Louis Milakofsky

Effect of Arginine Administration on Plasma and Brain Levels of Arginine and Various Related Amino Compounds in the Rat

Arginine (ARG) was injected (0.8 g/kg, i.p.) into rats and levels of ARG were determined in plasma and four brain areas in the morning and afternoon. In control rats, brain values for ARG and some amino compounds are lower in the afternoon than in the morning. After ARG administration, ARG levels increase about 10-fold in the plasma and 2- to 3-fold in the brain areas. Brain ARG levels follow plasma levels. Elevated ARG levels affect a number of related amino compounds both in the plasma and all brain areas most notably ornithine, phosphoserine, glycine, GABA and ammonia. An increase of citrulline after ARG administration suggests the possibility of ARG-stimulated nitric oxide formation in the midbrain. Thus, ARG shows a daily rhythm in the plasma and brain and its administration increases ARG brain levels which seem to follow plasma levels. In addition, ARG alters a number of other amino compounds most notably GABA, glycine, ornithine and ammonia, indicating that some pharmacological effects seen after ARG administration might be caused by elevated levels of ARG and/or changes in other amino compounds.

Comparison of amino acid levels in rat blood obtained by catheterization and decapitation

A novel, sensitive and highly resolving amino acid analysis procedure was developed and used to compare two methods of obtaining blood from experimental animals. The procedure, utilizing a meter long microbore HPLC column containing spherical cation-exchange resin and fluorescence detection following postcolumn reaction with o-phthaldialdehyde, was shown to reliably measure forty-one primary amine components in rat plasma. Comparison of values from blood obtained by decapitation and by catheterization documented the significant artifactual influence of the decapitation procedure on approximately half of the measured constituents.

Effects of repeated stress on plasma arginine levels in young and old rats

The effect of age and recurring stress responses on endogenous plasma arginine (ARG) concentrations were determined. Blood from catheterized young (3 months) and old (24 months) were used to ascertain the plasma concentrations of ARG under baseline conditions, during a 30-min immobilization experience and during a 3.5 h poststress period. Rats were again immobilized and blood drawn following a 1 day rest period (day 3) and then after an additional 3 day rest period (day 7). For young animals, ARG levels decrease significantly after 30 min of stress and remain suppressed during the entire poststress period (3.5 h). After a 1 day rest period, these young rats may have exhibited adaptation to stress on day 3. Old animals showed little or no reduction in plasma ARG concentrations during and after immobilization stress exposure on all three occasions. Baseline levels of old animals were lower in comparison to young animals and their stress responses were smaller. Thus, it is possible that significant changes in plasma ARG levels (as the nitric oxide precursor) during or after stress could serve as an indicator of impending pathology such as cardiovascular disease or immunocompetency.

Effects of acute ethanol administration on rat plasma amino acids and related compounds

Using high performance liquid chromatography (HPLC) with fluorometric detection, thirty-three amino acids (AA) and related compounds were measured in plasma obtained from catheterized rats over a 3-hr period following a 2 g/kg, i.p., injection of ethanol. The concentrations of twenty-three of these compounds had decreased significantly 15 min after the injection, and twenty-three remained depressed for the 3-hr period. Marked reductions were noted for alanine and arginine. Glutamic acid, 1-methylhistidine and 3-methylhistidine were unaffected by ethanol. During these studies individual differences were observed in that some rats showed marked biochemical changes, whereas other rats showed only minimal responses. These observations indicate that ethanol administration may have a significant and long-lasting impact on plasma amino acid biochemistry.

Levels of Taurine, Amino Acids and Related Compounds in Plasma, Vena cava, Aorta and Heart of Rats after Taurine Administration

A pharmacokinetic study on the fate of administered taurine in blood and some tissues and the effects on other amino compounds is presented. Injection of taurine (0.8 g/kg i.p.) causes markedly elevated plasma levels (70-fold at 15 min) which decrease later and approach baseline values after about 4 h. Concomitantly, other plasma amino compounds such as ornithine, threonine, asparagine, glutamine, alanine, citrulline, tyrosine, tryptophan, glycine, ammonia and arginine are reduced, whereas beta-alanine and phosphoserine are increased. At 30 min, tissue levels of taurine are roughly doubled in the vena cava and heart and tripled in the aorta. Other amino compounds affected are aspartic acid, serine, valine, methionine, tyrosine, ammonia, lysine, histidine, and arginine in the vena cava; aspartic acid, reduced glutathione, serine, and ammonia in the aorta; and reduced glutathione, alanine, citrulline and methionine in the heart. In most of these cases, plasma changes do not predict tissue changes which are generally substance- and tissue-specific. Thus, pharmacological effects seen after taurine administration could be caused by elevated taurine levels per se and/or taurine-induced changes in some of the amino acids and related compounds.

Effect of repeated stress on plasma catecholamines and taurine in young and old rats

The effect of age and multiple stress responses on plasma norepinephrine (NE), epinephrine (EPI), and taurine (TAU) levels were determined in F344 rats. Blood samples obtained from catheterized young (3 months) and old (24 months) animals were used to examine plasma levels of NE, EPI, and TAU under baseline conditions and in the same animals after a 30-min immobilization stress. Rats were again immobilized and blood drawn (Day 3) following a 1-day rest period and, after an additional 4-day rest period (Day 7). Age differences seen between young and old rats were not the same for the three neurochemicals measured but were relatively unique for the specific biochemicals. In old animals baseline values of NE but not EPI and TAU were higher then young animals and all three values did not change for the baseline during the two additional stress exposures. Initial stress responses were similar for all three biochemicals in both age groups. Although no signs of adaptation were evident in the old animals, adaptation to immobilization was seen for EPI and TAU but not NE on the third occasion in the young animals. Correlations seen between NE and EPI in young and old rats on the first day disappeared during the second stress period but were again seen during the third stress exposure suggesting subtle indicators of repeated stress subject to adaptation. TAU values for young but not old rats correlated positively with EPI concentrations during the first stress exposure and negatively after the immobilization was terminated indicating a regulatory interaction between EPI and TAU present in young but lost in old animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of ethanol stress on components of the allantoic fluid of the chicken embryo.

Recent studies showed that the allantoic fluid of the chicken embryo is a depot for stress-released catecholamines and many free amino acids and related compounds, and that it is separated from plasma and the amniotic fluid by selective barriers. To gain further insights into the functions of the allantois and its barriers, we studied the impact of stress (intra-allantoic injection of 0.1 ml ethanol) on 39 free amino acids and related compounds of the allantoic fluid. Using an HPLC-fluorometric method, we found that the concentration of seven substances was significantly increased 20 min after injection of ethanol, and back to control levels within 40 minutes. Five of these compounds (asparagine, alanine, leucine, tyrosine, lysine) had previously been shown to occur in plasma at concentrations above those in the allantoic fluid. However, taurine and phosphoethanolamine increased in the allantoic fluid even though their concentrations in plasma tended to be lower than in allantoic fluid. These findings (1) reveal the existence of complex embryonic/extraembryonic autoregulations, and (2) raise the question of the regulatory mechanisms involved in the transfer of substances across the allantoic barrier(s).

REGULATION OF SUBSTANCES IN ALLANTOIC AND AMNIOTIC FLUID OF THE CHICKEN EMBRYO

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.

A profile of amino acid and catecholamine levels during endotoxin-induced acute lung injury in sheep: searching for potential markers of the acute respiratory distress syndrome

The identification of plasma markers of the course of the acute respiratory distress syndrome (ARDS) is needed to improve its treatment and to further advance the development of new therapeutic agents. The status of markers of lung injury in ARDS is reviewed and some new potential markers are proposed. This study focused on plasma amino acids, related amino compounds, and catecholamine levels during the acute phase of endotoxin-induced lung injury in 8 sheep characterized by the onset of pulmonary edema caused by increased microvascular permeability. A number of significant changes from baseline values were found. During the sixth hour of a 12-hour period of endotoxin infusion, norepinephrine, epinephrine, and alanine levels increased whereas the isoleucine level decreased. During the sixth hour of the immediate postendotoxin period, the taurine level increased while the levels of arginine, citrulline, glycine, isoleucine, methionine, ornithine, serine, threonine, and tryptophan decreased. These findings are compared with prior studies in human subjects detailing the amino acid profile characteristic of advanced sepsis. We conclude that the present profile of catecholamine and amino acid changes during endotoxemia in sheep deserves further study in human subjects to determine its significance as a marker of the early stage of ARDS.

The effects of arginine administration on the levels of arginine, other amino acids and related amino compounds in the plasma, heart, aorta, vena cava, bronchi and pancreas of the rat

Arginine (0.8g/kg, ip) or a vehicle was administered to rats and the levels of arginine and a large number of related amino compounds++ were measured in plasma, heart, aorta, vena cava, pancreas and bronchi at specified time intervals. Arginine levels (nmol/ml) increased in the plasma from 237 to 3172 at 15 min, 1236 at 30 min and 509 at 120 min. Peak concentrations (nmol/g) of arginine are reached in the tissues at 15 or 30 minutes with control and postinjection values of 500 and 1769 in the heart, 314 and 1563 in the aorta, 575 and 2976 in the vena cava, 760 and 1943 in the bronchi, and 234 and 3638 in the pancreas. Arginine injection also affects a number of amino acids and related compounds in the plasma and tissues most notably ornithine, isoleucine, phosphoserine, leucine and ethanolamine. However, plasma level changes do not predict tissue level changes which are highly specific for an individual compound and tissue. There was no general indication that arginine injection stimulates nitric oxide (NO) formation in any tissue. Thus, arginine is rapidly absorbed from the abdominal cavity into the blood stream, is quickly taken up by the tissues studied and disappears after about 2 to 3 hours. The effects seen after arginine administration could be caused by arginine per se and/or changes in one or more of the related amino compounds but not by NO.