Leon D. Braun

KINETICS OF BLOOD‐BRAIN BARRIER TRANSPORT OF PYRUVATE, LACTATE AND GLUCOSE IN SUCKLING, WEANLING AND ADULT RATS

Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age‐groups. Data were analysed in terms of the Michaelis‐Menten equation with a component to allow for non‐saturable diffusion. Values are given for Km, Vmax and Kd. In suckling rats (15‐21 days) the Vmax values for both pyruvate and lactate were 2.0 μmol g−1 min−1. In 28‐day‐old rats the Vmax values had fallen to one‐half and in adults they were less than one‐tenth. Km, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28‐day‐old and adults although there was no difference with age in the Km values.

Carrier mediated blood-brain barrier transport of choline and certain choline analogs.

Blood‐brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a Km= 442 ± 60 μM and Vmax= 10.0 ± 0.6 nmol min‐1 g‐1. In 14–15‐day‐old suckling forebrains a similar Km (= 404 ± 88 μM) but higher Vmax (= 12.5 ± 1.5 nmol min‐1 g‐1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis‐Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex Km= 310 ± 103 μM and Vmax= 12.6 ± 2.8 nmol min‐1g‐1; paleocortex Km= 217 ± 76 μM and Vmax= 7.2 ± 1.5 nmol min‐1 g‐1).

Changes during development in transport processes of the blood-brain barrier

The permeability of the blood-brain barrier to several classes of compounds was studied in rats between the ages of 15 days and 9 weeks. 14C-labelled test substances were injected simultaneously with two reference isotopes, 3H2O and 113mIn-labelled EDTA, into the common carotid artery followed by decapitation 10 s later. There was evidence that a monocarboxylic acid transport system in 15 to 23 day-old rats had a capacity at least six times greater than that present in adult animals. L-Lactate and acetate showed the highest permeability. At all ages there was a constant ratio between L-lactate and (-)D-3-hyroxybutyrate values. D-Glucose permeability increased with age, while that of several amino acids tested was the same in young and adult rats.

Effect of albumin binding and amino acid competition on tryptophan uptake into brain.

Abstract— In the present study we examine the influence of pH, palmitate, and neutral amino acids on the passage of tryptophan from blood into brain during a single capillary pass, and on the partitioning of tryptophan between free and albumin‐bound forms. The results show that a considerable fraction of albumin‐bound tryptophan is stripped from albumin sites during passage, that uptake is concentration‐dependent, and that amino acid competition for carrier sites is quantitatively the most important factor in regulating tryptophan uptake into brain. The interaction between tryptophan concentration, tryptophan binding, and competing amino acids is of considerable influence on brain serotonin biosynthesis.

Dose dependent reduction of glucose utilization by pentobarbital in rat brain.

A new method of determining the rate of glucose utilization in brain regions of individual rats has been used to measure the dose dependency of the reduction of the metabolic activity of the cerebral cortex by pentobarbital. Cerebral cortical glucose utilization is depressed to a basal level of 44% of the control rate when cerebral pentobarbital levels exceed 50 μg per g of tissue. The major portion of this effect occurs between the cerebral pentobarbital range of 10-20 μg per g, which can be achieved by 1/5 to 1/10 the normal anesthetic intraperitoneal dosage. If a depression of brain metabolism is responsible for the previously reported protection of the brain from ischemic damage, these data suggest a substantial reduction of brain metabolic rate is achieved in the rat at a barbiturate dosage which may be therapeutically relevant in the human after acute brain ischemia.

[3H]Tryptamine and 3H-water as diffusible internal standards for measuring brain extraction of radio-labeled substances following carotid injection

A method has been described 4 and applied3, 5-9 which assesses blood-brain barrier permeability by injecting a radiolabeled test substance, usually 14C-labeled, into the rat common carotid artery mixed with a diffusible radiolabeled reference substance; usually 3H-water (THO). The rat was decapitated 15 sec later and the residual test label in brain measured by liquid scintillation counting relative to the amount of diffusible reference remaining in the brain. The percentage of residual test substance in brain relative to the reference substance was expressed as a brain uptake index (BUI):

Newborn Rabbit Blood–Brain Barrier Is Selectively Permeable and Differs Substantially from the Adult

Abstract: Examination of blood‐brain barrier (BBB) function by the intracarotid injection technique has been utilized in studies of newborn (6–30 h) and adult rabbits. The exclusion of mannitol (mol. wt. 182), dextran (mol. wt. 60,000–90,000) and indium‐bound EDTA indicate that the newborn BBB has restrictive properties similar to the adult. At birth, saturable, carrier‐mediated transport mechanisms are present, regulating the entry of glucose, amino acids, organic acids, purines, nucleosides and choline. No difference in brain uptake of glucose was observed between adult and newborn, but considerably higher uptake rates for arginine, choline and adenine were seen in the newborn. In contrast to suggestions of an immature barrier in young animals, these studies indicate that a sophisticated, selective BBB is operative at birth. Furthermore, the specific selectivity and dramatic increases seen for certain metabolites imply a vital function in the newborn for these carrier systems.

Kinetics of transport and phosphorylation of 2-fluoro-2-deoxy-D-glucose in rat brain.

Abstract: The kinetics of transport across the blood‐brain barrier and metabolism in brain (hemisphere) of [14C]2‐fluoro‐2‐deoxy‐d‐glucose (FDG) were compared to that of [3H]2‐deoxy‐d‐glucose (DG) and d‐glucose in the pentobarbital‐anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a Km of 6.9 ± 1.1 mM and a Vmax of 1.70 ± 0.32 μmol/min/g. The K1 for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k1) and efflux (K2) for FDG were calculated from the Km, Vmax, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k1 FDG/k1glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from 18FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).

The Interaction of Transport and Metabolism on Brain Glucose Utilization: A Reevaluation of the Lumped Constant

Abstract: The relative cerebral cortical metabolism of glucose (GLU) and 2‐deoxy‐D‐glucose (DG) was measured in vivo in control and insulin‐treated hypoglycemic rats. The ratio of the utilization rate constants for the two hexoses, i.e., KDG/KCLU is defined as the Hexose Utilization Index (HUI). The HUI was found to be invariant in rats whose cerebral glucose content exceeded 1 μmo1.g−1 wet weight (HUI = 0.48 ± 0.07). Severe hypoglycemia (plasma glucose <2 mM) effected a shift in the HUI to 1.04 ± 0.21. The results are consistent with a model in which the interpretation of the HUI is determined by the rate of transport into brain, or subsequent phosphorylation, as the rate‐limiting step for hexose utilization.

Developmental Modulations of Blood-Brain Barrier Permeability as an Indicator of Changing Nutritional Requirements in the Brain

Summary: The intracarotid injection technique has been utilized to examine blood-brain barrier function in studies of newborn (>24 h), 7, 14, 21 and 28 day-old, as well as adult rabbits. The age-related modulations in blood-brain barrier transport of adenine, arginine, choline, lactate and tryptophan were defined and demonstrated to be independent of each other. Lactic acid uptake was unusual in that the brain uptake index (BUI) was found to be greatest at 7 days postpartum. Elevated lactate uptake continues until 14 days and is then reduced. As indicated below, for all of the other metabolites examined, a maximal BUI was observed in the newborn brain and BUIs typically showed some sort of inverse relationship to animal age. The BUI of arginine is apparently halved in the first 7 days postnatally, and continues to decrease, reaching the value seen for the adult rabbit by an age of 21 days. In contrast, the brain uptake of adenine is unusual in that there appears to be a very gradual reduction in brain uptake occurring throughout the suckling period. A 3-fold decrease in the BUI of choline was observed during the first 2 wk postpartum. Tryptophan uptake undergoes a 4-fold reduction in the first 4 wk postnatally. Only minor variations in the uptakes of glucose and butanol (a reference substance which is completely cleared by brain over a wide range of blood flow rates) were observed over the range of ages examined. Therefore modulations in adenine, arginine, choline, lactate and tryptophan permeability are not attributable to blood flow alterations.Speculation: The brain uptake rates of the metabolites which gain access by way of specific carrier mediated transport mechanisms are presumed to reflect nutritional requirements of the developing brain. Thus the present study indicates that the inclusion of these metabolites (adenine, arginine, choline, tryptophan and monocarboxylic acids such as lactate) would be beneficial in clinical intravenous feeding of the neonate, to meet the apparent needs of the developing central nervous system.