relationships between CSF QUIN, L-kynurenine or kynurenic acid with the ratio of serum:CSF albumin concentrations, which indicates that the increases in CSF QUIN, L-kynurenine or kynurenic acid were not dependent on a breakdown of the blood-brain barrier. Kynurenic acid is also a kynurenine pathway metabolite that can attenuate the excitotoxic effects of QUIN when present in higher molar concentrations. While CSF kynurenic acid levels were increased in HIV-1-infected patients, the magnitude of the increases were smaller than those of QUIN and the molar concentrations of kynurenic acid were consistently lower than QUIN by at least one order of magnitude. We conclude that immune activation increases the levels of neuroactive kynurenines within the central nervous system of HIV-1-infected patients secondary to activation of indoleamine-2,3-dioxygenase.

Glutamate is a mediator of neurotoxicity in secretions of activated HIV-1-infected macrophages.

We sought to identify neurotoxin(s) secreted by HIV-1-infected mononuclear phagocytes that could contribute to the pathophysiology of HIV-1-associated dementia (HAD). Neurotoxic factors were characterized in batches of conditioned media (CM) from human monocyte-derived macrophages (MDM) infected with HIV-1(ADA) and/or activated with lipopolysaccharide (LPS). All of the neurotoxicity was: present in the <3000-Da fraction; blocked by 5 microM MK801; and not trypsin sensitive or extractable into polar organic solvents. Glutamate measured in CM accounted for all neurotoxic effects observed from HIV/LPS CM in astrocyte-poor neuronal cultures and may contribute to the pathophysiology of HIV-1-associated dementia.