Brief report: organic osmolytes in the brain of an infant with hypernatremia.
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Aspartic Acid↗
Biomedical subjects
B D Ross
Publications and source records attributed to B D Ross.
Measurement of pentose phosphate-pathway activity in a single incubation with [1,6-13C2,6,6-2H2]glucose.
The isotopically substituted molecule D-[1,6-13C2,6,6-2H2]glucose is introduced for measuring the relative activities of the pentose phosphate pathway (PPP) and glycolysis in a single incubation. PPP activity in cultured cells was determined by gas chromatography/mass spectrometric analysis of lactate produced by cells incubated with [1,6-13C2,6,6-2H2]glucose. Two other isotopes, [1,5,6-13C3]glucose and [6-13C,1,6,6-2H3]glucose, were not satisfactory for measurements of this activity. This method has four advantages over the traditional one in which 14CO2 production from [1-14C]glucose and [6-14C]glucose is compared: (1) repeated measurements can be made on a single set of cells, (2) only a single incubation is required, (3) extensive CO2 production by Krebs-cycle activity does not interfere with the measurements and (4) it is not necessary to measure the amount of glucose consumed in order to calculate relative activities of the PPP and glycolysis. Preliminary observation indicates that rat brain PPP activity may be measured in vivo with [1,6-13C2,6,6-2H2]glucose when combined with microdialysis techniques.
Animals↗
Abnormal cerebral metabolite concentrations in patients with probable Alzheimer disease.
To establish whether recently described abnormalities of peak ratios are the result of changes in metabolite concentrations, quantitative 1H magnetic resonance spectroscopy was performed in 10 patients with Alzheimer disease (AD) and seven normal elderly. CSF volumes, metabolite T1 and T2 relaxation rates, ratios and concentrations of N-acetyl residues, creatine, choline residues, myo-inositol, glutamine plus glutamate (Glx), and glucose were obtained. Difference spectroscopy and quantitative assays showed a 50% increase in myo-inositol (6.4-9.8 mM; P < 0.005) and a decrease in N-acetyl in occipital gray matter. A reduction in beta, gamma-Glx and a significant increase in intracerebral [glucose], greater than attributable to CSF, were defined. Choline concentration increased with age, but was not elevated above normal in AD patients. These findings indicate the need for quantitative 1H MRS to substantiate metabolite ratios. The increased myo-inositol concentration in AD is demonstrated by these studies.
Aged↗
In vitro and in vivo 13C and 31P NMR analyses of phosphocholine metabolism in rat glioma cells.
In vivo magnetic resonance spectroscopy (MRS) has revealed that phosphomonoesters (PME) such as phosphocholine (PCho) and phosphoethanolamine (PEth) are elevated in tumors and rapidly proliferating tissues. The regulation of PME levels and their relationship to proliferation are not well known. In the present study, we investigated the regulation of PCho and PEth levels in rat glioma cells grown in vivo and in vitro using 31P and 13C MRS. However, the ability of cells to produce choline endogenously is variable. To fully understand regulation of PCho levels, it is necessary to characterize the activity of the endogenous pathway, if it exists. This was first investigated by following the metabolic fate of 13C-labeled methionine of 9L glioma tumors in vivo. Our results indicate that there is a significant amount of de novo choline synthesis in vivo. However, similar experiments performed in vitro using cells cultured in bioreactors indicated that glioma cells themselves are unable to synthesize choline de novo, suggesting that the in vivo results were due to the involvement of extra-tumoral organs, e.g., liver. Further in vitro experiments demonstrated that the uptake and phosphorylation of physiologically relevant concentrations of exogenous choline is very active in these systems. Thus, it appears that the exogenous pathway for PCho biosynthesis predominates and regulates PCho levels in glioma cells. Our results also demonstrate that PCho levels are lowest, and PEth levels are highest, in non-proliferating cells. These observations indicate that there is a decrease in the biosynthesis of PCho concomitant with a reduction in culture growth. The source of the increased PEth is, as yet, undefined.
Animals↗
Metabolic loss of deuterium from isotopically labeled glucose.
The isotopically substituted molecule (6-13C, 1, 6, 6-2H3)glucose was evaluated to determine whether metabolic 2H loss would prevent its use in quantitating pentose phosphate pathway (PPP) activity. PPP activity causes the C1 of glucose to be lost as CO2, while C6 can appear in lactate. 2H NMR analysis of the lactate produced from this glucose can distinguish (3-2H)-lactate (from C1 of glucose) from (3-13C, 3, 3-2H2)lactate (from C6 of glucose). 2H NMR spectroscopic analysis of medium containing (6-13C, 1, 6, 6-2H3)glucose after incubation with cultured rat 9L glioma cells suggested a 30.8 +/- 2.1% PPP activity as compared with 6.0 +/- 0.8% from separate, parallel incubations with (1-13C)glucose and (6-13C)glucose. Subsequent experiments with other isotopically labeled glucose molecules suggest that this discrepancy is due to selective loss of 2H from the C1 position of glucose, catalyzed by phosphomannose isomerase. Failure to consider 2H exchange from the C1 and C6 positions of glucose can lead to incorrect conclusions in metabolic studies utilizing this and other deuterated or tritiated glucose molecules.
Animals↗
Scyllo-inositol depletion in hepatic encephalopathy.
Cerebral myo-inositol depletion is found in patients with hepatic encephalopathy and can be implicated in the pathogenesis of hepatic encephalopathy. We measured scyllo-inositol, a stereoisomer of myo-inositol, in brain extracts from patients dying in hepatic coma using HPLC and high resolution 1H MRS. The cerebral scyllo-inositol concentration, determined by both methods, in patients without hepatic encephalopathy was 0.41 +/- 0.11 mmol/kg wet weight. It decreased by 73% and 76%, respectively, as measured by HPLC and 1H MRS, in patients with hepatic encephalopathy. These findings indicate that myo-inositol depletion in patients with hepatic encephalopathy is not due to enhanced conversion of myo-inositol to scyllo-inositol or inhibition of myo-inositol transport by scyllo-inositol, but rather to the reduced biosynthesis or transport of both inositols.
Autopsy↗
Oxidation therapy: the use of a reactive oxygen species-generating enzyme system for tumour treatment.
Oxygen radicals induce cytotoxicity via a variety of mechanisms, including DNA damage, lipid peroxidation and protein oxidation. Here, we explore the use of a polyethylene glycol (PEG)-stabilised enzyme capable of producing reactive oxygen species (ROS), glucose oxidase (GO), for the purpose of harnessing the cytotoxic potential of ROS for treating solid tumours. PEG-GO (200 U), administered by two intratumoral injections 3 h apart, produced a significant growth delay in subcutaneous rat 9L gliomas as compared with control animals receiving heat-denatured PEG-GO. Rats were protected from systemic toxicity by subsequent i.v. administration of PEG-superoxide dismutase (PEG-SOD) and PEG-catalase. In vivo tumour metabolic changes, monitored using 31P magnetic resonance spectroscopy (31P-MRS) 6 h following initial administration of PEG-GO, revealed a 96 +/- 2% reduction in the ATP/Pi ratio and a 0.72 +/- 0.10 unit decline in intracellular pH. A 3-fold sensitisation of 9L glioma cells in vitro to hydrogen peroxide could be achieved by a 24 h preincubation with buthionine sulphoximine (BSO). This study suggests that oxidation therapy, the use of an intratumoral ROS-generating enzyme system for the treatment of solid tumours, is a promising area which warrants further exploration.
Animals↗
Subclinical hepatic encephalopathy: proton MR spectroscopic abnormalities.
PURPOSE: To determine whether hydrogen-1 magnetic resonance (MR) spectroscopy of the brain allows detection of subclinical hepatic encephalopathy (SCHE). MATERIALS AND METHODS: In a double-blind study, overt hepatic encephalopathy (HE) and SCHE (defined with clinical and neuropsychiatric tests) were compared by means of H-1 MR spectroscopic criteria--reduction in cerebral myo-inositol (< 2 standard deviations [SDs] from normal) and choline (< 2 SDs from normal) with or without increased cerebral glutamine (> 1 SD from normal)--in 20 patients with cirrhosis. RESULTS: Concordance between MR spectroscopic and neuropsychiatric test results was 94% (kappa = 0.84). MR spectroscopy allowed diagnosis of SCHE in nine of nine patients (100%) and of HE in seven of eight (88%). Myo-inositol depletion alone had 80%-85% sensitivity for detection of HE and SCHE. CONCLUSION: H-1 MR spectroscopy allows accurate diagnosis of SCHE, and the results suggest an important role for myo-inositol in psychomotor and visuopractic functions.
Adult↗
Oxidative stress in the central nervous system: monitoring the metabolic response using the pentose phosphate pathway.
We propose that monitoring the activity of the pentose phosphate pathway (PPP) may provide an opportunity to obtain unique information regarding the metabolic response to oxidative stress since glutathione peroxidase activity is coupled, via glutathione reductase, to the PPP enzyme glucose-6-phosphate dehydrogenase. PPP activity was quantitated from data obtained from gas chromatography/mass spectrometry analysis of released lactate following metabolic degradation of (1,6-13C2,6,6-2H2)glucose. The feasibility of this approach for in vitro studies is shown using cultured rat 9L gliosarcoma cells, primary mixed cerebrocortical and primary astrocytic cultures and in vivo using intracerebral microdialysis. Exposure of 9L gliosarcoma cells to increasing concentrations of phenazine methosulfate, diamide and H2O2 correlated with increasing stimulation of the PPP, revealing the coupling of the PPP to the glutathione pathway. In all cultured cell types, the activity of the PPP was stimulated in a concentration-dependent fashion by exposure to H2O2. In primary mixed and purified astrocytic cultures, PPP activity was stimulated with H2O2 from 2.0 to 22.5 and from 5.9 to 66.7%, respectively. H2O2-induced neuronal injury was evident before saturation of the PPP occurred. H2O2 toxicity was attenuated when neurons were preincubated with the iron chelator, deferoxamine, and did not occur until saturation of the PPP. In vivo measurements of PPP activity in the conscious rat forebrain revealed basal levels of 4.5%, which was stimulated to 16.9 and 35.7% when 1 mM H2O2 and 500 microM phenazine methosulfate were added to the perfusion solution, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Proton nuclear magnetic resonance urinalysis: coming of age.
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Animals↗
Decrease in cerebral inositols in rats and humans.
Rats with portacaval shunts and humans with hepatic encephalopathy show severe myo-inositol depletion in the brain. The portacaval-shunted rat may therefore be a useful model for the investigation of neurochemical pathways containing myoinositol, which are modulated not only in hepatic encephalopathy but also in diabetes mellitus and Alzheimer's disease.
Animals↗
15N n.m.r. measurement of the in vivo rate of glutamine synthesis and utilization at steady state in the brain of the hyperammonaemic rat.
The rate of glutamine synthesis and utilization at steady state was measured in vivo in the brains of hyperammonaemic rats by 15N n.m.r. in combination with biochemical techniques. Rats were given an intravenous 15NH4+ infusion at the rate of 4.8 +/- 0.3 mmol/h per kg body wt. for 3.5 +/- 0.2 h, followed by 14NH4+ infusion at the same rate for an additional 5.1 h (chase period). During the chase period, blood ammonia (0.61 +/- 0.015 mumol/g), brain ammonia (2.9 +/- 0.3 mumol/g), glutamate (9.4 +/- 0.8 mumol/g) and glutamine (15N + 14N; 14.4 +/- 1.3 mumol/g) were at steady state. The rate of change in the cerebral [5-15N]glutamine concentration was measured in vivo by 15N n.m.r. at 20.27 MHz. To estimate 15N enrichment of precursor ammonia for glutamine synthetase (GS) in astrocytes which are interposed between cerebral capillaries and neurons, 15N enrichments of blood and brain ammonia were measured by gas chromatography-mass spectrometry. The in vivo rate of glutamine synthesis, which is equal to the rate of glutamine utilization at steady state, was estimated, from the observed rate of change in [5-15N]glutamine concentration and 15N enrichment of brain glutamine, to be 4.8 +/- 1.1 mumol/h per g of brain if 15N enrichment of ammonia at the site of GS in astrocytes is equal to that of blood-borne ammonia, and 13.0 +/- 3.9 mumol/h per g if it is equal to that measured for the whole brain. The observed GS activity in vivo in the brain of the hyperammonaemic rat is 2-5% of the reported optimum activity in vitro measured at enzyme-saturating concentrations of all substrates. The result suggests that substrates and/or cofactors other than ammonia kinetically limit GS activity in vivo. The g.c. chromatogram and mass spectrum of ammonia-derived N-trifluoroacetyl-dibutylglutamate (TAB-glutamate) are shown in Supplementary Publication SUP 50170 (4 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire, U.K., from whom copies can be obtained on the terms indicated in Biochem. J.
Ammonia↗
Development of the human brain: in vivo quantification of metabolite and water content with proton magnetic resonance spectroscopy.
Cerebral metabolite concentrations and water content were measured by means of localized proton magnetic resonance spectroscopy in 50 children, while metabolite peak ratios in short echo time spectra were evaluated in 173 examinations. Normative curves for normal development were established for two cerebral locations. The current report presents the first study of absolute metabolite concentrations and T1- and T2- relaxation as a function of age. Myo-inositol was found dominating the spectra at birth (12 mmoles/kg), while choline is responsible for the strongest peak in older infants (2.5 mmoles/kg). Creatine and N-acetyl groups are at significantly lower concentrations in the neonate than in the adult (Cr: 6, NA: 5 mmoles/kg). NA and Cr are determined by gestational age, whereas the concentration of ml correlates best with postnatal age. Quantitative 1H MRS is expected to be of particular value in diagnosis and monitoring of pathology in infants, since metabolite ratios are often misleading.
Brain↗
Enhancement of hyperthermia effect in vivo by amiloride and DIDS.
PURPOSE: Intracellular pH is regulated mainly by Na+/H+ antiport and Cl-/HCO3- exchange through the cell membrane. Amiloride (3,5-diamino-6-chloro-N-(diaminomethylene)pyrazine carboxamide) is a diuretic drug that blocks Na+/H+ antiport and DIDS (4,4-diisothiocyanatostilbene-2,2'-disulfonic acid) is an inhibitor of Cl-/HCO3- exchange. We investigated the potency of these drugs to lower pHi and increase the thermosensitivity of tumors in vivo. MATERIALS AND METHODS: The cytocidal effect of heat in combination with drug effect in vivo was studied using the in vivo-in vitro clonogenic assay method and the tumor growth delay method with SCK tumors, a mammary adenocarcinoma, on the hind limbs of A/J mice. The effects of amiloride and DIDS on tumor pHi and high energy phosphate levels were investigated using 31P-NMR. RESULTS: We observed that amiloride or DIDS alone increased the effect of hyperthermia at 42.5 degrees C or 43.5 degrees C to suppress tumor growth. The thermosensitization was greater when the two drugs were combined. For example, hyperthermia at 43.5 degrees C alone resulted in a tumor growth delay of about 4 days. When 10 mg/kg amiloride or 25 mg/kg DIDS was injected prior to heating, the growth delay increased to about 6 days. When both drugs were injected prior to heating, a total growth delay of 8 days was obtained. In vivo-in vitro excision assays for cell survival demonstrated that these drugs enhanced the heat-induced tumor cell death. An i.p. injection of 10 mg/kg amiloride plus 25 mg/kg DIDS did not lower the tumor pHi over a 120 min interval. Heating the tumors at 42.5 degrees C for 1 hr significantly lowered the pHi and when the tumor-bearing mice were injected i.p with amiloride and DIDS, and the tumors were heated 1 hr later, the drop in pHi was greater relative to that by heating alone. Heating alone significantly lowered the tumor energy levels as indicated by PCr/Pi and beta-ATP/Pi ratios and an i.p. injection of 25 mg/kg amiloride prior to heating further reduced the energy status in the tumors. CONCLUSION: Amiloride or its analogs and DIDS may be useful in increasing the therapeutic efficacy of hyperthermia treatments by enhancing the reduction in tumor pHi.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Magnetic resonance methods for measurement of disease progression in rheumatoid arthritis.
Magnetic resonance imaging methods are described for measurement of disease activity in knee in rheumatoid arthritis patients. Measurements of cartilage thickness, joint effusion volume, and pannus volume have been made. The latter measurement relies on synthetic fractional enhancement images of the response to gadopentetate dimeglumine.
Adult↗
Alzheimer disease: depiction of increased cerebral myo-inositol with proton MR spectroscopy.
To define altered metabolites in the brain of patients with probable Alzheimer disease (AD) of two brain regions, localized in vivo hydrogen-1 magnetic resonance (MR) spectroscopy was performed with a short echo time (30 msec) in 11 elderly patients and 10 healthy age-matched subjects. The patients had mild to moderate dementia, assessed with standard neuropsychological tests. Two abnormalities in the patients' cerebral cortex were defined: When compared with healthy subjects, the patients showed a 22% increase (P = .005) (approximately equal to 1.5 mmol/kg) in myo-inositol (MI) and an 11% decrease (P = .005) in residues of N-acetyl (NA), a putative neuronal marker. The elevation of MI in patients with mild to moderate AD suggests that abnormalities in the inositol polyphosphate messenger pathway occur early in the natural history of AD. The combination of high MI and low NA at examination with H-1 MR spectroscopy shows promise as an early diagnostic test for AD.
Aged↗
Real-time study of the urea cycle using 15N n.m.r. in the isolated perfused rat liver.
1. Isolated rat liver was perfused with 10 mM-15NH4Cl, 5 mM-lactate and 1 mM-ornithine, or with 3 mM-[15N]alanine and 1 mM-ornithine, in haemoglobin-free medium. The liver was physiologically stable for over 3 h and synthesized urea at the rate of 1.15 mumol.min-1.g of liver-1 (15NH4(+)-perfused) or 0.41 mumol.min-1.g-1 ([15N]alanine-perfused). 2. The perfused liver was continuously monitored by 15N n.m.r. spectroscopy at 20.27 MHz for 15N. Well-resolved 15N resonances of precursors and intermediates of the urea cycle, present at tissue concentrations of 0.2-3.0 mumol/g, were observed from the intact liver in 5-40 min of acquisition. Key metabolites in liver extract and the final perfusion medium were analysed by n.m.r. and by biochemical assays to determine fractional 15N enrichment and the total 15N recovery. 3. In 15NH4(+)-perfused liver (n = 6), 15N incorporation into glutamate and alanine (1.0-1.3 mumol/g), as well as progressive formation of [15N2]urea, was observed during the first 2 h of perfusion. In the second and third hour, hepatic concentrations of [omega-15N]citrulline and [omega,omega'-15N]argininosuccinate increased to n.m.r.-detectable levels (0.3-0.9 mumol/g). The [15N]aspartate pool was large in the absence of added ornithine, but on its addition was rapidly incorporated into argininosuccinate (n = 3). 4. In [15N]alanine-perfused liver, major metabolites were [15N]glutamate, [gamma-15N]glutamine and [15N]urea. Urea-cycle intermediates were undetectable. 5. The results suggest that, in intact liver provided with excess ammonia, low concentrations of cytosolic argininosuccinate synthetase and argininosuccinate lyase limited the rate of metabolite flux in the urea cycle. By contrast, in alanine-perfused liver at a physiological rate of urea synthesis, mitochondrial carbamoylphosphate synthetase was rate-limiting. 6. The potential utility of 15N n.m.r. for study of metabolite channelling through urea-cycle enzymes in intact liver is discussed.
Alanine↗
Cerebral MRS in infant with suspected Reye's syndrome.
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Ammonia↗