Molecular crowding and viscosity as determinants of translational diffusion of metabolites in subcellular organelles.

The role of molecular crowding and viscosity on the apparent translational diffusion coefficient (ADC) of small metabolites was investigated in different subcellular organelles using the pulse-field gradient spin-echo 1H NMR technique. ADCs of metabolites with increasing radius of gyration (0.7 A < RG < 4.5 A) were measured in the cytoplasm of rat or chicken erythrocytes, in the nucleus of chicken erythrocytes, and in isolated rat liver mitochondria. Metabolite ADCs in these systems were compared with the corresponding ADCs determined in model solutions of increasing bulk viscosity but different molecular crowding. For solutions having the same viscosity, metabolite ADCs decreased with increasing concentration of cosolutes. This effect is adequately described by the modified Stokes-Einstein relationship, ADC = k/RG (1 + 2.5Phi), where k is a constant for a given temperature and Phi is an obstruction factor reporting the fractional volume of solution occupied by cosolutes, a measure of the molecular crowding in the solution. Cytoplasmic values of Phi for metabolites of different sizes did not depend exclusively on metabolite RG but on additional factors including the chemical nature of the metabolite, the presence of diffusional barriers, and metabolite-specific binding sites. In the case of water, nuclear Phi values approached those of the extracellular space while mitochondrial Phi values were significantly higher than those of the cytoplasm. Taken together, these results reveal important differences in molecular crowding within the different subcellular compartments, suggesting considerable diffusional heterogeneity for small metabolites within the different intracellular organelles.

Non-invasive studies of peripheral vascular compliance using a non-occluding photoplethysmographic method.

A non-invasive technique is implemented to measure a peripheral vascular compliance index Cindex, using an infrared photoplethysmographic waveform as an indicator of intravascular volume change and a continuous blood pressure monitor to measure the blood pressure during each heart-beat. The non-linear behaviour of Cindex with pressure and the effect of age on Cindex are studied in 62 males (15-73 years). Repeatability tests and the effect of ice-water exposure of a portion of a limb are studied in 10 and 14 subjects, respectively. For each individual, Cindex measurements are taken at discrete values of local mean arterial pressure (Pmean), and a Cindex against Pmean plot is obtained. There is a statistically significant difference (p < 0.05) in Cindex for the lower values of Pmean (60-100 mmHg) between two age groups formed (15-52 and 58-73 years). The cold-pressor test (CPT) shows a 68% median decrease in Cindex, with an inter-quartile range of 60-77%, in a matter of seconds. The results suggest that Cindex may be a useful noninvasive indicator of peripheral vascular compliance in humans.

Dynamics and environment of mitochondrial water as detected by 1H NMR.

The dynamics and environment of water in suspensions of isolated rat liver mitochondria have been investigated by 1H NMR. NMR longitudinal and transversal relaxation times (T1 and T2) were measured in the resuspension medium (2.65 s and 44.57 ms) and in mitochondrial suspensions (1.74 s and 23.14 s), respectively. Results showed monoexponential relaxation in both cases, suggesting a fast water exchange across the inner mitochondrial membrane. Ferromagnetically induced shift of the extramitochondrial water with nonpermeant ferromagnetic particles revealed no detectable water signal from the intramitochondrial compartment, confirming the fast exchange case. Simulations on a two-compartment model indicated that the intramitochondrial water residence time has an upper limit of approximately 100 microseconds. Calculated intramitochondrial relaxation times revealed that the intramitochondrial environment has an apparent viscosity 30 times larger than the resuspension medium and 15 times larger than the cytosol of erythrocytes. The higher apparent viscosity of the mitochondrial matrix could account for reductions of more than one order of magnitude in the diffusion coefficient of water and other substrates, limitations in the rate of enzymatic reactions which are diffusion controlled and a more favorable formation of multienzyme complexes.