Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

Creatine feeding does not enhance intramyocellular glycogen concentration during carbohydrate loading: an in vivo study by 31P- and 13C-MRS

The main aim of this study was to examine the hypothesis that creatine (Cr) feedingenhances myocellular glycogen storage in humans undergoing carbohydrateloading. Twenty trained male subjects were randomly assigned to have their dietssupplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CRGP,n=10) or placebo (PL-GP, n=10) for 5 days. Changes in resting myocellularglycogen and phosphocreatine (PCr) were determined with Magnetic ResonanceSpectroscopy (13C- and 31P-MRS, respectively). After CR-GP, the levels of intramyocellularglycogen increased from 147 ± 13 (standard error) mmol·(kg wet weight-1)to 172 ± 13 mmol·(kg wet weight)-1, while it increased from 134 ± 17 mmol·(kg wetweight)- to 182 ± 17 mmol·(kg wet weight)-1 after PL-GP; the increments in intramyocellularglycogen concentrations were not statistically different. The increment inthe PCr/ATP ratio after CR-GP (+ 0.20 ± 0.12) was significantly different comparedto PL-GP (- 0.34 ± 0.16) (p<0.05). The present results do not support the hypothesisthat Cr loading increases muscle glycogen storage (AU)

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Creatine feeding does not enhance intramyocellular glycogen concentration during carbohydrate loading: an in vivo study by 31P- and 13C-MRS.

The main aim of this study was to examine the hypothesis that creatine (Cr) feeding enhances myocellular glycogen storage in humans undergoing carbohydrate loading. Twenty trained male subjects were randomly assigned to have their diets supplemented daily with 252 g of glucose polymer (GP) and either 21 g of Cr (CR-GP, n = 10) or placebo (PL-GP, n = 10) for 5 days. Changes in resting myocellular glycogen and phosphocreatine (PCr) were determined with Magnetic Resonance Spectroscopy (13C- and 31P-MRS, respectively). After CR-GP, the levels of intramyocellular glycogen increased from 147 +/- 13 (standard error) mmol x (kg wet weight(-1)) to 172 +/- 13 m mol x (kg wet weight)(-1), while it increased from 134 +/- 17 mmol x (kg wet weight)(-) to 182 +/- 17 mmol x (kg wet weight)(-1) after PL-GP; the increments in intramyocellular glycogen concentrations were not statistically different. The increment in the PCr/ATP ratio after CR-GP (+ 0.20 +/- 0.12) was significantly different compared to PL-GP (- 0.34 +/- 0.16) (p < 0.05). The present results do not support the hypothesis that Cr loading increases muscle glycogen storage.

Identification and characterization of muscarinic acetylcholine receptor subtypes expressed in human skin melanocytes.

The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (ml through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9,000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the ml, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Noninvasive measurement of muscle high-energy phosphates and glycogen concentrations in elite soccer players by 31P- and 13C-MRS.

PURPOSE: The purpose of this study was to measure noninvasively the absolute concentrations of muscle adenosine triphosphate [ATP], phosphocreatine [PCr], inorganic phosphate (Pi), and glycogen [Gly] of elite soccer players. METHODS: Magnetic resonance spectroscopy (31P- and 13C-MRS) was used to measure the concentrations of metabolites in the calf muscles of 18 young male players [age = 17.5 +/- 1.0 (SD) yr]. RESULTS: Average muscle [PCr] and [ATP] were 17.8 +/- 3.3 and 6.0 +/- 1.2 mmol x (kg wet weight)(-1), respectively. The ratios of Pi/PCr and PCr/ATP were 0.15 +/- 0.05 and 3.00 +/- 0.26, respectively. The muscle [Gly] was 144 +/- 54 mmol x (kg wet weight)(-1). There was a high correlation (r = 0.93, P < 0.0001) between muscle ATP and PCr concentrations, but there was no correlation between [Gly] and [PCr] or [ATP]. The concentrations of the different metabolites determined in the present study with noninvasive MRS methods were within the ranges of values reported in human muscle from biochemical analysis of muscle biopsies. CONCLUSION: MRS methods can be utilized to assess noninvasively the muscle energetic status of elite soccer players during a soccer season. The high correlation between ATP and PCr might be indicative of fiber type differences in the content of these two metabolites.

Muscle glycogen degradation during simulation of a fatiguing soccer match in elite soccer players examined noninvasively by 13C-MRS.

PURPOSE: The purpose of this research project was to noninvasively determine individual muscle glycogen [Gly] degradation during a test intended to predict individual fatigue in intense soccer matches. METHODS: The [Gly] of the calf muscles of 17 elite soccer players [age = 17.4 +/- 0.8 (SD)] were measured with 13C-MRS before and after an alternating velocity test to exhaustion. Blood samples were taken before and 3 min after the test for determination of blood metabolites. RESULTS: Average muscle [Gly] was 135 +/- 53 mmol x (kg wet weight)(-1) before and 87 +/- 27 mmol x (kg wet weight)(-1) (P < 0.001) after exhaustion (42 +/- 25 min). There was a high correlation (r = 0.87, P < 0.0001) between muscle [Gly] at rest and net muscle [Gly] utilized. There was also a more moderate correlation (r = 0.62, P < 0.01) between net muscle [Gly] used and time to exhaustion during the soccer-specific test. There was some evidence of correlation (r = 0.42, P = 0.09) between resting [Gly] and time to exhaustion. Plasma lactate increased (P < 0.001) from 0.8 +/- 0.4 before the test to 2.5 +/- 1.0 mmol x L(-1) at exhaustion, whereas ammonia was raised (P < 0.0001) from 44.1 +/- 10.3 to 89.7 +/- 14.9 micromol x L(-1). Similarly, plasma free fatty acids were elevated (P < 0.0001) from 148 +/- 106 to 797 +/- 401 micromol x L(-1), and glycerol was increased (P < 0.0001) from 48.3 +/- 17.7 to 182.2 +/- 61.8 micromol x L(-1). Insulin levels (11.9 +/- 3.7 vs 11.7 +/- 4.8 microU x mL(-1)) remained the same. Creatine kinase levels increased (P < 0.0001) from 486 +/- 501 to 640 +/- 548 micromol x L(-1) after the test. CONCLUSIONS: We conclude that exhaustion during soccer-specific performance is related to the capacity to utilize muscle [Gly]. The results underline the importance of dietary counseling (glycogen loading and resynthesis strategies) and proper training to enhance the glycogen levels and glycogenolytic capacity of the players.

Human skin fibroblasts express m2, m4, and m5 subtypes of muscarinic acetylcholine receptors.

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChRs) are expressed by human skin fibroblasts (HSF). We have identified the molecular subtypes of these receptors by reverse transcription-polymerase chain reaction (RT-PCR), using m1-m5 subtype-specific primers. These experiments showed that only mRNAs for m2, m4, and m5 mAChR subtypes are present in HSF. The RT-PCR products were characterized by restriction analysis and Southern blotting. Northern blot analysis showed the presence of m2 and m4 mAChR RNA. Rabbit antibodies were raised using a synthetic peptide as immunogen corresponding to the C-terminus of the m2 protein and were used to visualize fibroblast mAChRs. Cell membranes of HSF in cell culture and specimens of normal human skin had a unique staining pattern specific for anti-m2 antibody, as well as for antibodies against m4 and m5. In Western blots of fibroblast proteins, the antibodies visualized the m2 receptor at 65 kDa, m4 at 70 kDa, and m5 at 95 kDa. The function of fibroblast mAChRs was examined by measuring muscarinic effects on intracellular free Ca2+ concentration ([Ca2+]i). Muscarine increased transiently [Ca2+]i in cultured HSF. This effect could be abolished by the muscarinic antagonist atropine. Thus, the results of this study showed that HSF express m2, m4, and m5 mAChR subtypes, and that fibroblast mAChRs are coupled to the regulation of [Ca2+]i.

Identification and mapping of keratinocyte muscarinic acetylcholine receptor subtypes in human epidermis.

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of cell-surface receptors, the nicotinic and the muscarinic cholinergic receptors. High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density. These receptors mediate effects of muscarinic drugs on keratinocyte viability, proliferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR and their location in human epidermis. Polymerase chain reaction amplification of cDNA sequences uniquely present within the third cytoplasmic loop of each subtype demonstrated the expression of the m1, m3, m4, and m5 mAChR subtypes. To visualize these mAChR, we raised rabbit anti-sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype. The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which could be abolished by preincubating the anti-serum with the peptide used for immunization. The immunofluorescent staining patterns produced by each antibody in the epidermis suggested that the profile of keratinocyte mAChR changes during epidermal turnover. The semiquantitative analysis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular cells mostly possessed m1. Thus, the results of this study demonstrate for the first time the presence of m1, m3, m4, and m5 mAChR in epidermal keratinocytes. Because keratinocytes express a unique combination of mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single cytotransmitter, acetylcholine, and muscarinic drugs may exert different biologic effects on keratinocytes at different stages of their maturation.

Diagnostic and prognostic value of cerebral 31P magnetic resonance spectroscopy in neonates with perinatal asphyxia.

The impact of depressed neonatal cerebral oxidative phosphorylation for diagnosing the severity of perinatal asphyxia was estimated by correlating the concentrations of phosphocreatine (PCr) and ATP as determined by magnetic resonance spectroscopy with the degree of hypoxic-ischemic encephalopathy (HIE) in 23 asphyxiated term neonates. Ten healthy age-matched neonates served as controls. In patients, the mean concentrations +/- SD of PCr and ATP were 0.99 +/- 0.46 mmol/L (1.6 +/- 0.2 mmol/L) and 0.99 +/- 0.35 mmol/L (1.7 +/- 0.2 mmol/L), respectively (normal values in parentheses). [PCr] and [ATP] correlated significantly with the severity of HIE (r = 0.85 and 0.9, respectively, p < 0.001), indicating that the neonatal encephalopathy is the clinical manifestation of a marred brain energy metabolism. Neurodevelopmental outcome was evaluated in 21 children at 3, 9, and 18 mo. Seven infants had multiple impairments, five were moderately handicapped, five had only mild symptoms, and four were normal. There was a significant correlation between the cerebral concentrations of PCr or ATP at birth and outcome (r = 0.8, p < 0.001) and between the degree of neonatal neurologic depression and outcome (r = 0.7). More important, the outcome of neonates with moderate HIE could better be predicted with information from quantitative 31P magnetic resonance spectroscopy than from neurologic examinations. In general, the accuracy of outcome predictability could significantly be increased by adding results from 31P magnetic resonance spectroscopy to the neonatal neurologic score, but not vice versa. No correlation with outcome was found for other perinatal risk factors, including Apgar score.

Isolation and expression of a cDNA clone encoding human kynureninase.

Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyridoxal-P)-dependent enzyme, catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse-transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G2) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P-binding site consensus sequence assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an approximately 2.0-kb mRNA species in all tissues tested. A second mRNA species (approximately 2.6 kb) was also detected in some tissues. After transfection of HEK-293 cells with the cDNA coding for kynureninase, the K(m) values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 +/- 37 microM and 13.2 +/- 2.0 microM, respectively.

Cloning and functional expression of a soluble form of kynurenine/alpha-aminoadipate aminotransferase from rat kidney.

Several aminotransferases with kynurenine aminotransferase (KAT) activity are able to convert L-kynurenine into kynurenic acid, a putative endogenous modulator of glutamatergic neurotransmission. In the rat, one of the described KAT isoforms has been found to correspond to glutamine transaminase K. In addition, rat kidney alpha-aminoadipate aminotransferase (AadAT) also shows KAT activity. In this report, we describe the isolation of a cDNA clone encoding the soluble form of this aminotransferase isoenzyme from rat (KAT/AadAT). Degenerate oligonucleotides were designed from the amino acid sequences of rat kidney KAT/AadAT tryptic peptides for use as primers for reverse transcription-polymerase chain reaction of rat kidney RNA. The resulting polymerase chain reaction fragment was used to screen a rat kidney cDNA library and to isolate a cDNA clone encoding KAT/AadAT. Analysis of the combined DNA sequences indicated the presence of a single 1275-base pair open reading frame coding for a soluble protein of 425 amino acid residues. KAT/AadAT appears to be structurally homologous to aspartate aminotransferase in the pyridoxal 5'-phosphate binding domain. RNA blot analysis of rat tissues, including brain, revealed a single species of KAT/AadAT mRNA of approximately 2.1 kilobases. HEK-293 cells transfected with the KAT/AadAT cDNA exhibited both KAT and AadAT activities with enzymatic properties similar to those reported for the rat native protein.

Magnetic resonance imaging of brain edema in the neonatal rat: a comparison of short and long term hypoxia-ischemia.

Diffusion-weighted and transversal relaxation time (T2)-weighted magnetic resonance imaging were used to study the relationship between the duration of hypoxia-ischemia [unilateral common carotid artery (CCA) ligation and exposure to 8% oxygen] and the in vivo visualization of brain edema in 7-d-old rats. After CCA ligation, 35 animals were divided into five groups according to the length of exposure to 8% oxygen: no exposure (n = 9), 15 min (n = 12), 30 min (n = 5), and 1 h (n = 9) exposure; six animals served as controls. Diffusion weighted images were acquired 2 h after the hypoxic-ischemic insult, sequential T2 weighted images were recorded for up to 7 d and the outcome was documented by histologic examination at 21 d. The apparent diffusion coefficient of water in the ipsilateral cortex was significantly decreased in all animals recovering from prolonged hypoxic-ischemic insult (30 min and longer), whereas this was the case in only 40% of animals exposed to 15 min of hypoxia. Moreover, T2 prolongation of brain tissue occurred only in the former group. These results indicate transient and reversible alterations of physiologic water compartmentation for short term hypoxia-ischemia, but irreversible edema formation for long term hypoxia-ischemia. They support the hypothesis that the duration of hypoxia-ischemia determines whether a vasogenic edema and infarction follows the initial cytotoxic edema.

Changes of liver metabolite concentrations in adults with disorders of fructose metabolism after intravenous fructose by 31P magnetic resonance spectroscopy.

A novel 31P magnetic resonance spectroscopy procedure allows the estimation of absolute concentrations of certain phosphorus-containing compounds in liver. We have validated this approach by measuring ATP, phosphomonesters, and inorganic phosphate (Pi) during fasting and after an i.v. fructose bolus in healthy adults and in three adults with disorders of fructose metabolism and by comparing results with known metabolic concentrations measured chemically. During fasting, the ATP concentration averaged 2.7 +/- 0.3 (SD, n = 9) mmol/L, which, after due correction for other nucleoside triphosphates, was 2.1 mmol/L and corresponded well with known concentrations. Fructose-1-phosphate (F-1-P) could not be measured during fasting; its concentration after fructose was calculated from the difference of the phosphomonester signals before (2.9 +/- 0.2 mmol/L) and after fructose. Pi was 1.4 +/- 0.3 mmol/L and represented the one fourth of Pi visible in magnetic resonance spectra. In the three healthy controls after fructose (200 mg/kg, 20% solution, 2.5 min), the fructokinase-mediated increase of F-1-P was rapid, reaching 4.9 mmol/L within 3 min, whereas the uncorrected ATP decreased from 2.7 to 1.8 mmol/L and the Pi from 1.4 to 0.3 mmol/L. The subsequent decrease of F-1-P, mediated by fructaldolase, was accompanied by an overshooting rise of Pi to 2.7 mmol/L. In the patient with essential fructosuria, the concentrations of F-1-P, ATP, and Pi remained unchanged, confirming that fructokinase was indeed inactive. In the patient with hereditary fructose intolerance, initial metabolic changes were the same as in the controls, but baseline concentrations were not yet reestablished after 7 h, indicating weak fructaldolase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of absolute metabolite concentrations in human tissue by 31P MRS in vivo. Part I: Cerebrum, cerebellum, cerebral gray and white matter.

Absolute metabolite concentrations were determined in four different brain regions using phosphorus magnetic resonance spectroscopy (31P MRS) on 10 healthy adult volunteers. Localized spectra were collected simultaneously from the cerebellum and the cerebrum and, later, from deep white matter and cortical gray matter by means of a two-volume ISIS pulse sequence and a Helmholtz-type RF-coli. Each brain spectrum was quantified with a calibration spectrum from a head-shaped simulation phantom. A time-domain fitting routine was used to process the fully relaxed data. Several metabolite concentrations (mmol/liter) differed significantly between the cerebrum and the cerebellum (PME = 3.2 +/- 0.3 and 4.0 +/- 0.6, PCr = 2.9 +/- 0.3 and 3.9 +/- 0.4, NTP = 2.9 +/- 0.2 and 2.6 +/- 0.2, respectively) and between cortical gray matter and deep white matter (PME = 3.1 +/- 0.4 and 4.3 +/- 0.8, PDE = 10.1 +/- 2.5 and 14.2 +/- 2.6, respectively). The concentration of free magnesium ion was found to be similar in all four brain regions (0.53 +/- 0.21 mmol/liter) but the intracellular pH was significantly higher in the cerebellum (7.04 +/- 0.03) than in the cerebrum (6.99 +/- 0.02).

Assessment of absolute metabolite concentrations in human tissue by 31P MRS in vivo. Part II: Muscle, liver, kidney.

Absolute metabolite concentrations were assessed in the muscle, the liver, and the kidney of healthy human volunteers by 31P MRS. Fully relaxed in vivo spectra were acquired with a surface coil and were localized with an adiabatic ISIS pulse sequence. The spectra were quantified with a subsequent measurement of a calibration phantom and were processed iteratively in the time domain. The following mean metabolite concentrations (mmol/liter) were measured in the resting male calf muscle (n = 9), in the fasting liver (n = 12), and in the orthotopic kidney (n = 5): [PME] = 2.0 +/- 0.6, 3.8 +/- 0.7, and 2.6 +/- 0.9, [Pi] = 2.9 +/- 0.3, 1.8 +/- 0.3, and 1.6 +/- 0.4, [PDE] = 3.8 +/- 0.8, 9.7 +/- 1.5, and 4.9 +/- 1.1, [PCr] = 22.0 +/- 1.2, 0, and 0, [NTP] = 5.7 +/- 0.4, 2.9 +/- 0.4, and 2.0 +/- 0.3, respectively. Several interesting findings are to be emphasized: The concentrations of Pi, PCr, and NTP were 20% lower in the muscle of women than of men. In addition, the pHi was significantly lower in female muscle (6.99 +/- 0.03) than in male muscle (7.05 +/- 0.03). The pHi in the liver (7.12 +/- 0.09) and in the kidney (7.09 +/- 0.08) were higher than in the muscle of both genders. The free magnesium concentration (mmol/liter) was higher in the liver (1.40 +/- 0.64) than in the kidney (0.79 +/- 0.39) and in the muscle (0.52 +/- 0.10).

Comparison of calibration strategies for the in vivo determination of absolute metabolite concentrations in the human brain by 31P MRS.

Cerebral concentrations of phosphorus metabolites can be assessed non-invasively by 31P MRS provided the metabolite signals are calibrated with the signal of a standard of known concentration. The reliability of the concentration estimates depends mainly on the strategy of calibration. Three strategies were compared by assessing the concentrations both in a test dummy and in the brain of volunteers. The first strategy utilized tissue water as an internal heteronuclear concentration standard. The second and third strategies used as phosphorus solution as an external homonuclear standard; this solution was either put into a reference bottle placed on top of the head or into a simulation phantom measured instead of the head. Localization was always achieved with the ISIS pulse sequence. The two external homonuclear strategies achieved a higher accuracy (mean error approximately 5%) and reproducibility (mean SD approximately 8%) of the concentration estimates than the internal heteronuclear strategy (mean error approximately 11%; mean SD approximately 15%).

Developmental changes of phosphorus metabolite concentrations in the human brain: a 31P magnetic resonance spectroscopy study in vivo.

Phosphorus magnetic resonance spectroscopy is a noninvasive method to investigate brain metabolism in vivo. ATP generally serves as an internal concentration standard for the quantification of the various phosphorus metabolites, because the ATP concentration in mammalian brains is assumed to be age independent. This presumption is based on observations made in the biochemical analysis of the developing rat brain. In the present study, metabolite concentrations were assessed with an external concentration standard. Each brain spectrum was quantified using a calibration spectrum that was acquired from a phantom after the in vivo brain measurement. Fully relaxed localized brain spectra were obtained from 16 neonates (2-28 d), 17 infants (6-20 mo), and 28 adults (22-58 y). The metabolite concentrations (in mmol/L) changed from neonates to adults: phosphomonoester from 4.5 to 3.5, inorganic phosphate from 0.6 to 1.0, phosphodiester from 3.2 to 11.7, phosphocreatine from 1.4 to 3.4, and ATP from 1.6 to 2.9. We conclude that 1) the ATP concentration in the human brain almost doubles between neonates and adults, and 2) ATP may not be used as an age-independent internal concentration standard.

Comparison of methods for the determination of absolute metabolite concentrations in human muscles by 31P MRS.

In order to determine metabolite concentrations in human skeletal muscles by in vivo 31P MRS, different quantification methods were analyzed with regard to the accuracy and reproducibility of results and the simplicity of handling. Each quantification method comprised a calibration strategy and a localization technique. Extensive in vivo and in vitro tests showed that homonuclear phantom-based calibration strategies yielded significantly more accurate (lower systematic errors) and more reproducible (lower statistical errors) concentration estimates than heteronuclear strategies using internal water as a concentration standard. Additionally, the former strategies are easier to handle than the latter. Localization with the volume-selective sequence ISIS yielded slightly more reproducible results than localization by surface coil. We conclude that phosphorus metabolite concentrations are determined most accurately with phantom-based calibration strategies in combination with ISIS localization (measurement errors approximately 5-7%).