Pharmacokinetics of a single inhalation of hydrogen gas in pigs.

The benefits of inhaling hydrogen gas (H2) have been widely reported but its pharmacokinetics have not yet been sufficiently analyzed. We developed a new experimental system in pigs to closely evaluate the process by which H2 is absorbed in the lungs, enters the bloodstream, and is distributed, metabolized, and excreted. We inserted and secured catheters into the carotid artery (CA), portal vein (PV), and supra-hepatic inferior vena cava (IVC) to allow repeated blood sampling and performed bilateral thoracotomy to collapse the lungs. Then, using a hydrogen-absorbing alloy canister, we filled the lungs to the maximum inspiratory level with 100% H2. The pig was maintained for 30 seconds without resuming breathing, as if they were holding their breath. We collected blood from the three intravascular catheters after 0, 3, 10, 30, and 60 minutes and measured H2 concentration by gas chromatography. H2 concentration in the CA peaked immediately after breath holding; 3 min later, it dropped to 1/40 of the peak value. Peak H2 concentrations in the PV and IVC were 40% and 14% of that in the CA, respectively. However, H2 concentration decay in the PV and IVC (half-life: 310 s and 350 s, respectively) was slower than in the CA (half-life: 92 s). At 10 min, H2 concentration was significantly higher in venous blood than in arterial blood. At 60 min, H2 was detected in the portal blood at a concentration of 6.9-53 nL/mL higher than at steady state, and in the SVC 14-29 nL/mL higher than at steady state. In contrast, H2 concentration in the CA decreased to steady state levels. This is the first report showing that inhaled H2 is transported to the whole body by advection diffusion and metabolized dynamically.

[Different types of molecular hydrogen donors and their pharmacokinetics in vivo].

Molecular hydrogen (H2) has been shown to have diverse biomedical effects. As a small molecular gas, hydrogen can be diffused to the target without hindrance. A variety of related hydrogen products used in medical research and public health have been developed. There are various methods of administration of H2, mainly including inhaling hydrogen gas, drinking hydrogen water, injecting hydrogen-saline, orally taking solid-state H2 sustained-release agents, and stimulating intestinal microbiomes to produce hydrogen. Pharmacokinetics of H2 in vivo vary with methods of administration and thus influence its biomedical effects. This review summarizes the types of H2 donors and their pharmacokinetics in vivo.

Hydrogen gas distribution in organs after inhalation: Real-time monitoring of tissue hydrogen concentration in rat.

Hydrogen has therapeutic and preventive effects against various diseases. Although animal and clinical studies have reported promising results, hydrogen distribution in organs after administration remains unclear. Herein, the sequential changes in hydrogen concentration in tissues over time were monitored using a highly sensitive glass microsensor and continuous inhalation of 3% hydrogen gas. The hydrogen concentration was measured in the brain, liver, kidney, mesentery fat and thigh muscle of rats. The maximum concentration, time to saturation, and other measurements representing the dynamics of distribution were obtained from the concentration curves, and the results obtained for different organs were compared. The time to saturation was significantly longer (20.2 vs 6.3-9.4 min. P = 0.004 in all cases) and increased more gradually in muscle than in the other organs. The maximum concentration was the highest in liver and the lowest in the kidney (29.0 ± 2.6 vs 18.0 ± 2.2 µmol/L; P = 0.03 in all cases). The concentration varied significantly depending on the organ (P = 0.03). These results provide the fundamentals for elucidating the mechanisms underlying the in vivo favourable effects of hydrogen gas in mammalian systems.

Changes in IL-4 and IL-13 expression in allergic-rhinitis treated with hydrogen-rich saline in guinea-pig model

Background: Medical gas hydrogen (H2) has a special role in airway inflammation; however, the effect of H2on allergic rhinitis (AR) remains unclear. This study explored the possible roles of H2 on the pathogenesis of AR and observed the influences of H2 on cytokines IL-4 and IL-13. Methods: An AR guinea pig model was established by nasal ovalbumin sensitisation. Eighteen guinea pigs were divided into three groups, namely, saline control, AR-sensitised, and hydrogen-rich saline (HRS)-treated groups, with each group having six guinea pigs. The frequencies of sneezing and scratching were recorded. The IgE level and cytokine (IL-4 and IL-13) levels in the serum were measured. The expression levels of IL-4 and IL-13 mRNA and protein in the nasal mucosa were also determined by real-time reverse transcriptase-polymerase chain reaction and Western blot. We also observed the infiltration of cytokine (IL-4 and IL-13) in nasal mucosa by immunofluorescence. Results: The frequencies of sneezing and scratching, as well as the levels of IgE, IL-4, and IL-13, in the serum were higher in the AR group than in the control group (p < 0.01), whereas all these parameters were decreased significantly after HRS treatment (p < 0.05). The expression levels of IL-4 and IL-13 mRNA and protein in the nasal mucosa were also lower in guinea pigs treated with HRS than those in the AR group (p < 0.05). Conclusions: HRS could affect anti-inflammation in AR and decreased the expression of IL-4 and IL-13 (AU)

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Immersing lungs in hydrogen-rich saline attenuates lung ischaemia-reperfusion injury.

Objectives: Anti-oxidant effects of hydrogen have been reported in studies examining ischaemia-reperfusion injury (IRI). In this study, we evaluated the therapeutic efficacy of immersing lungs in hydrogen-rich saline on lung IRI. Methods: Lewis rats were divided into three groups: (i) sham, (ii) normal saline and (iii) hydrogen-rich saline. In the first experiment, the left thoracic cavity was filled with either normal saline or hydrogen-rich saline for 1 h. Then, we measured the hydrogen concentration in the left lung using a sensor gas chromatograph ( N = 3 per group). In the second experiment, lung IRI was induced by occlusion of the left pulmonary hilum for 1 h, followed by reperfusion for 3 h. During the ischaemic period, the left thoracic cavity was filled with either normal saline or hydrogen-rich saline. After reperfusion, we assessed lung function, histological changes and cytokine production ( N = 5-7 per group). Results: Immersing lungs in hydrogen-rich saline resulted in an elevated hydrogen concentration in the lung (6.9 ± 2.9 µmol/1 g lung). After IRI, pulmonary function (pulmonary compliance and oxygenation levels) was significantly higher in the hydrogen-rich saline group than in the normal saline group ( P < 0.05). Similarly, pro-inflammatory cytokine levels (interleukin-1ß and interleukin-6) in the left lung were significantly lower in the hydrogen-rich saline group than in the normal saline group ( P < 0.05). Conclusions: Immersing lungs in hydrogen-rich saline delivered hydrogen into the lung and consequently attenuated lung IRI. Hydrogen-rich solution appears to be a promising approach to managing lung IRI.

Protective effects of hydrogen-rich medium on lipopolysaccharide-induced monocytic adhesion and vascular endothelial permeability through regulation of vascular endothelial cadherin.

We observed the effect of hydrogen-rich medium on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs), hyaline leukocyte conglutination, and permeability of the endothelium. Endotheliocytes were inoculated on 6-well plates and randomly divided into 4 groups: control, H2, LPS, LPS+H2, H2, and LPS+H2 in saturated hydrogen-rich medium. We applied Wright's stain-ing to observe conglutination of hyaline leukocytes and HUVECs, flow cytometry to determine the content of vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), enzyme-linked immunosorbent assay to measure the E-selectin concentration in the cell liquor, the transendothelial electrical resistance (TEER) to test the permeability of endothelial cells, and Western blot and immunofluorescence to test the expression and distribution of vascular endothelial (VE)-cadherin. Compared with control cells, there was an increase in endothelium-hyaline leukocyte conglutination, a reduction in VCAM-1, ICAM-1, and E-selectin, and the TEER value increased obviously. Compared with LPS, there was an obvious reduction in the conglutination of LPS+H2 cells, a reduction in VCAM-1, ICAM-1, and E-selectin levels, and a reduction in the TEER-resistance value, while the expression of VE-cadherin increased. Fluorescence results showed that, compared with control cells, the VE-cadherin in LPS cells was in-complete at the cell joints. Compared with LPS cells, the VE-cadherin in LPS+H2 cells was even and complete at the cell joints. Liquid rich in hydrogen could reduce LPS-induced production of adhesion molecules and endothelium-hyaline leukocyte conglutination, and influence the expression and distribution of VE-cadherin to regulate the permeability of the endothelium.

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury.

Maternal inflammation is associated with spontaneous preterm birth and respiratory impairment among premature infants. Recently, molecular hydrogen (H2) has been reported to have a suppressive effect on oxidative stress and inflammation. The aim of this study was to evaluate the effects of H2 on fetal lung injury caused by maternal inflammation. Cell viability and the production of interleukin-6 (IL-6) and reactive oxygen species (ROS) were examined by treatment with lipopolysaccharide (LPS) contained in ordinal or H2-rich medium (HM) using a human lung epithelial cell line, A549. Pregnant Sprague Dawley rats were divided into three groups: Control, LPS, and HW + LPS groups. Rats were injected with phosphate-buffered saline (Control) or LPS intraperitoneally (LPS) on gestational day 19 and provided H2 water (HW) ad libitum for 24 h before LPS injection (HW + LPS). Fetal lung samples were collected on day 20, and the levels of apoptosis, oxidative damage, IL-6, and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry. The number of apoptotic cells, and levels of ROS and IL-6 were significantly increased by LPS treatment, and repressed following cultured with HM in A549 cells. In the rat models, the population positive for cleaved caspase-3, 8-hydroxy-2'-deoxyguanosine, IL-6, and VEGF was significantly increased in the LPS group compared with that observed in the Control group and significantly decreased in the HW + LPS group. In this study, LPS administration induced apoptosis and oxidative damage in fetal lung cells that was ameliorated by maternal H2 intake. Antenatal H2 administration may decrease the pulmonary mobility associated with inflammation in premature infants.

Estimation of the hydrogen concentration in rat tissue using an airtight tube following the administration of hydrogen via various routes.

Hydrogen exerts beneficial effects in disease animal models of ischemia-reperfusion injury as well as inflammatory and neurological disease. Additionally, molecular hydrogen is useful for various novel medical and therapeutic applications in the clinical setting. In the present study, the hydrogen concentration in rat blood and tissue was estimated. Wistar rats were orally administered hydrogen super-rich water (HSRW), intraperitoneal and intravenous administration of hydrogen super-rich saline (HSRS), and inhalation of hydrogen gas. A new method for determining the hydrogen concentration was then applied using high-quality sensor gas chromatography, after which the specimen was prepared via tissue homogenization in airtight tubes. This method allowed for the sensitive and stable determination of the hydrogen concentration. The hydrogen concentration reached a peak at 5 minutes after oral and intraperitoneal administration, compared to 1 minute after intravenous administration. Following inhalation of hydrogen gas, the hydrogen concentration was found to be significantly increased at 30 minutes and maintained the same level thereafter. These results demonstrate that accurately determining the hydrogen concentration in rat blood and organ tissue is very useful and important for the application of various novel medical and therapeutic therapies using molecular hydrogen.

Protective effect of hydrogen-rich saline on ischemia/reperfusion injury in rat skin flap.

OBJECTIVE: Skin damage induced by ischemia/reperfusion (I/R) is a multifactorial process that often occurs in plastic surgery. The mechanisms of I/R injury include hypoxia, inflammation, and oxidative damage. Hydrogen gas has been reported to alleviate cerebral I/R injury by acting as a free radical scavenger. Here, we assessed the protective effect of hydrogen-rich saline (HRS) on skin flap I/R injury. METHODS: Abdominal skin flaps of rats were elevated and ischemia was induced for 3 h; subsequently, HRS or physiological saline was administered intraperitoneally 10 min before reperfusion. On postoperative Day 5, flap survival, blood perfusion, the accumulation of reactive oxygen species (ROS), and levels of cytokines were evaluated. Histological examinations were performed to assess inflammatory cell infiltration. RESULTS: Skin flap survival and blood flow perfusion were improved by HRS relative to the controls. The production of malondialdehyde (MDA), an indicator of lipid peroxidation, was markedly reduced. A multiplex cytokine assay revealed that HRS reduced the elevation in the levels of inflammatory cytokines, chemokines and growth factors, with the exception of RANTES (regulated on activation, normal T-cell expressed and secreted) growth factor. HRS treatment also reduced inflammatory cell infiltration induced by I/R injury. CONCLUSIONS: Our findings suggest that HRS mitigates I/R injury by decreasing inflammation and, therefore, has the potential for application as a therapy for improving skin flap survival.

Hydrogen inhalation ameliorated mast cell-mediated brain injury after intracerebral hemorrhage in mice.

OBJECTIVE: Hydrogen inhalation was neuroprotective in several brain injury models. Its mechanisms are believed to be related to antioxidative stress. We investigated the potential neurovascular protective effect of hydrogen inhalation especially effect on mast cell activation in a mouse model of intracerebral hemorrhage. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: One hundred seventy-one 8-week-old male CD-1 mice were used. INTERVENTIONS: Collagenase-induced intracerebral hemorrhage model in 8-week-old male CD-1 mice was used. Hydrogen was administrated via spontaneous inhalation. The blood-brain barrier permeability and neurologic deficits were investigated at 24 and 72 hours after intracerebral hemorrhage. Mast cell activation was evaluated by Western blot and immuno-staining. The effects of hydrogen inhalation on mast cell activation were confirmed in an autologous blood injection model intracerebral hemorrhage. MEASUREMENT AND MAIN RESULTS: At 24 and 72 hours post intracerebral hemorrhage, animals showed blood-brain barrier disruption, brain edema, and neurologic deficits, accompanied with phosphorylation of Lyn kinase and release of tryptase, indicating mast cell activation. Hydrogen treatment diminished phosphorylation of Lyn kinase and release of tryptase, decreased accumulation and degranulation of mast cells, attenuated blood-brain barrier disruption, and improved neurobehavioral function. CONCLUSION: Activation of mast cells following intracerebral hemorrhage contributed to increase of blood-brain barrier permeability and brain edema. Hydrogen inhalation preserved blood-brain barrier disruption by prevention of mast cell activation after intracerebral hemorrhage.

Comparison of CCD-equipped laser speckle flowgraphy with hydrogen gas clearance method in the measurement of optic nerve head microcirculation in rabbits.

The aim of this study was to verify the correlation between mean blur rate (MBR) obtained with CCD-equipped laser speckle flowgraphy (LSFG) and capillary blood flow (CBF) obtained by the hydrogen gas clearance method in rabbit optic nerve head (ONH). Using Japanese white rabbits under systemic anesthesia, a hydrogen electrode was inserted an area of the ONH free from superficial capillaries. MBR was measured with LSFG near the hydrogen electrode. CBF and MBR were measured in the range of 32.4-83.5 mL/min/100 g and 3.5-6.0, respectively. MBR and CBF were significantly correlated (r = 0.73, P < 0.01, n = 14). After inhalation of carbon dioxide (CO(2)) or intravenous administration of endothelin-1 (ET-1), MBR and CBF were changed in the relative range of 0.74-1.27 and 0.76-1.35, respectively. The relative changes in MBR and CBF induced by CO(2) and ET-1 were also significantly correlated (r = 0.67, P < 0.01). The current results suggest that MBR may correlate with CBF and also change with CBF, as an index of blood flow in the ONH, linearly.

²H kinetic isotope effects and pH dependence of catalysis as mechanistic probes of rat monoamine oxidase A: comparisons with the human enzyme.

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A. The pH profile of k(cat) for rat MAO A shows a pK(a) of 8.2 ± 0.1 for the benzylamine ES complex and pK(a) values of 7.5 ± 0.1 and 7.6 ± 0.1 for the ES complexes with p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine, respectively. In contrast to the human enzyme, the rat enzyme exhibits a single pK(a) value (8.3 ± 0.1) with k(cat)/K(m) for benzylamine versus pH and pK(a) values of 7.8 ± 0.1 and 8.1 ± 0.2 for the ascending limbs, respectively, of k(cat)/K(m) versus pH profiles for p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine and 9.3 ± 0.1 and 9.1 ± 0.2 for the descending limbs, respectively. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A has large deuterium kinetic isotope effects on k(cat) and on k(cat)/K(m). These effects are pH-independent and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log k(cat) with the electronic substituent parameter (σ) at pH 7.5 and 9.0 show a dominant contribution with positive ρ values (1.2-1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues for rat MAO A shows an increased van der Waals volume (V(w)) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits functional properties similar but not identical with those of the human enzyme and provide additional support for C-H bond cleavage via a polar nucleophilic mechanism.

Kinetic study of the prooxidant effect of alpha-tocopherol. Hydrogen abstraction from lipids by alpha-tocopheroxyl radical.

A kinetic study of the prooxidant effect of alpha-tocopherol was performed. The rates of allylic hydrogen abstraction from various unsaturated fatty acid esters (ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) by alpha-tocopheroxyl radical in toluene were determined, using a double-mixing stopped-flow spectrophotometer. The second-order rate constants (k (p)) obtained are <1 x 10(-2) M(-1 )s(-1) for 1, 1.90 x 10(-2) M(-1 )s(-1) for 2, 8.33 x 10(-2 )M(-1 )s(-1) for 3, 1.92 x 10(-1) M(-1 )s(-1) for 4, and 2.43 x 10(-1 )M(-1 )s(-1) for 5 at 25.0 degrees C. Fatty acid esters 3, 4, and 5 contain two, four, and six -CH(2)- hydrogen atoms activated by two pi-electron systems (-C=C-CH(2)-C=C-). On the other hand, fatty acid ester 2 has four -CH(2)- hydrogen atoms activated by a single pi-electron system (-CH(2)-C=C-CH(2)-). Thus, the rate constants, k (abstr)/H, given on an available hydrogen basis are k (p)/4 = 4.75 x 10(-3 )M(-1 )s(-1) for 2, k (p)/2 = 4.16 x 10(-2) M(-1 )s(-1) for 3, k (p)/4 = 4.79 x 10(-2 )M(-1 )s(-1) for 4, and k (p)/6 = 4.05 x 10(-2 )M(-1 )s(-1) for 5. The k (abstr)/H values obtained for 3, 4, and 5 are similar to each other, and are by about one order of magnitude higher than that for 2. From these results, it is suggested that the prooxidant effect of alpha-tocopherol in edible oils, fats, and low-density lipoproteins may be induced by the above hydrogen abstraction reaction.

Measurement of bone blood flow using the hydrogen washout technique-part II: Validation by comparison to microsphere entrapment.

Accurate and reproducible measurement of bone blood flow has important clinical and experimental applications. Hydrogen washout is simple, safe, and widely used, but its use in bone tissue has not been validated. To this end, we have compared cortical bone blood flow measurements obtained by radioactive-labeled microsphere entrapment with those from hydrogen washout. Blood flow was measured in tibial cortical bone of 12 New Zealand White rabbits by radioactive microsphere entrapment and by hydrogen washout. Besides a control group (n = 6), four animals were treated with systemic epinephrine (0.8 microg/kg/min) (group 2) and two with nitroprusside (100 microg/kg/min) (group 3). Furthermore, nine femora from seven rats were isolated on their vascular pedicles and repeated bone blood flow measurements were made at each location with the hydrogen washout method to confirm reproducibility of blood flow determinations by hydrogen washout. An average flow of 2.3 +/- 2.0 mL/min/100 g was obtained with the microsphere method and 2.0 +/- 0.5 mL/min/100 g with the hydrogen washout method. There was a significant correlation and agreement: R(2) = 0.97 (p < 0.01). No consistent flow variations were found with systemic vasoactive drug administration. Hydrogen washout provided reproducible results and showed high sensitivity to flow changes. Hydrogen washout is both sensitive and reproducible in measuring bone blood flow. Results agree well with flow values obtained by labeled microsphere entrapment.

Measurement of bone blood flow using the hydrogen washout Technique-Part I: quantitative evaluation of tissue perfusion in the laboratory rat.

The measurement of blood flow in bone is of interest in both clinical and experimental settings. Such determinations would ideally provide accurate, quantitative, and reproducible data with relatively simple and safe technology, even in the small bones of experimental animals. Methods that provide absolute or quantitative measurements include positron emission tomography, "isotope fractioning" using radioactive or fluorescent-labeled microspheres, and measurement of the rate of washout of diffusible tracers delivered either by injection or inhalation. In this article, we describe in detail a modification of the original Whiteside hydrogen washout technique, using modern hydrogen sensors, a micromanipulator for probe placement, and custom software that greatly simplifies blood flow determination and is effective in the small bones of experimental animals.

Carbon nanoscrolls: a promising material for hydrogen storage.

A multiscale theoretical approach was used for the investigation of hydrogen storage in the recently synthesized carbon nanoscrolls. First, ab initio calculations at the density functional level of theory (DFT) were performed in order to (a) calculate the binding energy of H2 molecules at the walls of nanoscrolls and (b) fit the parameters of the interatomic potential used in Monte Carlo simulations. Second, classical Monte Carlo simulations were performed for estimating the H2 storage capacity of "experimental size" nanoscrolls containing thousands of atoms. Our results show that pure carbon nanoscrolls cannot accumulate hydrogen because the interlayer distance is too small. However, an opening of the spiral structure to approximately 7 A followed by alkali doping can make them very promising materials for hydrogen storage application, reaching 3 wt % at ambient temperature and pressure.

Hydrogen breath test for diagnosis of lactose malabsorption: the importance of timing and the number of breath samples.

BACKGROUND: The hydrogen breath test (H(2)BT) is the most widely used procedure in the diagnostic workup of lactose malabsorption and lactose intolerance. AIM: To establish whether a simplified two- or three-sample test may reduce time, costs and staff resources without reducing the sensitivity of the procedure. PATIENTS AND METHODS: Data from 1,112 patients (292 men, 820 women) with a positive 4 h, nine-sample H(2)BT were retrospectively analyzed. Patients were stratified according to the degree of lactose malabsorption, the occurrence and type of symptoms. Loss of sensitivity in the procedure was evaluated taking into account two-sample tests (0 min and 120 min or 0 min and 210 min) or three-sample tests (0 min, 120 min and 180 min or 0 min, 120 min and 210 min). RESULTS: Using a two-sample test (0 min and 120 min or 0 min and 210 min) the false-negative rate was 33.4% and 22.7%, respectively. With a three-sample test (0 min, 120 min and 180 min or 0 min, 120 min or 210 min), lactose malabsorption was diagnosed in 91.2% (1,014 of 1,112) patients and in 96.1% (1,068 of 1,112) patients, respectively. Of 594 patients with abdominal symptoms, 158 (26.6%) and 73 (12.2%) would have false-negative results with 0 min and 120 min or 0 min and 210 min two-sample tests, respectively. The three-sample tests, 0 min, 120 min and 180 min or 0 min, 120 min and 210 min, have a false-negative rate of 5.9% and 2.1%, respectively. CONCLUSIONS: A three-sample H(2)BT is time- and cost-sparing without significant loss of sensitivity for the diagnosis both of lactose malabsorption and lactose intolerance.

[Non-invasive investigation of muscle function using 31P magnetic resonance spectroscopy and 1H MR imaging]. / Explorations de la fonction musculaire par spectrométrie et imagerie de résonance magnétique.

31P MRS and 1H MRI of skeletal muscle have become major new tools allowing a complete non invasive investigation of muscle function both in the clinical setting and in basic research. The comparative analysis of normal and diseased muscle remains a major requirement to further define metabolic events surrounding muscle contraction and the metabolic anomalies underlying pathologies. Also, standardized rest-exercise-recovery protocols for the exploration of muscle metabolism by P-31 MRS in healthy volunteers as well as in patients with intolerance to exercise have been developed. The CRMBM protocol is based on a short-term intense exercise, which is very informative and well accepted by volunteers and patients. Invariant metabolic parameters have been defined to characterize the normal metabolic response to the protocol. Deviations from normality can be directly interpreted in terms of specific pathologies in some favorable cases. This protocol has been applied to more than 4,000 patients and healthy volunteers over a period of 15 years. On the other hand, MRI investigations provide anatomical and functional information from resting and exercising muscle. From a diagnostic point of view, dedicated pulse sequences can be used in order to detect and quantify muscle inflammation, fatty replacement, muscle hyper and hypotrophy. In most cases, MR techniques provide valuable information which has to be processed in conjunction with traditional invasive biochemical, electrophysiological and histoenzymological tests. P-31 MRS has proved particularly useful in the therapeutic follow-up of palliative therapies (coenzyme Q treatment of mitochondriopathies) and in family investigations. It is now an accepted diagnostic tool in the array of tests which are used to characterize muscle disorders in clinical routine. As a research tool, it will keep bringing new information on the physiopathology of muscle diseases in animal models and in humans and should play a role in the metabolic characterization of gene and cell therapy.

Changes in masseter muscle blood flow during voluntary isometric contraction in humans.

The effect of jaw clenching on local blood flow in the masseter muscle was measured using the hydrogen clearance method in 13 healthy subjects. Sustained isometric masseter-muscle contraction levels of 25 and 50% of maximum voluntary contraction (MVC) were investigated. The blood flow at 25% MVC before contraction, during contraction and after contraction was 12.3 +/- 10.9, 19.2 +/- 12.1 and 78.8 +/- 63.9 mL min(-1) (100 g)(-1) (mean +/- s.d.), respectively. At 50% MVC, it was 14.2 +/- 12.9, 18.6 +/- 10.0 and 80.1 +/- 61.8, respectively. The volume of blood flow was significantly greater after contraction as compared with before contraction at both levels (P < 0.0001) and there was no significant difference between before and during contraction periods (P = 0.17: 25% MVC; P = 0.38: 50% MVC). At 50% MVC blood flow before contraction and the difference in blood flow before and during contraction showed significant negative correlation (r = -0.636, P < 0.02). When the volume of blood flow was low before contraction it tended to increase during contraction and decreased when it was high before contraction. These findings indicate that blood flow in the masseter muscle during sustained isometric contraction is affected by the condition of contraction and may be influenced by the muscle region. It was also indicated that the blood flow during high level contraction was influenced by the volume of blood flow before contraction. Clinically, our findings may help to understand pathological changes which may lead to chronic masticatory muscle pain.