Oliver E. Pratt

Kinetics of thiamine transport across the blood-brain barrier in the rat.

1. By measurement of the rate of disappearance of injected tracer thiamine from the bloodstream, a programme for the continuous injection of thiamine at a variable rate has been devized by which a steady raised level can be achieved rapidly and maintained in the circulation. By this means the flux of radioactive thiamine across the blood‐brain barrier has been measured. 2. In separate experiments progressively higher levels of thiamine were maintained in the bloodstream. Evidence was obtained that the transport of thiamine across the blood‐brain barrier is a carrier‐mediated process which can be saturated by raised levels of thiamine. 3. The saturation of the transport process was incomplete: kinetic analysis showed that there was a non‐saturable component of the transport which was probably due to passive diffusion. 4. The contribution of the non‐saturable component was normally small and is probably insufficient to meet the needs of the brain for the vitamin unless the concentration of the vitamin in the blood is raised considerably above normal. 5. This two‐component transport process had substantially similar kinetic parameters in different regions of the brain.

The influence of brain acetaldehyde on oxidative status, dopamine metabolism and visual discrimination task

The toxic effect of acetaldehyde on brain oxidative capacity and dopamine metabolism has been investigated in rat brains after a single intraperitoneal injection of acetaldehyde (5 mmol/kg) and the results compared with those from chronically ethanol fed rats. Acetaldehyde was present in rat brain 120 hr after a single dose of acetaldehyde, confirming that it is able to cross the blood-brain barrier. Brain catalase increased significantly after acetaldehyde or chronic ethanol administration although there were no other significant changes in the total brain activity of superoxide dismutase, glutathione peroxidase or glutathione reductase. Dopamine turnover was increased in both experimental groups. The acute dose of acetaldehyde reduced the ability of the rats to relearn a computer visual discrimination task.

Decrease in oligodendrocyte carbonic anhydrase activity preceding myelin degeneration in cuprizone induced demyelination.

Both immunohistochemical and biochemical evidence is presented to show for the first time that carbonic anhydrase II (CA II) activity falls in the brain of mice in cuprizone (bis(cyclohexanone)oxalyldihydrazone) induced demyelination well before demyelination develops. This fall began during the first week, whereas the first signs of myelin degeneration induced by cuprizone did not appear until 3 weeks and demyelination in the superior cerebellar peduncle in the mouse took 6-8 weeks to develop. The findings suggest that oligodendrocyte CA II activity is essential either for the survival of oligodendrocytes or for the maintenance of central myelin.

The vasculature of experimental brain tumours: Part 4. The quantification of vascular permeability

In order to quantify changes in vessel permeability seen previously in experimental astrocytomas produced in rats by an intracerebral injection of cultured neoplastic glial cells, the flux of mannitol across the vascular endothelium from the blood into the normal brain or tumour tissue was measured using a specially devised technique by which a steady level of radioactively labelled mannitol can be achieved rapidly and maintained in the bloodstream. This is done by a continuous injection given at a rate which is adjusted by a predetermined programme so as to replace the tracer at the rate at which it has been found to leave the circulation in previous experiments. In separate experiments on both tumour-bearing and control rats steady levels of the tracer were maintained in the circulation for progressively longer times of up to 30 min. The kinetic parameters of the process gave estimates for the apparent transfer constant of mannitol across the vascular endothelium and of the size of the extravascular extracellular mannitol space in the tumours. The apparent transfer constant for the movement of mannitol across the blood-brain barrier was increased more than a hundred-fold in the region of the tumour compared to the values for the brain of control rats or that of tumour-bearing rats remote from the tumour site. The extracellular extravascular space within the tumour was estimated to be 22%, somewhat larger than accepted normal values.

The activation of red blood cell transketolase in groups of patients especially at risk from thiamin deficiency

Erythrocyte transketolase activation by thiamin diphosphate has been studied in elderly patients with moderate or severe chronic dementia, acute alcoholic admissions and chronic alcoholics with evidence of brain damage, mostly of the Wernicke-Korsakoff type. Significantly more patients in each group than controls showed abnormal activation of transketolase, not only by 0.3 mM thiamin diphosphate (TDP) but also in further activation by increase to 3 mM. This indicated the presence in a proportion of the alcoholic and the demented patients of an abnormal enzyme variant, similar to that previously found in vitro. The modified transketolase activation test may warn not only of marginal thiamin deficiency but also independently, of susceptibility to brain damage in patients at risk.

Hyperosmolar Opening of the Blood-Brain Barrier in the Energy-Depleted Rat Brain. Part 1. Permeability Studies

A simple saline perfusion system was used to investigate the effects of hyperosmolar solutions of arabinose and mannitol upon the permeability of the blood-brain barrier. The small, polar molecule [14C]mannitol and the larger, visual marker Evans blue were used as indicators of barrier integrity in the perfused energydepleted brain. One-minute perfusion of hyperosmolar solutions consistently opened the barrier suggesting that the mechanism of osmotic barrier opening is independent of energy-producing metabolism. The accumulation of radiolabel in the brain was expressed as the ratio of tissue to perfusate radioactivity (Rt/Rp) and, for cerebrum, this increased from a control value of 0.0022 ± 0.0007 (mean ± SEM; n = 4) to a value of 0.0124 ± 0.0008 (n = 4) following 0.9 M arabinose and to 0.0495 ± 0.0072 (n = 4) following 1.8 M arabinose. There was a significant reduction of water content of hyperosmolar perfused brains. These findings support the hypothesis that osmotic barrier opening is the result of the passive shrinkage of endothelial cells and the surrounding tissue.

Maintenance of the integrity of the blood-brain barrier in the rat during an in situ saline-based perfusion.

Integrity of the blood-brain barrier to the small polar tracer mannitol was maintained for up to 30 min during an in situ perfusion of the brain with a saline-based solution containing the metabolic inhibitor 2,4-dinitrophenol. The patency of the capillary bed after perfusion was demonstrated by injecting a solution of Indian ink and gelatin, and ultrastructural examination showed the microvasculature to be well preserved. These findings suggest that the blood-brain barrier can be studied under conditions that are independent of normal cerebral function and metabolism.

Valine entry into rat brain after diet-induced changes in plasma amino acids

Abstract: Passage of amino acids across the blood‐brain barrier is modified by the amino acid composition of the blood. Because blood amino acid concentrations respond to changes in protein intake, we have examined associations among diet, plasma amino acid patterns, and the rate of entry of threonine into the brain. Rats were adapted for 8 h/ day for 7–10 days to diets containing 6, 18, or 50% casein before receiving a single, independently varied, final meal of a diet containing 0, 6, 18, or 50% casein. After 4–7 h, they were anesthetized and infused intravenously with [14C]threonine for 5 min before plasma and brain samples were taken for determination of radioactivity and amino acid content. Plasma and brain threonine concentrations decreased as protein content increased in the diets to which the rats had been adapted. Plasma threonine concentrations increased twofold, from 1.6 to 3.0 mM, when rats adapted to 6% casein meals received a single 50% casein meal rather than a nonprotein meal; a fivefold increase, from 0.13 to 0.69 mM, occurred when rats had been previously adapted to 50% casein meals. Increasing the protein content of the final meal did not increase brain threonine concentrations. Highest and lowest rates of threonine entry into the brain occurred, respectively, in rats adapted to 6 and 50% casein meals. Changes in plasma threonine concentrations and threonine flux into brain reflected protein content of both pretreatment and final meals.

Reduced Stability of Rat Brain Transketolase After Conversion to the Apo Form

The stability of rat brain transketolase, whether measured at 37 or 0°C, was reduced after conversion to the apo form by removal of thiamine diphosphate, as shown by a decline in the activity recovered when assayed in the presence of thiamine diphosphate. Both the shape of the breakdown curve and the failure to recover the full activity, even after incubation with thiamine diphosphate, showed that the breakdown of the apotrans‐ketolase was complex. The initial rate of breakdown of the apoenzyme was sharply pH dependent, being minimal at 37°C at a pH value of 7.6, close to that likely to exist in vivo. The rate rose sharply with deviation of the pH in either direction. The stability of the enzyme on storage at 0°C showed a similar pattern of pH dependence, provided that allowance is made for temperature effects on dissociation constants. These findings provide further support for the hypothesis that differences in brain transketolase may play a part in the etiology of Wernicke‐Korsakoffs syndrome.

The effect of dexamethasone on vascular permeability of experimental brain tumours

SummaryThe vessels of experimental gliomas show an abnormally high permeability to small polar molecules, such as mannitol. To establish whether this change in vessel permeability is modified by treatment with the corticosteroid dexamethasone, the kinetics of [14C]mannitol transfer into rat astrocytomas were estimated in both steroid- and saline-treated, tumourbearing animals. This was achieved by injecting [14C]mannitol i.v., using a specially devised technique, so as to maintain a constant concentration of tracer in the blood plasma. In separate experiments steady levels of the tracer were maintained in the circulation from 1 to 30 min. Mean plasma and tumour radioactivity were measured, and the apparent transfer constant of mannitol across the vascular endothelium and the size of the extravascular extracellular mannitol space in the tumours were calculated.Despite a significant clinical improvement in the treated animals and adequate circulating levels of dexamethasone at the time of the permeability studies, no difference in either the apparent transfer constant for the movement of mannitol into the tumours or the fractional extracellular mannitol space was detected between these animals and the controls. With steroid treatment both tumour-bearing and non-tumour bearing animals lost weight, and in the latter there was no consistent change in routine biochemical or haematological parameters. It was concluded that under these conditions it is unlikely that clinical improvement with dexamethasone therapy was due to a non-specific reduction in tumour vessel permeability to polar substances.