M. Fuchs

Quantitative determination of free intracellular α-keto acids in neutrophils

For the first time, a procedure is described for the quantitative analysis of free α-keto acid content in human neutrophils (PMNs) relative to single cell number by reversed-phase fluorescence high-performance liquid chromatography. The procedure is minimally invasive and is unsurpassed in the quality of PMN separation, ease of sample preparation as well as sample stability. This method can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular α-keto acid analysis in particularly for the surveillance of severe diseases as well as cellular or organ dysfunction.

Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and α-keto acid homeostasis or immunocompetence

Summary.We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, β-alanine and DFMO on neutrophil amino and α-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of •NO-synthase [L-NAME], an •NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [β-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.

Benzodiazepine receptor-dependent modulation of neutrophil (PMN) free amino- and α-keto acid profiles or immune functions

Summary.We have examined the effects of midazolam, Ro 5-4864 (agonist for “peripheral” [p] benzodiazepine receptors [BR]), PK 11195 (antagonist for pBR), flumazenil (antagonist for “central” BR), naloxone (antagonist for opiate receptors) and the combination of midazolam and Ro 5-4864, PK 11195, flumazenil or naloxone on intracellular amino- and α-keto acids and the immune function markers superoxide anion (O2−), hydrogen peroxide (H2O2) and released myeloperoxidase (MPO) activity in neutrophils (PMN). Only midazolam and Ro 5-4864 led to significant changes in the dynamic PMN free amino- and α-keto acid pools. Concerning PMN immune function markers, midazolam and Ro 5-4864 significantly decreased O2− and H2O2 formation and released MPO. When midazolam and Ro 5-4864 were applied together they appeared to act additively. Pre-incubation with PK 11195 partially neutralized the midazolam effects whereas flumazenil or naloxone showed no effects. We therefore believe that pBR are involved in the signal transmission of anesthetic-induced cellular metabolic changes in PMN.

Effects of propofol and taurine on intracellular free amino acid profiles and immune function markers in neutrophils in vitro.

Abstract We have examined the effects of propofol, taurine, and the combination of propofol and taurine on amino acid profiles and the immune function markers superoxide anion (O2 −), hydrogen peroxide (H2O2), and released myeloperoxidase (MPO) activity in neutrophils (PMN). Propofol led to significant changes in the dynamic PMN-free amino acid pool. Exogenous taurine significantly reduced PMN neutral amino acid and α-aminobutyrate (α-aba) as intracellular taurine increased. Incubation with propofol plus taurine resulted in lower intracellular taurine levels and elevated α-aba and neutral amino acid concentrations compared to propofol alone. Concerning PMN immune function markers, propofol significantly decreased O2 −-and H formation and released MPO. Taurine led to an increased release of MPO and concomitant significantly reduced O2 − and H2O2 levels. When propofol and taurine were applied together they appeared to act additively with regard to superoxide and hydrogen peroxide formation. In the case of MPO, taurine neutralized propofols effects, supporting the idea that MPO activity may be regulated by taurine. We believe therefore that taurine is important for strengthening PMN host defense capability, although the mechanisms are not yet clear. Moreover, taurine appears to act primarily by altering the PMN osmotic balance, while propofol seems to affect PMN amino acid metabolism and/or uptake and release.

Effects of ornithine on neutrophil (PMN) free amino acid and α-keto acid profiles and immune functions in vitro

Summary.The objective of this study was to determine the effects of ornithine on polymorphonuclear leucocyte (PMN) free amino- and α-keto acid profiles, superoxide anion (O2−) generation, hydrogen peroxide (H2O2) formation and released myeloperoxidase acitivity (MPO). Exogenous ornithine significantly increased PMN asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, α-ketoglutarate and pyruvate as intracellular ornithine increased. Concerning PMN immune function markers ornithine increased H2O2-generation and MPO acitivity while O2−-formation was decreased. We believe therefore that ornithine is important for affecting PMN “susceptible free amino- and α-keto acid pool” although the mechanisms are not yet clear. This may be one of the determinants in PMN nutrition considerably influencing and modulating PMN host defense capability.

Effects of α-ketoglutarate on neutrophil intracellular amino and α-keto acid profiles and ROS production

The aim of this study was to determine the effects of α-ketoglutarate on neutrophil (PMN), free α-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O2−), hydrogen peroxide (H2O2)] and released myeloperoxidase (MPO) acitivity. Exogenous α-ketoglutarate significantly increased PMN α-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular α-ketoglutarate changes, increases in O2– formation, H2O2-generation and MPO acitivity have also been observed. We therefore believe that α-ketoglutarate is important for affecting PMN “susceptible free amino- and α-keto acid pools” although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic α-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.

Conscientious metabolic monitoring on a patient with hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome undergoing anaesthesia.

Summary. Currently we know not more than 50 patients who show an interesting combination of increased plasma ornithine concentrations, postprandial hyperammonemia, and homocitrullinuria (HHH-syndrome). Since exact knowledge of this severe, although rare syndrome is important for any perioperative or intensive medical treatment concerning therapy and progression of the disease, we report a comprehensive study on a 32-year old woman with this rare multifaceted disorder who had to undergo general anaesthesia. For the first time amino acid status in plasma, urine, cerebrospinal fluid and especially polymorphonuclear leucocytes, which in the investigation showed to be valuable tool for evaluating amino acid metabolism in nucleated cells in HHH-syndrome, and further important pathophysiologic indicators of cellular and metabolic function have been conscientiously investigated and compared. The pathophysiological repercussions of our results as well as the recommendations for conscientious therapeutical management are discussed.

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

For the first time the immunonutritional role of pyruvate on neutrophils (PMN), free α-keto and amino acid profiles, important reactive oxygen species (ROS) produced [superoxide anion (O2−), hydrogen peroxide (H2O2)] as well as released myeloperoxidase (MPO) acitivity has been investigated. Exogenous pyruvate significantly increased PMN pyruvate, α-ketoglutarate, asparagine, glutamine, aspartate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Moreover, increases in O2− formation, H2O2-generation and MPO acitivity in parallel with intracellular pyruvate changes have also been detected. Regarding the interesting findings presented here we believe, that pyruvate fulfils considerably the criteria for a potent immunonutritional molecule in the regulation of the PMN dynamic α-keto and amino acid pools. Moreover it also plays an important role in parallel modulation of the granulocyte-dependent innate immune regulation. Although further research is necessary to clarify pyruvate’s sole therapeutical role in critically ill patients’ immunonutrition, the first scientific successes seem to be very promising.

Which mechanisms are involved in taurine-dependent granulocytic immune response or amino- and α-keto acid homeostasis?

Summary.We examined the effects of β-alanine (taurine analogue and taurine transport antagonist), taurine (regarding its role in neutrophil (PMN) immunonutrition) and taurine combined either with L-NAME (inhibitor of •NO-synthase), SNAP (•NO donor), DON (glutamine-analogue and inhibitor of glutamine-requiring enzymes), DFMO (inhibitor of ornithine-decarboxylase) and β-alanine on neutrophil amino- and α-keto acid profiles or important PMN immune functions in order to establish whether taurine transport-, nitric oxide-, glutamine- or ornithine-dependent mechanisms are involved in any of the taurine-induced effects. According to the present findings, the taurine-mediated effect appears to be based primarily on a modulation of important transmembraneous transport mechanisms and only secondarily on directly or indirectly induced modifications in intragranulocytic amino- and α-keto acid homoeostasis or metabolism. Although a direct relation to the parallel observed immunological modifications can only be presumed, these results show very clearly that compositional modifications in the free intragranulocytic amino- and α keto-acid pools coinciding with changes in intragranulocytic taurine levels are relevant metabolic determinants that can significantly influence the magnitude and quality of the granulocytic immune response.

The effects of thiopentone on free intracellular amino acids in polymorphonuclear leucocytes

Summary. Previous studies have shown the inhibitory effects of thiopentone on polymorphonuclear leucocyte (PML) function. However, major biochemical mechanisms which have been involved are still unknown. The aim of this study was therefore to investigate thiopentones effects on intracellular amino acid metabolism in PML using both advanced PML separation – and HPLC techniques, especially developed for this purpose and precisely validated in our institute. Overall, our study indicates important dose-dependent alterations of free intracellular amino acid metabolism following thiopentone treatment and draw attention to the biochemical mechanisms which may be involved in both thiopentone-induced modulation in PML function and cellular immunocompetence.