Effect of mild carboxy-hemoglobin on exercising skeletal muscle: intravascular and intracellular evidence.

We studied muscle blood flow, muscle oxygen uptake (VO(2)), net muscle CO uptake, Mb saturation, and intracellular bioenergetics during incremental single leg knee-extensor exercise in five healthy young subjects in conditions of normoxia, hypoxia (H; 11% O(2)), normoxia + CO (CO(norm)), and 100% O(2) + CO (CO(hyper)). Maximum work rates and maximal oxygen uptake (VO(2 max)) were equally reduced by approximately 14% in H, CO(norm), and CO(hyper). The reduction in arterial oxygen content (Ca(O(2))) (approximately 20%) resulted in an elevated blood flow (Q) in the CO and H trials. Net muscle CO uptake was attenuated in the CO trials. Suprasystolic cuff measurements of the deoxy-Mb signal were not different in terms of the rate of signal rise or maximum signal attained with and without CO. At maximal exercise, calculated mean capillary PO(2) was most reduced in H and resulted in the lowest Mb-associated PO(2). Reductions in ATP, PCr, and pH during H, CO(norm), and CO(hyper) occurred earlier during progressive exercise than in normoxia. Thus the effects of reduced Ca(O(2)) due to mild CO poisoning are similar to H.

Evolving techniques for the investigation of muscle bioenergetics and oxygenation.

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) are both powerful, non-invasive methodologies and, as such, offer great potential to investigate both human biochemistry and human physiology, and ultimately to contribute significantly to the field of medicine. Consequently there has been much effort devoted to fostering the evolution of these methodologies into distinct and applicable techniques. Here we will highlight several MRI and MRS techniques for the assessment of human biochemistry and physiology that ultimately may provide useful clinical assessments and diagnoses of various muscular and cardiovascular pathologies. Specifically, the evolving techniques that will be discussed are: (1) (1)H MRS of myoglobin to assess the intracellular partial pressure of O(2), (2) (31)P MRS to assess metabolic capacity, and (3) the combination of (31)P chemical shift imaging to assess local metabolic demand (oxygen uptake; .VO(2)) with arterial spin labelling to assess local perfusion (blood flow; .Q), in an effort to characterize the elusive spatial matching of skeletal muscle (.Q/.VO(2)).

Skeletal muscle intracellular PO(2) assessed by myoglobin desaturation: response to graded exercise.

The relationship between skeletal muscle intracellular PO(2) (iPO(2)) and progressive muscular work has important implications for the understanding of O(2) transport and utilization. Presently there is debate as to whether iPO(2) falls progressively with increasing O(2) demand or reaches a plateau from moderate to maximal metabolic demand. Thus, using (1)H magnetic resonance spectroscopy of myoglobin (Mb), we studied cellular oxygenation during progressive single-leg knee extensor exercise from unweighted to 100% of maximal work rate in six active human subjects. In all subjects, the Mb peak at 73 ppm was not visible at rest, whereas the peak was small or indistinguishable from the noise in the majority of subjects during progressive exercise from unweighted to 50-60% of maximum work rate. In contrast, beyond this exercise intensity, a Mb peak of consistent magnitude was discernible in all subjects. When a Mb half saturation of 3.2 Torr was used, the calculated skeletal muscle PO(2) was variable before 60% of maximum work rate but in general was relatively high (>18 Torr, the measurable PO(2) with the poorest signal-to-noise ratio, in the majority of cases), whereas beyond this exercise intensity iPO(2) fell to a relatively uniform and invariant level of 3.8 +/- 0.5 Torr across all subjects. These results do not support the concept of a progressive linear fall in iPO(2) across increasing work rates. Instead, this study documents variable but relatively high iPO(2) from rest to moderate exercise and again confirms that from 50-60% of maximum work rate iPO(2) reaches a plateau that is then invariant with increasing work rate.

Preferential incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants.

OBJECTIVE: To determine the metabolic fate of glucosamine (GlcN) in intact articular cartilage tissue. METHODS: Intact articular cartilage explants were cultured for up to 13 days in Dulbecco's modified Eagle's medium supplemented with 1) 1-13C-labeled GlcN, 2) 1-13C-labeled glucose (Glc), or 3) no labeling. Every 3-4 days, samples were removed and frozen in liquid nitrogen for carbon-13 magnetic resonance spectroscopic (MRS) analysis. The metabolic products of the labeled precursors were determined from the MRS data based on resonance positions and comparison with known standards and published values. RESULTS: GlcN was taken up by the chondrocytes and incorporated selectively into the hexosamine, but not the hexuronic acid, components of the glycosaminoglycan chains of articular cartilage proteoglycan. The data also demonstrated that GlcN is the substrate of choice for the galactosamine moieties of the chondroitin sulfates, incorporating at levels 300% higher than with an equivalent amount of labeled Glc. CONCLUSION: The results indicate that GlcN facilitates the production of proteoglycan components that are synthesized through the hexosamine biochemical pathway.

Impaired muscle oxygen transfer in patients with chronic renal failure.

We hypothesized that impaired O2 transport plays a role in limiting exercise in patients with chronic renal failure (CRF). Six CRF patients (25 +/- 6 yr) and six controls (24 +/- 6 yr) were examined twice during incremental single-leg isolated quadriceps exercise. Leg O2 delivery (QO2(leg)) and leg O2 uptake (VO2(leg)) were obtained when subjects breathed gas of three inspired O2 fractions (FI(O2)) (0.13, 0.21, and 1.0). On a different day, myoglobin O2 saturation and muscle bioenergetics were measured by proton and phosphorus magnetic resonance spectroscopy. CRF patients, but not controls, showed O2 supply dependency of peak VO2 (VO2(peak)) by a proportional relationship between peak VO2(leg) at each inspired O2 fraction (0.59 +/- 0.20, 0.47 +/- 0.10, 0.43 +/- 0.10 l/min, respectively) and 1) work rate (933 +/- 372, 733 +/- 163, 667 +/- 207 g), 2) QO(2leg) (0.80 +/- 0.20, 0.64 +/- 0.10, 0.59 +/- 0.10 l/min), and 3) cell PO2 (6.3 +/- 5.4, 1.7 +/- 1.3, 1.2 +/- 0.7 mmHg). CRF patients breathing 100% O2 and controls breathing 21% O2 had similar peak QO2(leg) (0.80 +/- 0.20 vs. 0.79 +/- 0.10 l/min) and similar peak VO2(leg) (0.59 +/- 0.20 vs. 0.57 +/- 0.10 l/min). However, mean capillary PO2 (47.9 +/- 4.0 vs. 38.2 +/- 4.6 mmHg) and the capillary-to-myocite gradient (40.7 +/- 6.2 vs. 34.4 +/- 4.0 mmHg) were both higher in CRF patients than in controls (P < 0.03 each). We conclude that low muscle O2 conductance, but not limited mitochondrial oxidative capacity, plays a role in limiting exercise tolerance in these patients.

17O-decoupled (1)H spectroscopy and imaging with a surface coil: STEAM decoupling.

(17)O-decoupled (1)H spin-echo imaging has been reported as a means of indirect (17)O detection, with potential application to measurement of blood flow and metabolism. In its current form, (17)O decoupling requires large RF amplitudes and a 180 degrees refocusing pulse, complicating its application in volume and surface coils, respectively. To overcome this problem, we have developed an (17)O-decoupled proton stimulated echo sequence ("STEAM decoupling") to allow (17)O detection with a surface coil. A high B(1) amplitude is easily generated, allowing complete decoupling of (17)O and (1)H. Slice-selective, (17)O-decoupled (1)H imaging is readily performed and the sequence is easily adapted for localized spectroscopy. Intrinsic correction for variations in B(1) and further compensation for B(1) inhomogeneity are discussed.

Sodium and proton MR properties of cartilage during compression.

Proton and sodium MR relaxation times of bovine articular cartilage specimens were measured as a function of proteoglycan (PG) depletion and as a function of mechanical compression. Proton and sodium relaxation times of normal cartilage were compared with relaxation times of PG-depleted cartilage to evaluate the significance of PG depletion-induced changes in MR relaxation parameters. These comparisons were conducted for both uncompressed and mechanically compressed states. The mechanical compressions were performed with an MR-compatible pressure cell and evaluated dynamically via interleaved one-dimensional proton and sodium MR projection imaging. The comparisons indicate that sodium relaxation parameters are sensitive to PG depletion when cartilage is in a mechanically compressed state or an uncompressed state. In contrast, proton relaxation parameters do not change significantly with PG depletion when cartilage is in an uncompressed state. However, during mechanical compression, proton T2 becomes sensitive to PG depletion. These results support the potential of sodium magnetic resonance imaging (MRI) as a possible modality for obtaining imaging contrast related to PG depletion. The results also indicate the potential of proton MRI to provide such contrast if the image acquisition is conducted in conjunction with a mechanical compression via physical exercise.J. Magn. Reson Imaging 10:961-967, 1999.

Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise.

Vascular endothelial growth factor (VEGF) is involved in extracellular matrix changes and endothelial cell proliferation, both of which are precursors to new capillary growth. Angiogenesis is a vital adaptation to exercise training, and the exercise-induced reduction in intracellular PO2 has been proposed as a stimulus for this process. Thus we studied muscle cell PO2 [myoglobin PO2 (MbPO2)] during exercise in normoxia and in hypoxia (12% O2) and studied the mRNA levels of VEGF in six untrained subjects after a single bout of exercise by quantitative Northern analysis. Single-leg knee extension provided the acute exercise stimulus: a maximal test followed by 30 min at 50% of the peak work rate achieved in this graded test. Because peak work rate was not affected by hypoxia, the absolute and relative work rates were identical in hypoxia and normoxia. Three pericutaneous needle biopsies were collected from the vastus lateralis muscle, one at rest and then the others at 1 h after exercise in normoxia or hypoxia. At rest (control), VEGF mRNA levels were very low (0.38 +/- 0.04 VEGF/18S). After exercise in normoxia or hypoxia, VEGF mRNA levels were much greater (16.9 +/- 6.7 or 7.1 +/- 1.8 VEGF/18S, respectively). In contrast, there was no measurable basic fibroblast growth factor mRNA response to exercise at this 1-h postexercise time point. Magnetic resonance spectroscopy of myoglobin confirmed a reduction in MbPO2 in hypoxia (3.8 +/- 0.3 mmHg) compared with normoxia (7.2 +/- 0.6 mmHg) but failed to reveal a relationship between MbPO2 during exercise and VEGF expression. This VEGF mRNA increase in response to acute exercise supports the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis but questions the importance of a reduced cellular PO2 as a stimulus for this response.

Cellular PO2 as a determinant of maximal mitochondrial O(2) consumption in trained human skeletal muscle.

Previously, by measuring myoglobin-associated PO(2) (P(Mb)O(2)) during maximal exercise, we have demonstrated that 1) intracellular PO(2) is 10-fold less than calculated mean capillary PO(2) and 2) intracellular PO(2) and maximum O(2) uptake (VO(2 max)) fall proportionately in hypoxia. To further elucidate this relationship, five trained subjects performed maximum knee-extensor exercise under conditions of normoxia (21% O(2)), hypoxia (12% O(2)), and hyperoxia (100% O(2)) in balanced order. Quadriceps O(2) uptake (VO(2)) was calculated from arterial and venous blood O(2) concentrations and thermodilution blood flow measurements. Magnetic resonance spectroscopy was used to determine myoglobin desaturation, and an O(2) half-saturation pressure of 3.2 Torr was used to calculate P(Mb)O(2) from saturation. Skeletal muscle VO(2 max) at 12, 21, and 100% O(2) was 0.86 +/- 0.1, 1.08 +/- 0.2, and 1.28 +/- 0.2 ml. min(-1). ml(-1), respectively. The 100% O(2) values approached twice that previously reported in human skeletal muscle. P(Mb)O(2) values were 2.3 +/- 0.5, 3.0 +/- 0.7, and 4.1 +/- 0.7 Torr while the subjects breathed 12, 21, and 100% O(2), respectively. From 12 to 21% O(2), VO(2) and P(Mb)O(2) were again proportionately related. However, 100% O(2) increased VO(2 max) relatively less than P(Mb)O(2), suggesting an approach to maximal mitochondrial capacity with 100% O(2). These data 1) again demonstrate very low cytoplasmic PO(2) at VO(2 max), 2) are consistent with supply limitation of VO(2 max) of trained skeletal muscle, even in hyperoxia, and 3) reveal a disproportionate increase in intracellular PO(2) in hyperoxia, which may be interpreted as evidence that, in trained skeletal muscle, very high mitochondrial metabolic limits to muscle VO(2) are being approached.

Lactate efflux from exercising human skeletal muscle: role of intracellular PO2.

It remains controversial whether lactate formation during progressive dynamic exercise from submaximal to maximal effort is due to muscle hypoxia. To study this question, we used direct measures of arterial and femoral venous lactate concentration, a thermodilution blood flow technique, phosphorus magnetic resonance spectroscopy (MRS), and myoglobin (Mb) saturation measured by 1H nuclear MRS in six trained subjects performing single-leg quadriceps exercise. We calculated net lactate efflux from the muscle and intracellular PO2 with subjects breathing room air and 12% O2. Data were obtained at 50, 75, 90, and 100% of quadriceps maximal O2 consumption at each fraction of inspired O2. Mb saturation was significantly lower in hypoxia than in normoxia [40 +/- 3 vs. 49 +/- 3% (SE)] throughout incremental exercise to maximal work rate. With the assumption of a PO2 at which 50% of Mb-binding sites are bound with O2 of 3.2 Torr, Mb-associated PO2 averaged 3.1 +/- 0.3 and 2.3 +/- 0.2 Torr in normoxia and hypoxia, respectively. Net blood lactate efflux was unrelated to intracellular PO2 across the range of incremental exercise to maximum (r = 0.03 and 0.07 in normoxia and hypoxia, respectively) but linearly related to O2 consumption (r = 0.97 and 0.99 in normoxia and hypoxia, respectively) with a greater slope in 12% O2. Net lactate efflux was also linearly related to intracellular pH (r = 0.94 and 0.98 in normoxia and hypoxia, respectively). These data suggest that with increasing work rate, at a given fraction of inspired O2, lactate efflux is unrelated to muscle cytoplasmic PO2, yet the efflux is higher in hypoxia. Catecholamine values from comparable studies are included and indicate that lactate efflux in hypoxia may be due to systemic rather than intracellular hypoxia.

Sodium MRI of human articular cartilage in vivo.

Preliminary results from in vivo sodium MRI of human patellar articular cartilage are presented. Sodium images generated of an in vitro bovine patella clearly distinguish the region of proteoglycan depletion from the region of healthy cartilage. This provides the first evidence that sodium imaging may be used to detect changes due to osteoarthritis in vivo. The process of optimizing imaging time and signal-to-noise ratio, as well as potential implications in the detection of osteoarthritic change, are discussed.

A simplified sequence for observing deoxymyoglobin signals in vivo: myoglobin excitation with dynamic unexcitation and saturation of water and fat (MEDUSA).

This paper describes a new, simplified pulse sequence for observing NMR signals from deoxymyoglobin in vivo. Paramagnetically shifted resonances from deoxymyoglobin can be exploited to noninvasively calculate intracellular oxygen tension in striated muscle. However, special sequences are required to observe these weak signals against the larger water and fat signals encountered in vivo. The pulse sequence described here, which is based on inversion recovery sequences, efficiently suppresses both water and fat resonances and can be implemented with short repetition rates. Moreover, it is perfectly suited for studies with surface coils, where RF inhomogeneities render other popular suppression sequences ineffective.

In vivo sodium multiple quantum spectroscopy of human articular cartilage.

The authors report, for the first time, sodium properties of human articular cartilage in vivo using sodium multiple-quantum-filtered methods with a surface coil. A flip angle-independent, phase-cycled pulse sequence was used to obtain triple-quantum-filtered spectra as a function of preparation time. Biexponential relaxation rates were calculated by fitting the triple-quantum-filtered spectral amplitudes to a theoretical expression. Theoretical analysis of the flip angle dependence of even rank two-quantum coherence (T2[2]), odd rank two-quantum coherence (T2[3]), and triple-quantum coherence are presented and verified against experimental results on a cartilage specimen. Sodium multiple-quantum-filtered spectral lineshapes obtained in vivo correlate well with those observed on in vitro specimens. Relaxation rates obtained from asymptomatic volunteers were found to be: T(2rise) = 1.0 + 0.12 ms, T(2decay) = 12.0 +/- 0.75 ms (mean +/- SD). The diagnostic potential of this method in detecting early changes in articular cartilage is described.

Lipid composition changes in normal breast throughout the menstrual cycle.

The detection of breast cancer in women using magnetic resonance imaging (MRI) is increasingly used as a supplement to X-ray mammography. Furthermore, proton MR spectroscopy (1H MRS) has detected alterations in lipid profiles that are linked with tumor development and progression in human biopsy tissue. Because normal "resting" breast is highly active, it is necessary to consider that any alterations observed in lipid profiles may not be indicative of breast tumor development. The purpose of this study was to assess the changes in lipid composition in the breast throughout the menstrual cycle in "normals" using MRS at 4.0 T. Five women with no known history of breast disease were subject to biweekly MRS breast examinations. MRS results showing water and fat resonances revealed cyclic changes in the lipid content throughout the duration of the menstrual cycle. In particular, intensity changes were seen in methylene (-CH2-) and allylic methylene (CH2CH2*CH=) resonances at 2.1 ppm and 1.3 ppm, respectively. These intensity changes assumed a similar cyclic trend for each subject over the 28 days that correlate with the follicular, ovulatory, and luteal phases of the menstrual cycle. The results obtained may indicate cell synthesis or metabolic activity in the breast during the menstrual cycle and provide valuable information pertinent to lipid responses associated with breast disease.

Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport.

The assumption that cellular oxygen pressure (PO2) is close to zero in maximally exercising muscle is essential for the hypothesis that O2 transport between blood and mitochondria has a finite conductance that determines maximum O2 consumption. The unique combination of isolated human quadriceps exercise, direct measures of arterial, femoral venous PO2, and 1H nuclear magnetic resonance spectroscopy to detect myoglobin desaturation enabled this assumption to be tested in six trained men while breathing room air (normoxic, N) and 12% O2 (hypoxic, H). Within 20 s of exercise onset partial myoglobin desaturation was evident even at 50% of maximum O2 consumption, was significantly greater in H than N, and was then constant at an average of 51 +/- 3% (N) and 60 +/- 3% (H) throughout the incremental exercise protocol to maximum work rate. Assuming a myoglobin PO2 where 50% of myoglobin binding sites are bound with O2 of 3.2 mmHg, myoglobin-associated PO2 averaged 3.1 +/- .3 (N) and 2.1 +/- .2 mmHg (H). At maximal exercise, measurements of arterial PO2 (115 +/- 4 [N] and 46 +/- 1 mmHg [H]) and femoral venous PO2 (22 +/- 1.6 [N] and 17 +/- 1.3 mmHg [H]) resulted in calculated mean capillary PO2 values of 38 +/- 2 (N) and 30 +/- 2 mmHg(H). Thus, for the first time, large differences in PO2 between blood and intracellular tissue have been demonstrated in intact normal human muscle and are found over a wide range of exercise intensities. These data are consistent with an O2 diffusion limitation across the 1-5-microns path-length from red cell to the sarcolemma that plays a role in determining maximal muscle O2 uptake in normal humans.

Sensitivity of in vivo MRS of the N-delta proton in proximal histidine of deoxymyoglobin.

The sensitivity of in vivo MRS of the N-delta proton of the proximal histidine of deoxymyoglobin in human skeletal muscles is discussed. Longitudinal relaxation time T1 of this deoxymyoglobin signal was measured in cuffed human forearms at 1.5 T and found to be 9.9 ms. Deoxymyoglobin spectra can be obtained from a forearm in seconds. The detection sensitivity of deoxymyoglobin in fully ischemic skeletal muscles and that of 31P MRS of PCr in normal resting muscles are compared.

In vivo MRS measurement of deoxymyoglobin in human forearms.

In vivo MRS measurement of deoxymyoglobin (deoxy-Mb) in human forearms was performed by observing the N-delta proton of F8 proximal histidine. The concentration of deoxy-Mb reflects the oxygen level in the muscle. In resting muscles, the deoxy-Mb level was below the detection sensitivity. When ischemia was introduced by arterial occlusion, the time-resolved NMR spectra (with 1 min resolution) was recorded to follow the change in the deoxy-Mb signal. The result shows that deoxy-Mb signal builds up and levels off within 6 min. This technique may be used to study bioenergetics of muscle during exercise and under pathological conditions.

Phosphorus 31 nuclear magnetic resonance examination of female reproductive tissues.

Nuclear magnetic resonance spectroscopy is a powerful method of investigating the relationship between metabolism and function in living tissues. We present evidence that the phosphorus 31 spectra of myometrium and placenta are functions of physiologic state and gestational age. Specific spectroscopic abnormalities are observed in association with disorders of pregnancy and gynecologic diseases. Our results suggest that noninvasive nuclear magnetic resonance spectroscopy examinations may sometimes be a useful addition to magnetic resonance imaging examinations, and that nuclear magnetic resonance spectroscopy of biopsy specimens could become a cost-effective method of evaluating certain biochemical abnormalities.