Accuracy of real-time MR temperature mapping in the brain: A comparison of fast sequences

PurposeTo compare magnetic resonance (MR) thermometry based on the proton resonance frequency (PRF) method using a single shot echoplanar imaging (ss EPI) sequence to both of the standard sequences, gradient echo (GRE) and segmented echoplanar imaging (seg EPI) in the in vivo human brain, at 1.5T and 3T.Material and methodsRepetitive MR thermometry was performed on the brain of six volunteers using GRE, seg EPI, and ss EPI sequences on whole-body 1.5T and 3T clinical systems using comparable acquisition parameters. Phase stability and temperature data precision in the human head were determined over 12 min for the three sequences at both field strengths. An ex-vivo swine skeletal muscle model was used to evaluate temperature accuracy of the ss EPI sequence during heating by high intensity focused ultrasound (HIFU).ResultsIn-vivo examinations of brain revealed an average temperature precision of 0.37 °C/0.39 °C/0.16 °C at 3T for the GRE/seg EPI/ss EPI sequences. At 1.5T, a precision of 0.58 °C/0.63 °C/0.21 °C was achieved. In the ex-vivo swine model, a strong correlation of temperature data derived using ss EPI and GRE sequences was found with a temperature deviation <1 °C.ConclusionThe ss EPI sequence was the fastest and the most precise sequence for MR thermometry, with significantly higher accuracy compared to GRE.     

Neurofilament proteins NF-L, NF-M and NF-H in brain of patients with Down syndrome and Alzheimer's disease

Summary. Neurofilaments (NFs) are integral constituents of the neuron playing a major role in brain development, maintenance, regeneration and the pattern of expression for NFs suggests their contribution to plasticity of the neuronal cytoskeleton and creating and maintaining neuronal architecture. Using immune-histochemical techniques the altered expression of NFs in Down syndrome (DS) and Alzheimer's disease (AD) has been already published but as no corresponding systematic immune-chemical study has been reported yet, we decided to determine proteins levels of three NFs in several brain regions of DS and AD brain. We evaluated immunoreactive NF-H, NF-M and NF-L levels using Western blotting in brain regions temporal, occipital cortex and thalamus of patients with DS (n = 9), AD (n = 9) and controls (n = 12). We found significantly increased NF-H in temporal cortex (controls: means 0.74 ± 0.39 SD; DS: means 3.01 ± 2.18 SD) of DS patients and a significant decrease of NF-L in occipital cortex of DS and AD patients (controls: means 1.19 ± 0.86 SD; DS: means 0.35 ± 0.20; AD: 0.20 ± 0.11 SD). We propose that the increase of NF-H in temporal cortex of DS brain is due to neuritic sprouting as observed in immune-histochemical studies. The increase may not be caused by the known accumulation of NFs in plaques, tangles or Lewy bodies due to our solubilization protocol. The decrease of NF-L in occipital cortex of DS and AD patients may well be reflecting neuronal loss. Altogether, however, we suggest that NFs are not reliable markers for neuronal death, a hallmark of both neurodegenerative diseases, in DS or AD. The increase of NF-H in DS or the decrease of NF-L in DS and AD leaves the other NFs unchanged, which points to dysregulation in DS and AD and raises the question of impaired structural assembly of neurofilaments. Received July 19, 2000 Accepted July 28, 2000     

Dosimetric analysis of Tomotherapy-based intracranial stereotactic radiosurgery of brain metastasis

PurposeThis paper analyzes Tomotherapy-based intracranial stereotactic radiosurgery (HTSRS) of brain metastasis targeting two end-points: 1) evaluation of dose homogeneity, conformity and gradient scores for single and multiple lesions and 2) assay of dosimetric criticality of completion of HTSRS procedures.Methods42 treatment plans of 33 patients (53 brain lesions) treated with HTSRS were analyzed. Dose to healthy brain, homogeneity, conformity and gradient indexes were evaluated for each lesion. Influence of Field Length and multiple lesions cross-talk effect were assessed. Treatment interruption and completion was investigated using radiochromic films in order to examine the delivered dose and its robustness to patient intrafraction movement.ResultsThe average dose homogeneity index was 1.04 ± 0.02 (SD). Average dose conformity and gradient score indexes were 1.4 ± 0.2 and 50 ± 14 respectively. We found a strong correlation of the dose to healthy brain and conformity and gradient indexes with target(s) volume for which analytical functions were obtained. Field Length and cross-talk effect were significantly correlated with poor gradient scores, but were found not to affect dose conformity.ConclusionsHomogeneity and conformity of HTSRS plans achieved excellent scores, while dose falloff and dose to healthy brain were slightly larger when compared with non-coplanar SRS techniques. Care should be given if treating large (>3 cc) or multiple near in-plane lesions in order to reduce dose to healthy brain. Analysis of interrupted treatments suggests splitting HTSRS treatments in two consecutive fractions in order to prevent target miss and overdosage due to patient intrafraction movement.     

Effects of the murine skull in optoacoustic brain microscopy

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small‐animal neuroimaging methods, a variety of acoustic and light‐related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high‐resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically‐ and optically‐determined resolution scenarios. It is shown that strong low‐pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

(a) Experimental setup for hybrid acoustic and optical resolution optoacoustic microscopy. (b) Transcranial scan of an adult mouse brain using the optical resolution mode. Scale bar is 375 µm.     

Pilot feasibility study of in vivo intraoperative quantitative optical coherence tomography of human brain tissue during glioma resection

This study investigates the feasibility of in vivo quantitative optical coherence tomography (OCT) of human brain tissue during glioma resection surgery in six patients. High‐resolution detection of glioma tissue may allow precise and thorough tumor resection while preserving functional brain areas, and improving overall survival. In this study, in vivo 3D OCT datasets were collected during standard surgical procedure, before and after partial resection of the tumor, both from glioma tissue and normal parenchyma. Subsequently, the attenuation coefficient was extracted from the OCT datasets using an automated and validated algorithm. The cortical measurements yield a mean attenuation coefficient of 3.8 ± 1.2 mm^?1 for normal brain tissue and 3.6 ± 1.1 mm^?1 for glioma tissue. The subcortical measurements yield a mean attenuation coefficient of 5.7 ± 2.1 and 4.5 ± 1.6 mm^?1 for, respectively, normal brain tissue and glioma. Although the results are inconclusive with respect to trends in attenuation coefficient between normal and glioma tissue due to the small sample size, the results are in the range of previously reported values. Therefore, we conclude that the proposed method for quantitative in vivo OCT of human brain tissue is feasible during glioma resection surgery.     

Microvascular characteristics of the acoustic fats: Novel data suggesting taxonomic differences between deep and shallow‐diving odontocetes

Odontocetes have specialized mandibular fats, the extramandibular (EMFB) and intramandibular fat bodies (IMFB), which function as acoustic organs, receiving and channeling sound to the ear during hearing and echolocation. Recent strandings of beaked whales suggest that these fat bodies are susceptible to nitrogen (N₂) gas embolism and empirical evidence has shown that the N₂ solubility of these fat bodies is higher than that of blubber. Since N₂ gas will diffuse from blood into tissue at any blood/tissue interface and potentially form gas bubbles upon decompression, it is imperative to understand the extent of microvascularity in these specialized acoustic fats so that risk of embolism formation when diving can be estimated. Microvascular density was determined in the EMFB, IMFB, and blubber from 11 species representing three odontocete families. In all cases, the acoustic tissues had less (typically 1/3 to 1/2) microvasculature than did blubber, suggesting that capillary density in the acoustic tissues may be more constrained than in the blubber. However, even within these constraints there were clear phylogenetic differences. Ziphiid (Mesoplodon and Ziphius, 0.9 ± 0.4% and 0.7 ± 0.3% for EMFB and IMFB, respectively) and Kogiid families (1.2 ± 0.2% and 1.0 ± 0.01% for EMFB and IMFB, respectively) had significantly lower mean microvascular densities in the acoustic fats compared to the Delphinid species (Tursiops, Grampus, Stenella, and Globicephala, 1.3 ± 0.3% and 1.3 ± 0.3% for EMFB and IMFB, respectively). Overall, deep‐diving beaked whales had less microvascularity in both mandibular fats and blubber compared to the shallow‐diving Delphinids, which might suggest that there are differences in the N₂ dynamics associated with diving regime, phylogeny, and tissue type. These novel data should be incorporated into diving physiology models to further understand potential functional disruption of the acoustic tissues due to changes in normal diving behavior.     

A pilot study to assess the effect of acute exercise on brain glutathione

The brain is highly susceptible to oxidative stress due to its high metabolic demand. Increased oxidative stress and depletion of glutathione (GSH) are observed with aging and many neurological diseases. Exercise training has the potential to reduce oxidative stress in the brain. In this study, nine healthy sedentary males (aged 25?±?4 years) undertook a bout of continuous moderate intensity exercise and a high-intensity interval (HII) exercise bout on separate days. GSH concentration in the anterior cingulate was assessed by magnetic resonance spectroscopy (MRS) in four participants, before and after exercise. This was a pilot study to evaluate the ability of the MRS method to detect exercise-induced changes in brain GSH in humans for the first time. MRS is a non-invasive method based on nuclear magnetic resonance, which enables the quantification of metabolites, such as GSH, in the human brain in vivo. To add context to brain GSH data, other markers of oxidative stress were also assessed in the periphery (in blood) at three time points [pre-, immediately post-, and post (～1?hour)-exercise]. Moderate exercise caused a significant decrease in brain GSH from 2.12?±?0.64?mM/kg to 1.26?±?0.36?mM/kg (p?=?.04). Blood GSH levels increased immediately post-HII exercise, 580?±?101?µM to 692?±?102 µM (n?=?9, p?=?.006). The findings from this study show that brain GSH is altered in response to acute moderate exercise, suggesting that exercise may stimulate an adaptive response in the brain. Due to the challenges in MRS methodology, this pilot study should be followed up with a larger exercise intervention trial.     

A radioenzymatic assay for normetanephine in brain tissue

A simple, rapid and sensitive radioenzymatic assay for measurement of normetanephrine (NMN) in the brain has been described. The assay which is based on conversion of NMN to its N-methylated tritiated derivative metanephrine (³N-MN), by partially purified bovine adrenal phenyl-ethanolamine-N-methyl represents an extension of a previously developed procedure for measurement of urinary NMN. The sensitivity of the assay is 50 pg and results can be obtained in less than 4 hours. In rat brain, NMN concentration was 61 ± 3.4 ng/gm for hypothalamus and 82 ± 4.2 for brain stem at level of obex in male rats; 74 ±11 and 139 ± 10, respectively in female rats. Measurement of NMN in different areas of the brain may help to elucidate possible involvement of central nervous system in the pathophysiology of disease states such as hypertension.The role of central catecholamines in the pathogenesis of disease states has been investigated by a variety of techniques. These include methods which estimate relative concentrations of catecholamines in different parts of the neurones (1,2) and absolute concentrations in specific brain nuclei (3). In addition, estimates been made of catecholamine synthesis (4,%) an dturnover rates (6). Neurotransmitter actually released into the synaptic cleft cannot be measured. Moreover, part of what is released is taken up again by the neurone. The remainder is subject to catabolism and the resulting metabolic products may well reflect the amount of physiologically active transmitter. This report describes a rapid specific and sensitive assay for measurement of the O-methylated metabolite of norepinephrine, normetanephrine (NMN) in brain tissue, which is an extension of a previously developed procedure for urinary NMN (31). The metabolite is stable and readily extracted into solvents.     

N-terminal brain natriuretic propeptide levels correlate with procalcitonin and C-reactive protein levels in septic patients

The aim of this study was to find the relationship between N-terminal brain natriuretic propeptide (NT-proBNP), procalcitonin (PCT) and C-reactive protein (CRP) plasma concentrations in septic patients. This was a prospective study, performed at Medical University Hospital No. 5 in łódź. Twenty patients with sepsis and severe sepsis were included in the study. N-terminal brain natriuretic propeptide, procalcitonin and C-reactive protein concentrations, and survival were evaluated. In the whole studied group (128 measurements), the mean NT-proBNP, procalcitonin and C-reactive protein concentrations were, respectively: 140.80±84.65 pg/ml, 22.32±97.41 ng/ml, 128.51±79.05 mg/l. The correlations for the NT-proBNP level and procalcitonin and C-reactive protein levels were 0.3273 (p<0.001) and 0.4134 (p<0.001), respectively. NT-proBNP levels correlate with PCT and CRP levels in septic patients. In the survivor subgroup, the mean NT-proBNP plasma concentrations were significantly lower than in the non-survivor subgroup.     

Cognitive disorder in brain concussion

This paper presented original study results concerning the prevalence and clinical characteristics of cognitive impairment associated with brain concussion. The cognitive functions of 80 consecutive patients （mean age = 37.40±1l.74years; 50 men and 30 women） admitted to the hospital with brain concussions were evaluated. Their cognitive scores were compared with 40 age- and education-matched healthy volunteers without history of cranial trauma. Cognitive impairment without dementia was found in 93% of the patients. Cognitive impairment in brain concussion was also characterized by prominent cognitive slowness （bradyphrenia）, concentration decrease, free recall insufficiency, and visual-spatial dysfunction. Age and severity of anxiety significantly influence the cognitive performance of patients.     

Biosynthesis of brain 2-phenylethylamine: influence of decarboxylase inhibitors and D-amphetamine

2-Phenylethylamine (PEA) is an endogenous brain amine which probably modulates affective behavior. Using a gas-liquid chromatographic method for the quantification of PEA (as its dinitrophenyl-sulfonic acid derivative), we found in rabbits 340.9 ± 45.8 ng of PEA/g of wet brain. Brain PEA levels were markedly decreased by the ip administration of 200 mg/Kg, 4 hrs before sacrifice, of the L-aromatic amino acid decarboxylase inhibitors α-methyldopa (28.2 ± 5.1 ng/g), L-α-methyldopa hydrazine (MK-486 [66.9 ± 13.0 ng/g]) or a combination of both (30.0 ± 3.3 ng/g). Since MK-486 inhibits only peripheral decarboxylase, brain PEA must be in part of peripheral origin. Another decarboxylase inhibitor, RO 4-4602 mg/Kg, 4 hrs before sacrifice) failed to affect brain PEA content. D-amphetamine (10 mg/Kg) induced a small depletion of PEA after 30 min in untreated animals; when given in combination with RO 4-4602, brain PEA content was markedly decreased. This supports the view that amphetamine releases PEA and stimulates its synthesis.     

The efficacy of RGD modified liposomes loaded with vinorelbine plus tetrandrine in treating resistant brain glioma

Brain glioma is one of the most common and devastating intracranial malignancies with a high mortality. Chemotherapy for brain glioma is not ideal due to blood brain barrier (BBB) and multidrug resistance (MDR). The objectives of the present study were to develop a kind of RGD (Arg-Gly-Asp) tripeptide modified vinorelbine plus tetrandrine liposomes to achieve BBB transportation, MDR reversion and glioma cell targeting simultaneously. The studies were performed on glioma cells, resistant glioma cells and glioma-bearing mice. Results showed that the constructed liposomes with suitable physicochemical properties could significantly enhance the transport across BBB, obviously accumulate in glioma cells, and exhibit evident capabilities in diminishing brain glioma in mice. Action mechanism studies indicated that the enhanced anticancer efficacy could be attribute to the follows: prolonged elimination half-life (7.093?±?1.311?h); increased AUC_0–24?h (28.92?±?2.66?mg/L*h); transporting across BBB; enhanced cellular uptake; down-regulation on P-gp (0.49?±?0.06 fold); inducing apoptosis via activating caspase 8, 9, and 3 (2.40?±?0.22, 3.57?±?0.29, and 4.33?±?0.30 folds, respectively). In conclusion, the RGD modified vinorelbine plus tetrandrine liposomes may offer a promising therapeutic strategy for treatment of brain glioma.     

Characterization of a voltage-dependent L-type calcium channel from rabbit and turtle brain

The binding of [³H]nitrendipine to membrane preparation from turtle and rabbit brain was studied. A single population of [³H]nitrendipine binding sites was detected in both species. [³H]nitrendipine bound with high affinity to brain membrane from both rabbit and turtle, revealing a significant population of binding sites (K _d values of 0.55±0.05 nM and 0.56±0.04 nM and B_max values of 122±11 and 275±18 fmol/mg of protein, respectively). Displacement studies showed a similar order of potency of various unlabeled ligands against [³H]nitrendipine both in rabbit or in turtle: nitrendipine > nifedipine ≥ nicardipine ≫ verapamil ≥ diltiazem. Our results show that a two fold increment of [³H]nitrendipine binding sites exists in the turtle brain respect to the rabbit.     

Simultaneous determination of mechanical properties and physiologic parameters in living rat brain

Mechanical properties were determined in living adult rat brain. Reconstructed magnetic resonance images of the rat brain before and after 2 mm compression with a 4.06 mm diameter vinyl screw showed the total volumetric strain was maximal at the site of indentation. The pressure response to stepwise brain compression showed a linear relationship to the point of respiratory compromise. Instrumented indentation was performed on live brain with intact dura using a 4-mm-diameter flat punch indenter to a maximum depth 1.2 mm at loading–unloading rates not exceeding 0.34 N/min and 1.2 mm/min. The calculated elastic modulus showed no consistent change after death. Creep deformation over 15 s was 7.86±1.6% in live brain and 27.0±0.24% after death. In constant multicycle indentation, displacement from 1st to 10th cycle increased 8.0±1.7% in life and 12.9±2.8% after death. The results suggest that elastic properties of rat intracranial contents do not change immediately after death, while changes in the viscous properties are substantial. The process of measuring these properties can alter physiological parameters.     

Selenium and mercury are redistributed to the brain during viral infection in mice

As part of the general host response to coxsackievirus B3 (CB3) infection, the concentration of essential and nonessential trace elements changes in different target organs of the infection. Essential (e.g., Se) and nonessential (e.g., Hg) trace elements are known to interact and affect inflammatory tissue lesions induced by CB3 infection. However, it is unknown whether these changes involve the brain. In the present study, the brain Hg and Se contents were measured through inductively coupled plasma-mass spectrometry and their distribution investigated by means of nuclear microscopy in the early phase (d 3) of CB3 infection in normally fed female Balb/c mice. Because of the infection, the concentration of Hg (4.07±0.46 ng/g wet wt) and Se (340±16 ng/g wet wt) in the brain increased twofold for Hg (8.77±1.65 ng/g wet wt, p<0.05) and by 36% for Se (461±150 ng/g wet wt, ns). Nuclear microscopy of brain sections from mice having elevated Se and Hg concentrations failed to find localized levels of the elements high enough to make detection possible, indicating approximately homogeneous tissue distribution. Although the pathophysiological interpretation of these findings requires further research, the increase of Hg in the brain during infection might have an influence on the pathogenesis of the disease.     

Kinin-forming activity in rat brain

The present study shows that rat brain contains a kinin-forming activity which is distinguishable from plasma kallikrein. Kinin-forming activity was found in an acetone powder of frozen brain tissue (between 27 and 175.5 ng generated bradykinin/g fresh brain tissue/h). Analysis by high pressure liquid chromatography (HPLC) indicated that the kinin formed chromatographed like true bradykinin (BK). After subcellular fractionation using differential centrifugation of homogenized fresh brain tissue the kinin-forming activity was found mainly in a microsomal (P-3) fraction after preincubation with 2 μM melittin. Further fractionation of P-3 fraction using discontinuous sucrose gradient centrifugation identified activity in both the 1 M sucrose layer (5.8 ± 3.1 ng kinin/mg protein/h) and at the interface between the 0.8 and 0.3 sucrose layers (9.4 ± 4 ng kinin/mg protein/h). Melittin pretreatment did not change these values. The distribution pattern of the kallikrein-like activity was different from that of cathepsin d-like acid protease. The two kinin-forming activities were equally sensitive to treatment with various trypsin inhibitors but were clearly distinguishable from plasma kallikrein: brain activity was inhibited completely by Trasylol but not by soybean trypsin inhibitor (SBTI) or ovomucoid while plasma kallikrein was completely inhibited by SBTI and partially by ovomucoid and Trasilol. Our results clearly distinguish between plasma kallikrein, brain cathepsin d-like acid protease activity and an apparent brain kinin-forming activity, but do not by themselves establish a central biosynthetic pathway for kinin generation.     

Electroencephalographic markers of the spontaneous recovery of the vertical posture in patients with consequences of traumatic brain injury

Nine patients (mean age, 23.6 ± 3.15 years) with a severe traumatic brain injury (TBI) associated with a loss of the ability to maintain vertical posture were enrolled in a comprehensive clinical and electroencephalographic study during the spontaneous recovery of vertical posture (VP). The patients were divided into three groups according to their functional deficit assessed on the international MPAI, FIM, and MMSE scales, which was determined by the severity of the TBI. The EEG data were compared to those of ten healthy subjects (mean age, 22.8 ± 0.67 years). In patients with a moderate impairment of the brain and a rapid recovery of VP (over two weeks), a change in the sitting position revealed EEG signs similar to reactive EEG restructurings of healthy individuals during the maintenance of VP in the form of a predominant EEG coherence increase in the right hemisphere for most of the rhythm bands; however, at this stage of VP recovery, the EEG restructurings retained pathological signs. In patients with a more severe functional deficit, spontaneous recovery of VP was accompanied by hyperreactive EEG restructurings for all rhythm bands without regional specificity. This finding showed up irrespective of the lateralization of the brain injury, which could be considered as a marker of positive dynamics of VP recovery. In patients with the most severe impairment and functional deficit and long-term (more than three months) absence of spontaneous VP recovery, the absence of reactive EEG restructurings was revealed in their passive verticalization, which could be used as a marker of negative prognosis.     

Rat and mouse brain histamine N-methyltransferase: modulation by methylated indoleamines

A purification procedure for rat and mouse brain histamine N-methyltransferase (HMT, EC 2.1.1.8) is described which achieves the preparation of 87-fold purified rat brain and 166-fold purified mouse brain enzyme. The purified HMT (MW 29,000) is inhibited by a number of physiologically and pharmacologically active amines, among them several methylated indoleamines, at concentrations above 5 ± 10^-6M. At concentrations below 1 ± 10^-7M, most of the methylated indoleamines stimulate HMT , provided histamine is maintained at, or close to, its optimal concentration as an HMT substrate, namely 1 ± 10^-5M. A study of the nature of the inhibitory process revealed a non-competitive inhibition of HMT by dopamine as against a competitive inhibition of the enzyme by most methylated indoleamines. Increasing the concentration of histamine beyond the optimal value, i.e. to inhibitory levels, resulted in less stimulation. The findings support the notion that MSO elicits the formation in selected brain cells of supranormal amounts of several methylated indoleamines which are able to stimulate HMT (and possibly other methyltransferases, see Salas et al., 1977), thereby causing the depletion of the cerebral levels of S-adenosyl-L-methionine, reported previously (Schatz & Sellinger , 1975b).     

Microglial cells of the rat brain in postnatal period (comparative immunocytochemical analysis)

In the course of the brain’s development, distribution of microglial cells was studied in rats using immunocytochemical detection. To identify the microglial cells, antibodies to lipocortin 1 (LC1) and phosphotyrosine (PT) were used. On postnatal day 1, LC1-positive microglial cells of an ameboid shape were distributed mainly in the subventricular zone; their mean density was 31±8 cells/mm² (counted across the total area of frontal sections). On postnatal day 7, microglial cells of an intermediate type were located throughout the whole brain; their density was 54±15 cells/mm². On the 15th day, LC1-positive cells were of a ramified shape, and their density reached 104±20 cells/mm² (the microglial cell density in the mature normal brain was 103±3 cells/mm²). On postnatal day 7, PT-positive cells were similar in their morphology to LC1-positive cells of an intermediate type, while their mean density was 32 cells/mm². In the mature brain, the density of PT-positive microglia was 53±5 cells/mm²; the shape of the cells in the white and gray matter of the brain was, on the whole, similar to that of LC1-positive microglia. Therefore, LC1 is a specific marker for different types of microglial cells in the developing brain. Our data about 3D distribution and morphological peculiarities of microglial cells at different stages of postnatal development are consistent with the hypothesis on the neuroectodermal origin of microglia.     

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K_D = 25 ± 4 μM and K′_D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?_b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?_b = 3.5 ± 0.4 for tight binding sites and an average ?_b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s^?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.