A Quantitative Regional Analysis of Amino Acids Involved in Rat Brain Protein Synthesis by High Performance Liquid Chromatography

Summary: Leucine influx into six brain regions was de termined in anesthetized rats with the in situ brain per fusion technique using either saline or plasma perfusate.This technique has several advantages over other methods such as the brain uptake index (BUI) technique.The concentration dependence of L-Ieucine influx was best described by a model with a saturable and a nonsat urable component. For the saturable component, Villa, equaled 10,67 ± 0.21 x 10-4 fLmol S-l g I and Kill equaled 0.0257 ± 0.0009 fLmol ml-I, whereas the con stant of non saturable diffusion (Kd) equaled 0.957 ± 0.067 x 10-4 S -I in the parietal lobe during saline per fusion. V rna' was higher in the cortical lobes than in other brain areas, probably owing to a greater capillary surface area. There were no regional differences in Kill or Kd. The apparent Kill for L-Ieucine during plasma perfusion wasThe cerebrovascular endothelium. in addition to restricting the exchange of ions. proteins, and hy drophilic nonelectrolytes, has specific transport systems for rapid transfer of essential nutrients from plasma to brain (Rapoport, 1976 port (Takasato et aI., [984). First, the perfusion technique is 100 times more sensitive than the BU[ technique for the measurement of cerebrovascular permeability. Second, the rat� 01' regional cerebral perfusion is measured directly during perfusion, whereas the cerebral flow rate of the B UI intraca rotid bolus has never been determined experimen tally. Third, with the perfusion technique, there is negligible mixing of perfusate with blood before the perfusion fluid reaches the brain capillaries (Taka sato et aI., [984). In contrast, the assumption that the B U [ intracarotid bolus does not mix with blood before reaching the capillaries has never been ver ified experimentally (Fenstermacher et aI., 198 I).Minimal mixing is important, owing to the sensi tivity of cerebrovascular amino acid transport to competition effects (Rapoport, [976; Pard ridge . 1977a). Fourth, the perfusion technique, unlike the intravenous injection method, allows virtually ab solute control of solute concentrations in brain cap illaries. And last, the perfusion method, unlike the intravenous injection method, is not subject to er-

Section: Discussionmentioning

confidence: 98%

Regional Cerebrovascular Transport of Leucine as Measured by the in situ Brain Perfusion Technique

Smith

Takasato

Sweeney

et al. 1985

“…as described before (Bodsch and Hossmann, 1983). Blood samples were taken during the incorporation period, deproteinized, and subjected to HPLC amino acid analysis.…”

Section: Protein Synthesismentioning

confidence: 99%

“…The second part was treated with periodate (Johnson and Chou. 1973) for isolation of aminoacyl-tRNA, which in turn were alkaline treated and analyzed for the amino acid content by HPLC (Bodsch and Hossmann. 1983).…”

Section: Protein Synthesismentioning

confidence: 99%

Recovery of Monkey Brain after Prolonged Ischemia. II. Protein Synthesis and Morphological Alterations

Bodsch

Barbier

Oehmichen

et al. 1986

Recovery of protein synthesis following 1 h of complete ischemia of the monkey brain was assessed by 3H-labeled amino acid incorporation in vivo at various postischemic periods between 1.5 and 24 h. The regional autoradiographic patterns obtained were compared on the basis of precursor-product relationships determined biochemically at the end of the tracer incorporation studies. Shortly after ischemia, protein synthesis was severely inhibited, but it gradually recovered with increasing recirculation times. In the cerebellum it returned to almost normal levels within 3 h and in the cortex within 24 h. Hippocampal and thalamic regions, however, did not recover control levels of protein synthesis at 24 h. His-toautoradiographic evaluation of amino acid incorporation in individual neurons revealed recovery of pyramidal neurons in the CA1 and CA3 sectors of the hippocampus within 6 h of recirculation, which, however, was followed by secondary inhibition after longer recirculation. Neurons in cortical layer 5 steadily recovered to near control within 24 h, with the exception of those located in arterial border zones, which returned to only 50% of control at 24 h. Incomplete recovery was also observed in thalamic neurons and Purkinje cells. The regional and histoauto-radiographic pattern of protein synthesis correlated with the morphological appearance of cells. Ischemic cell changes (mainly of the dark type with microvacuolization and perineuronal glial swelling) were marked after short recirculation times but gradually disappeared in parallel with the return of protein synthesis in most regions of the brain. Only in pyramidal cells of the hippocampus, thalamic neurons, and Purkinje cells were changes not reversed during the observation period. The results obtained corroborate the electrophysiological observations reported in the first part of this investigation and support the notion that the majority of the neurons of monkey brain survive complete cerebrocirculatory arrest of 1 h for at least 1 day.

“…Twenty-five minutes after [I4CJDG bolus injection, a mixture of tritiated amino acids (phenylalanine, tyrosine, isoleucine, leucine, and methionine; total concentration 50 j..L Cil100 g body weight) was infused over 30 s to mea sure cerebral protein synthesis (Bodsch and Hossmann, 1983). Arterial blood samples were collected to trace 3 H plasma radioactivity.…”

Section: Time Course Of Tracer Applicationmentioning

confidence: 99%

Triple-Tracer Autoradiography of Cerebral Blood Flow, Glucose Utilization, and Protein Synthesis in Rat Brain

Mies

Bodsch

Paschen

et al. 1986

A triple-tracer autoradiographic technique is described that permits the simultaneous measurement of cerebral blood flow, glucose consumption, and protein synthesis using 131I-iodoantipyrine (131I-IAP), [14C]deoxyclusively from 14C disintegrations without contamination by either 131I or 3H and that represented regional glucose utilization. Brain sections were then wash-incubated for 12 h to remove [14C]DG, [14C]DG-6-phosphate, and free 3H-amino acids from the tissue, and exposed to 3H-sensitive LKB Ultro-film for 2 weeks for autoradiography of 3H-amino acid incorporation into proteins. 14C radioactivity remaining in the tissue section after wash-incubation was determined by exposing sections again for 2 weeks to Kodak NMB film; the resulting contribution to the blackening of 3H-autoradiograms was corrected for by means of digital subtraction using an image-processing system. The triple-tracer autoradiographic technique was validated in rats under various physiological and pathophysiological conditions. In intact animals extinction correction was necessary only for 3H-autoradiograms. Under pathophysiological conditions, however, significant contamination of 131I by 14C occurred in regions with low blood flow and increased glucose utilization rate; this also required correction by digital subtraction. The interpretation of triple-tracer autoradiographic results is limited by the same restrictions as single-tracer autoradiography, but the simultaneous assessment of the three parameters considerably facilitates the interpretation of the flow/metabolic relationship, particularly under pathological conditions.