NMR and cDNA array analysis prior to heart failure reveals an increase of unsaturated lipids, a glutamine/glutamate ratio decrease and a specific transcriptome adaptation in obese rat heart.

Obesity is a risk factor for heart failure through a set of hemodynamic and hormonal adaptations, but its contribution at the molecular level is not clearly known. Therefore, we investigated the kinetic cardiac transcriptome and metabolome in the Spontaneous Hypertensive Heart Failure (SHHF) rat. The SHHF rat is devoid of leptin signaling when homozygous for a mutation of the leptin receptor (ObR) gene. The ObR-/- SHHF rat is obese at 4 months of age and prone to heart failure after 14 months whereas its lean counterpart ObR-/+ is prone to heart failure after 16 months. We used a set of rat pangenomic high-density macroarrays to monitor left ventricle cardiac transcriptome regulation in 4- and 10-month-old, lean and obese animals. Comparative analysis of left ventricle of 4- and 10-month-old lean rat revealed 222 differentially expressed genes while 4- and 10-month-old obese rats showed 293 differentially expressed genes. (1)H NMR analysis of the metabolome of left ventricular extracts displayed a global decrease of metabolites, except for taurine, and lipid concentration. This may be attributed to gene expression regulation and likely increased extracellular mass. The glutamine to glutamate ratio was significantly lower in the obese group. The relative unsaturation of lipids increased in the obese heart; in particular, omega-3 lipid concentration was higher in the 10-month-old obese heart. Overall, several specific kinetic molecular patterns act as a prelude to heart failure in the leptin signaling deficient SHHF obese rat.

A glucuronidation pathway of capecitabine occurs in rats but not in mice and humans.

Glucuronidation of 5'-DFCR, a metabolite of capecitabine, was confirmed in experimental models from rats whereas 5'-DFCR glucuronide was detected neither in bile or liver from mice nor in liver microsomes from human. Metabolic interactions at the level of the glucuronidation pathway between CAP and other drugs are unlikely in patients.

Metabolism of a novel nucleoside analogue, OGT 719, in the isolated perfused rat liver model, in rats, in tumour models and in patients.

1. The metabolic pathway(s) of OGT 719, a novel nucleoside analogue in which galactose is covalently attached to the N1 of 5-fluorouracil (FU), have been investigated with (19)F-NMR spectroscopy in (1) the isolated perfused rat liver (IPRL) model, (2) normal rats, (3) rats bearing the HSN LV10 sarcoma, (4) nude mice xenografted with the human hepatoma HepG2 and (5) urine from patients. 2. The administration of OGT 719 results in the formation of small amounts of FU. IPRL experiments with OGT 719 in combination with asialofetuin, a natural asialoglycoprotein receptor (ASGP-r), suggest competitive binding of OGT 719 to the ASGP-r. 3. The data obtained in non-tumour rats also demonstrated an extremely low metabolization of OGT 719 into FU and alpha-fluoro-beta-alanine, the well-known major metabolite of FU. 4. A comparison of tumour extracts from rats bearing the HSN LV10 sarcoma treated with FU or OGT 719 showed the incorporation of FU into RNA in rats treated with FU but not in rats treated with OGT 719; nevertheless, the incorporation of FU into RNA was observed in the liver from rats treated with OGT 719. 5. In a human hepatoma xenografted to nude mice, both the OGT 719 and FU contents of the tumour were markedly higher than in the corresponding liver, suggesting a tumour-specific trapping of OGT 719 in hepatoma. 6. The metabolism of OGT 719 was also extremely low in patients. 7. In conclusion, the present study shows the value of (19)F-NMR for demonstrating for the first time that OGT 719 is a prodrug of FU although very poorly metabolized.

Catabolism of adenine nucleotides and its relation with intracellular phosphorylated metabolite concentration during ethanol oxidation in perfused rat liver.

Ethanol-induced perturbations in the energy metabolism and in the catabolism of adenine nucleotides were investigated by 31P NMR spectroscopy and HPLC analyses in perfused rat liver. Ethanol oxidation reduced the redox potential of the hepatocyte, leading to an intracellular accumulation of sn-glycerol 3-phosphate. This accumulation, in turn, led to a cytosolic P(i) depletion with stoichiometric relationship close to 1/1 for an initial period of 2 min. The concentration of nucleoside 5'-triphosphates (83 +/- 4% of ATP) was decreased during ethanol oxidation, reaching about 66% of its control value [2.88 +/- 0.02 mumol.(g of liver wet wt)-1] at high ethanol doses (10 and 70 mM). The depletion of P(i) relieved the inhibition exerted by this compound on AMP deaminase, key enzyme in the catabolism of adenine nucleotides. The degradation of AMP was monitored by HPLC analyses of the adenine nucleosides and bases released in the effluents. Integration over time of the total release of these metabolites accounted for the depletion of ATP recorded in the same time by 31P NMR spectroscopy. This result suggests that ATP depletion occurring during ethanol oxidation originated from an enhanced degradation of adenine nucleotides. There was a strong linear correlation (r2 = 0.92) between cytosolic P(i) level and allantoin release rate during ethanol perfusion. Cytosolic P(i) and allantoin release exhibited biphasic behavior, the recovery toward the initial levels being related to the release of P(i) in the cytoplasm during the complete catabolism of adenine nucleotides. Finally, the depletion of P(i) affected the glycogenolysis pathway, with a maximal inhibition of ca. 19% of the initial level.

Twenty-four-hour hypothermic preservation of rat liver with Euro-Collins and UW solutions. A comparative evaluation by 31P NMR spectroscopy, biochemical assays, and light microscopy.

A comparative study of 24 hr preservation at 4 degrees C of excised rat livers with Euro-Collins and hydroxyethyl starch-free University of Wisconsin (UWm) solutions has been conducted based on the assessment of (1) the cellular energy status determined by 31P NMR spectroscopy and (2) cellular injury estimated from the loss of purine compounds (inosine, hypoxanthine, xanthine, and uric acid) during cold ischemia and reperfusion measured by HPLC, the leakage of intracellular enzymes, and the modifications of parenchyma established by light microscopy. Recovery of nucleosides di- and triphosphate was greater in the UWm group (80 +/- 6% vs. 58 +/- 6%) while inorganic phosphate formation was comparatively reduced. During hypothermic storage, the UWm groups generated a higher amount of inosine and hypoxanthine (in relation to the presence of adenosine in the protective solution) while no xanthine or uric acid was detected due to the inhibitory effect of allopurinol. Conversely, large quantities of xanthine and uric acid were found in the reperfusate of the EC group, pinpointing the cytotoxic role of oxygen-derived free radicals in the generation of cellular damage, as also illustrated by a higher aspartate aminotransferase leakage in the EC group (devoid of allopurinol and glutathione. Light microscopy indicated no histological alterations in the UWm group and mild alterations in the EC group that showed ballooning of hepatocytes (no lactobionate and raffinose in EC) and an alternation of clarifications and eosinophilic condensations. This study clearly confirms and illustrates the overall superiority of UWm solution in liver transplant preservation.

The effects of ethanol concentration on glycero-3-phosphate accumulation in the perfused rat liver. A reassessment of ethanol-induced inhibition of glycolysis using 31P-NMR spectroscopy and HPLC.

The dose-dependent effect of ethanol on the hepatic metabolism of the perfused rat liver has been investigated by (a) 31P-NMR spectroscopy for the follow-up of intracellular phosphorylated metabolites and (b) HPLC for compounds released in the effluents. Perfusion of livers from fed rats with ethanol induced an increase in the level of sn-glycerol 3-phosphate and net accumulations of 3.30 +/- 0.33 and 0.69 +/- 0.15 mumol x g-1 wet liver were reached after 20 min, for 70 mM and 0.5 mM ethanol, respectively. sn-Glycerol-3-phosphate accumulation was fully detected by 31P NMR as indicated by comparing quantitations based on NMR and biochemical assays. Ethanol administration up to a concentration of 10 mM induced a dose-dependent decrease in the release of lactate + pyruvate by the liver. Lactate release decreased from 1129 +/- 39 to 674 +/- 84 nmol x min-1 x g-1, while pyruvate decreased from 230 +/- 9 to 6.2 +/- 0.4 nmol x min-1 x g-1, after 20 min of perfusion with 10 mM ethanol. Nevertheless, the flux through 6-phosphofructo-1-kinase, as measured by both the accumulation of sn-glycerol 3-phosphate and release of lactate + pyruvate, was not affected in the early phase of ethanol oxidation. Finally, data obtained from oxygen consumption, the release of acetate and the accumulation of sn-glycerol 3-phosphate do not support the involvement of the microsomal ethanol-oxidizing system in the catalysis of ethanol oxidation, even at high doses of alcohol.

In vitro correlation between force and energy metabolism in porcine malignant hyperthermic muscle studied by 31P NMR.

A comparative study of mechanical and energetic parameters of superfused muscle strips from normal pigs and malignant hyperthermia susceptible (MHS) pigs has been conducted. Phosphorus nuclear magnetic resonance spectroscopy at 80.9 MHz and mechanical measurements were used to assess muscle metabolic state. At rest, biceps femoris biopsies of MHS pigs displayed reduced phosphocreatine level, higher inorganic phosphate, and a more acidic internal pH. In normal stimulated fibers, caffeine infusion (8 or 16 mM) induced twitch potentiation and contracture while twitch tension was reduced and contracture more pronounced in malignant fibers. In normal and malignant fibers, calcium ionophore A23187 produced effects similar to those of caffeine, with the exception of twitch potentiation, which was not observed. With caffeine or A23187, the ATP level remained constant throughout the rest-stimulation-recovery protocol for normal and malignant fibers but phosphocreatine dropped to undetectable levels upon stimulation of malignant fibers. In both treatments some heterogeneity in the resonances of inorganic phosphate was observed in malignant fibers together with a more severe acidosis which might play a role in the impairment of the excitation-contraction process.

Simple cation-exchange high-performance liquid chromatography optimized to the measurement of metabolites in the effluents from perfused rat livers using refractive index and ultraviolet detectors.

A high-performance liquid chromatographic method designed to analyse effluents from perfused organs is described. In the case of rat liver, compounds released by the liver are readily separated and quantitated, using a strong cation exchanger (Aminex HPX 87H), two detectors connected in series (ultraviolet detector at 210 nm and refractive index detector), and by optimizing the concentration of sulphuric acid in the mobile phase. Chromatographic conditions described in the present work enable the quantitation, in a single run, of metabolites derived from the tricarboxylic acid cycle, glycolysis, ketogenesis, adenine nucleotides catabolism and ethanol oxidation. The advantage of this method stems from its ease of implementation, sensitivity and flexibility.

Application of reverse-DEPT polarization transfer pulse sequence to study the metabolism of carbon-13-labeled substrates in perfused organs by 1H NMR spectroscopy.

Metabolism of 13C-enriched metabolites can be advantageously studied by reverse-polarization transfer methods. In this work an improved reverse-DEPT sequence has been applied for the first time on perfused organs in a 20-mm probe. The metabolic fate of 99% enriched [2-13C]acetate perfused in excised rat liver and heart has been documented.

Effect of ethanol on hepatic energy metabolism and intracellular pH in chronically ethanol-treated rats. A 31P NMR study of normoxic or hypoxic perfused liver.

31P Nuclear magnetic resonance spectroscopy has been used to investigate the combined effect of 70 mM ethanol administration and hypoxia on the energetic metabolism and cytosolic pH of the perfused liver isolated from naive, fasted or chronically ethanol-treated rats. Ethanol-treatment or fasting induce a similar 25% reduction in ATP levels, and a differential enhancement of Pi levels to 130% and 225% of control values, respectively. In the naive rats, acute ethanol administration triggers profound variations in high-energy phosphorylated metabolite levels with, in particular, a 300% increase in phosphomonoesters, mostly due to sn-glycerol-3-phosphate (G3P) accumulation. A 4 min hypoxic period in the presence of ethanol induced a further accumulation of G3P. In the livers from chronically ethanol-treated rats, the G3P level remains low during acute ethanol administration, despite the presence of glycogen, indicating an attenuation of the ethanol-induced redox shift. The combination of 70 mM ethanol and hypoxia leads to an increased G3P level in the livers of chronically ethanol-treated rats which is consistent with a sustained alcohol dehydrogenase activity. The subsequent cytosolic acidosis is significantly less pronounced in the chronically ethanol-treated rats than in naive rats. These observations illustrate the adaptation of metabolic processes occurring in the livers of chronically ethanol-treated rats, which results in an increased resistance to acute ethanol exposure and hypoxic stress.

A homonuclear shift correlated and spatially localized spectroscopy using stimulated echoes.

A new method which combines localized high-resolution proton NMR spectroscopy with two-dimensional correlated spectroscopy using stimulated echoes is presented. Stimulated-echo correlated spectroscopy (STECSY) is a straightforward extension of the STEAM method. Experiments with phantoms illustrate the efficacy of STECSY. An in situ application on rat adipose tissue demonstrates that STECSY is a helpful tool with which to select and assign resonances in complex 1H NMR spectra.

Liposome-mediated delivery of gadolinium-diethylenetriaminopentaacetic acid to hepatic cells: a P-31 NMR study.

Delivery of gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA) (a magnetic resonance imaging contrast agent) to hepatic cells via liposomes used as carriers was demonstrated and monitored by P-31 NMR spectroscopy at 162 MHz on a perfused rat liver model. The penetration of Gd-DTPA into hepatic cells was reflected by a 41% reduction in the longitudinal relaxation time of the beta phosphorus atom of intrahepatic ATP used as an index of the contrast agent presence. The perfusion of free Gd-DTPA did not affect the relaxation index nor did liposomes devoid of paramagnetic agent. There was no indication of interference of Gd-DTPA, free or encapsulated in liposomes, with liver energetic metabolism. Alterations of hepatic magnetic resonance imaging signal induced by Gd-DTPA containing liposomes might help to assess the functional state of the parenchyma and pinpoint the intracellular compartment as opposed to the intravascular space.

[Nuclear magnetic resonance spectroscopy and hepatic metabolism. Real time study of cell energy and intermediate metabolism in the isolated and perfused rat liver]. / Spectroscopie de résonance magnétique nucléaire et métabolisme hépatique. Etude en temps réel de l'énergétique cellulaire et du métabolisme intermédiaire sur le foie de rat isolé et perfusé.

Nuclear magnetic resonance spectroscopy is increasingly used to study cellular metabolism in a manner respecting cell integrity. The contribution of phosphorus-31 and carbon-13 NMR is discussed and illustrated by specific examples taken from work on hepatic metabolism carried out in this laboratory. Particular emphasis is laid on metabolite identification, quantitation and fluxes as studied by phosphorus-31 NMR which provides a direct insight of energy metabolism. The analysis of perfused rat liver by naturally abundant carbon-13 NMR illustrates the potential of the method to study non invasively lipid and carbohydrate metabolism in living systems. The use of carbon-13 enriched substrates to pinpoint a specific pathway of the intermediary metabolism is described in the case of excised rat liver perfused with (2-13C)-acetate.

Hepatic metabolism during acute ethanol administration: a phosphorus-31 nuclear magnetic resonance study on the perfused rat liver under normoxic or hypoxic conditions.

The effect of ethanol metabolism on the energetic parameters and intracellular pH of the isolated perfused rat liver from fed rats was studied by phosphorus-31 nuclear magnetic resonance spectroscopy. This technique allowed us to analyze nondestructively and in real time the role of low oxygen tension on the possible injurious effect of ethanol on the liver cells. A quantitative analysis of nuclear magnetic resonance data recorded on a perfused rat liver within a 30 mm diameter probe has been performed at 80.9 MHz. Under normoxic and normothermic conditions, the levels of phosphorylated metabolites detected by nuclear magnetic resonance were 2.8, 0.3 and 2 mumoles per gm liver wet weight for ATP, ADP and inorganic orthophosphate, respectively. The cytosolic pH was 7.25 +/- 0.05. During a period of 4 min of hypoxia induced by reducing the perfusion flow rate to 25% of its initial value (i.e., from 12 ml to 3 ml per min per 100 gm body weight), the level of ATP dropped to 2.2 mumoles per gm liver wet weight. Concomitantly, ADP and inorganic orthophosphate increased to 0.6 and 3.3 mumoles per gm liver wet weight. Cytosolic pH fell to 7.02 +/- 0.05. Perfusion of the liver with a Krebs medium containing 70 mM (0.4%) ethanol induced a sharp decrease in intracellular inorganic orthophosphate to reach 1.3 mumole per gm liver wet weight and after a lag time of 4 to 6 min, a decrease in ATP level (2.15 mumoles per gm liver wet weight). A large increase in phosphomonoesters (mainly sn-glycerol 3-phosphate) up to 6 mumoles per gm liver wet weight was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorus-31 nuclear-magnetic-resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver.

A quantitative analysis of the phosphorus-31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30-mm sample cell. The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well-oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25 +/- 0.05 and free magnesium concentration 0.5 mM. Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn-glycerol 3-phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occurred during ethanol oxidation. No significant pH variation is observed during ethanol infusion. In perfused liver of rats subjected to 48-h fasts, the concentrations of cytosolic phosphorylated metabolites are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%. During a 4-min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min-1 (100 g body weight)-1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02 +/- 0.05 at the end of the 4-min hypoxic period.

31P nuclear magnetic resonance study of a human colon adenocarcinoma cultured cell line.

31P nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the energy metabolism in a human colon adenocarcinoma cell line (HT 29). NMR spectra were recorded at 80.9 MHz on approximately 2.5 X 10(8) cells continuously perfused with culture medium within a 20-mm NMR sample tube. Typical NMR spectra display a series of well-resolved resonances assigned to nucleoside triphosphates (mainly adenosine 5'-triphosphate), uridine diphosphohexose derivatives (uridine 5'-diphosphate-N-acetylglucosamine, uridine 5'-diphosphate-N-acetylgalactosamine, uridine 5'-diphosphate-glucose), intra- and extracellular inorganic phosphate, and phosphomonoesters (mainly phosphorylcholine and glucose 6-phosphate). Measurement of phosphorylated metabolite concentrations from the intensity of NMR signals is in good agreement with the results provided by conventional biochemical assays. 31P NMR allows to follow noninvasively the effect of anoxia on HT 29 cells. The results indicate that the cells are able to maintain about 60% of their initial nucleoside triphosphate level after 2 h of anaerobic perfusion. Cells accumulate inorganic phosphate during anoxia and the intracellular-extracellular pH gradient increases from 0.5 in well-oxygenated cells to more than 1 pH unit under anoxic conditions. The value of intracellular pH of well-oxygenated HT 29 cells is 7.1. The effect of glucose starvation upon energy metabolism has also been examined in real time by NMR: a rapid decline of adenosine 5'-triphosphate down to 10% of the initial value is observed over a period of 2 h. In contrast, the level in uridine diphosphohexoses reaches a new steady state value representing 60% of the initial one. Refeeding the cells with 25 mM glucose leads to a dramatic drop of internal pH reflecting the activation of the glycolytic pathway.

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver.

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.

Glutamate-glutamine metabolism in the perfused rat liver. 13C-NMR study using (2-13C)-enriched acetate.

13C-NMR has been used to follow the metabolism of 13C-enriched substrates in isolated perfused rat liver. The fate of 90% enriched [2-13C]acetate has been studied in the perfused liver in order to investigate mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of 13C into glutamine can be linked to the operation of the glutamate-glutamine antiport and to the high activity of cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is evidence of the existence of an active glutamine-glutamate futile cycle.

[Nuclear magnetic resonance spectroscopy. New tool for investigating cellular metabolism in living systems]. / La spectroscopie de résonance magnétique nucléaire. Un nouvel outil d'investigation du métabolisme cellulaire dans les systèmes vivants.

Nuclear magnetic resonance spectroscopy is increasingly used to study cellular metabolism in a manner respecting cell integrity. The contribution of phosphorus-31 and carbon-13 NMR is discussed and illustrated by specific examples taken from work carried out in this laboratory or from the literature. A particular emphasis is layed on metabolite identification, quantitation and fluxes as studied by phosphorus-31 NMR which provides a direct insight of energy metabolism. The analysis of perfused rat liver by natural abundance carbon-13 NMR illustrates the potential of the method of study non-invasively lipid and carbohydrate metabolism in living systems. The use of carbon-13 enriched substrates to pinpoint a specific pathway of the intermediary metabolism is described in the case of excised rat heart and liver perfused with (2-C13)-acetate. Clinical applications of NMR spectroscopy are rapidly reviewed.