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Taurine: new implications for an old amino acid

Georgia B. Schuller-Levis, Eunkyue Park
DOI: http://dx.doi.org/10.1016/S0378-1097(03)00611-6 195-202 First published online: 1 September 2003

Abstract

Taurine is a semi-essential amino acid and is not incorporated into proteins. In mammalian tissues, taurine is ubiquitous and is the most abundant free amino acid in the heart, retina, skeletal muscle, brain, and leukocytes. In fact, taurine reaches up to 50 mM concentration in leukocytes. Taurine has been shown to be tissue-protective in many models of oxidant-induced injury. One possibility is that taurine reacts with hypochlorous acid, produced by the myeloperoxidase pathway, to produce the more stable but less toxic taurine chloramine (Tau-Cl). However, data from several laboratories demonstrate that Tau-Cl is a powerful regulator of inflammation. Specifically, Tau-Cl has been shown to down-regulate the production of pro-inflammatory mediators in both rodent and human leukocytes. Taurolidine, a derivative of taurine, is commonly used in Europe as an adjunctive therapy for various infections as well as for tumor therapy. Recent molecular studies on the function of taurine provide evidence that taurine is a constituent of biologic macromolecules. Specifically, two novel taurine-containing modified uridines have been found in both human and bovine mitochondria. Studies investigating the mechanism of action of Tau-Cl have shown that it inhibits the activation of NF-κB, a potent signal transducer for inflammatory cytokines, by oxidation of IκB-α at Met45. Key enzymes for taurine biosynthesis have recently been cloned. Cysteine sulfinic acid decarboxylase, a rate-limiting enzyme for taurine biosynthesis, has been cloned and sequenced in the mouse, rat and human. Another key enzyme for cysteine metabolism, cysteine dioxygenase (CDO), has also been cloned from rat liver. CDO has a critical role in determining the flux of cysteine between cysteine catabolism/taurine synthesis and glutathione synthesis. Taurine transporter knockout mice show reduced taurine, reduced fertility, and loss of vision due to severe apoptotic retinal degeneration. Apoptosis induced by amino chloramines is a current and important finding since oxidants derived from leukocytes play a key role in killing pathogens. The fundamental importance of taurine in adaptive and acquired immunity will be unveiled using genetic manipulation.

Keywords
  • Taurine
  • Taurine chloramine
  • Inflammatory mediator
  • Nitric oxide
  • Tumor necrosis factor α
  • Cysteine sulfinic acid decarboxylase
  • Taurolidine

1 Introduction

Taurine, a sulfur-containing amino acid present in high concentrations in mammalian plasma and cells, plays an important role in several essential biological processes such as development of the central nervous system (CNS) and the retina, calcium modulation, membrane stabilization, reproduction, and immunity [13]. In fact, taurine is the single most abundant amino acid in leukocytes (20–50 mM) [4]. Taurine, although not incorporated into proteins, is considered to be an essential amino acid for felines and a conditionally indispensable amino acid for humans and non-human primates [2]. The level of cysteine sulfinic acid decarboxylase (CSD), an enzyme required for biosynthesis of taurine, is very low in the cat and low in humans and primates. For this reason, taurine has been added to infant formula as well as to parenteral solutions. Taurine occurs naturally in food, especially in seafood and meat. The mean daily intake from omnivore diets was determined to be around 58 mg. Taurine-containing health drinks, usually containing about 1 g of taurine, are marketed worldwide for the treatment of various conditions, for improvement of athletic performance and for general well being [5]. Animal studies have not indicated toxicity due to taurine. In light of recent evidence on the role of taurine in immunity, risk assessment studies on the effect of these drinks in immunocompromised patients, children, and pregnant women should be performed.

2 Taurine and taurolidine as adjunct therapy for infections, endotoxemia, and tumors

Several recent papers describe the role of taurolidine (Geislick Pharma, AG, Woljusen, Switzerland) in infection [6]. Taurolidine is a derivative of taurine and is commonly used in Europe, the UK, Ireland and the USA as adjunctive therapy for various infections. Taurolidine is chemically designated as bis-(1,1-dioxyperhydro-1,2,4-thiadiazinyl-4) methane and consists of two taurolidine rings derived from taurine and three molecules of formaldehyde combining to form a two-ringed structure bridged by a methylene group [6]. Taurolidine, which is stable, has a short half-life, is non-toxic, metabolizes to taurine, CO2 and H2O, and irreversibly inactivates lipopolysaccharide (LPS). Recent reports include anti-endotoxin, anti-bacterial, and anti-adherence activities for taurolidine. Taurolidine is now included in a new catheter lock solution (Neutrolin; Biolink, Norwell, MA, USA) to prevent catheter-related infections. Bedrosian et al. [7] attribute the activity of taurolidine to blocking the production of interleukin (IL)-1 and tumor necrosis factor (TNF). Taurolidine may have anti-bacterial action that is independent of the resultant taurine metabolites. While both taurine and taurolidine can down-regulate inflammation, it is unclear whether taurine is anti-bacterial and would be useful in a serious infection. Tissue damage could be minimized by taurine's anti-inflammatory properties, but a possible lack of anti-microbial function, associated with enhancement of macrophage and polymorphonuclear leukocyte (PMN) proinflammatory activity, would be detrimental to elimination of pathogens.

In studies by De Costa et al. [8] taurolidine enhanced survival in an animal model of melanoma. Natural killer cells and lymphocyte-activated killer cells were functional in the taurolidine-treated group compared to untreated animals with melanoma, perhaps accounting for the increased survival in the treated group. Taurolidine also inhibited the growth of a rat metastatic colorectal tumor cell line in vitro and in vivo [9]. These studies suggest that taurolidine may have value in management of patients with tumors. Egan et al. [10] have shown in sheep that taurolidine had a therapeutic role in preventing endotoxin-induced lung injury. In this model i.v. taurine (300 mg kg−1), given 1 h before i.v. endotoxin, significantly reduced lung injury.

Although reports of decreased plasma levels of taurine in trauma, sepsis and critical illness are available, very little is known about the relationships among changes in plasma taurine, other amino acid levels, and metabolic variables. A large series of plasma amino acid profiles were obtained in 250 trauma patients with sepsis who were undergoing total parenteral nutrition [11]. The results, which characterized the relationships between plasma taurine and other amino acid levels in sepsis, provide evidence that the more severe decreases in plasma taurine correlate with the worsening of metabolic and cardiorespiratory patterns.

3 Immunologic consequences of taurine deficiency versus supplementation

For cats and primates, deficiency of dietary taurine results in abnormalities in development of the CNS, retinal and tapetal degeneration, as well as significant changes in the cardiovascular and reproductive systems. These changes are also accompanied by abnormalities in the immune system [3]. A lack of taurine in the diet of cats resulted in a significant leukopenia, a shift in the percentage of polymorphonuclear and mononuclear leukocytes, an increase in the absolute count of mononuclear leukocytes, and a change in the sedimentation characteristics of white cells. Functional studies of polymorphonuclear cells isolated from cats fed taurine-free diets demonstrated a significant decrease in the respiratory burst as measured by chemiluminescence as well as a decrease in phagocytosis of Staphylococcus epidermidis compared to cats fed the same diet containing taurine. In addition, serum γ-globulin in cats fed taurine-free diets was significantly increased compared to taurine-supplemented cats, indicating that other immune cells may be affected by taurine deficiency. Histological examination of lymph nodes and spleen revealed regression of follicular centers with depletion of reticular cells, mature and immature lymphocytes as well as mild extravascular hemolysis [3]. These results indicate there are profound immunologic abnormalities in cats with prolonged taurine deficiency.

Reports indicate an increased incidence of pediatric problems in children from vegan communities that eat little to no taurine [12]. These problems are usually attributed to malnutrition but a role for immunologic and other consequences of taurine deficiency cannot be ruled out.

Taurine is found in particularly high concentrations in tissues exposed to elevated levels of oxidants. Several in vivo models of oxidant-induced damage have been studied using taurine as a protectant against inflammation. Hamsters pretreated with supplemented dietary taurine and then exposed to NO2 did not show morphological alterations typical of NO2 damage [13]. Wang et al. [14] demonstrated that taurine and niacin reduced the inflammation and fibrosis caused by bleomycin. This group also reported that taurine and niacin blocked the bleomycin-induced increased production of nitric oxide in bronchoalveolar lavage fluid, as well as the overexpression of iNOS mRNA and NOS protein in lung tissue [15]. Rats treated with guanidinoethanesulfonate, which is a competitive inhibitor of taurine binding and transport and depletes cellular taurine levels, showed enhanced lung pathology after treatment with both bleomycin and paraquat [16]. Thus, maintenance of tissue taurine levels was critical to the prevention of oxidant-induced lung injury.

We performed studies to determine if ozone-induced lung inflammation was modified by pretreatment of 5% taurine in the drinking water for 10 days prior to ozone (O3) exposure (2 ppm for 3 h). The number of inflammatory cells and hydroxyproline levels in the bronchoalveolar lavage of taurine-treated rats was significantly reduced compared to untreated rats exposed to O3[17]. Light microscopy revealed a significant inflammatory infiltrate in the lungs of rats 48 h after exposure to O3 followed by focal hyperplasia in the terminal and respiratory bronchioles (72 h) (Fig. 1). Rats pretreated with taurine in the drinking water for 10 days and then exposed to O3 showed none of these alterations (Fig. 1). These results show that supplemental taurine protects rats from acute ozone-induced lung inflammation and hyperplasia.

Figure 1

Left: Light micrograph of rat lung 48 h after ozone exposure (pretreated with taurine). Note that there is no evidence of a macrophage infiltrate. 170×. Right: Light micrograph of rat lung 48 h after ozone exposure (water only). Note many vacuolated macrophages (arrow) present in the alveolar spaces into the respiratory bronchiole (RB), alveolar duct (AD), and surrounding alveoli. 340×.

Bleomycin-induced lung injury results in dysregulated matrix remodeling, leading to thickened alveolar walls, alveolar collapse and scarring [18]. Fibrosis culminates in the overproduction of interstitial collagen. Fibrosis is strikingly absent and inflammation is reduced in the lung of rats pretreated with 5% taurine in the drinking water for 10 days prior to bleomycin instillation [18]. Significantly more intercellular adhesion molecule (ICAM) was demonstrated in the bleomycin-treated group compared to the taurine-treated bleomycin group, indicating that ICAM correlated with lung damage. Those cells which do enter the lung in the taurine-treated group do not appear to ‘stick and stay’ which may be one mechanism for the absence of fibrosis in this group.

Other evidence supporting the ‘stick and stay’ idea is the data of Abdih et al. [19] and Egan et al. [20]. Abdih et al. [19] demonstrated that taurine prevents IL-2-induced acute lung injury, in part, by decreasing neutrophil interactions. Data from Egan et al. [20] demonstrate that following administration of LPS there was an increase in leukocyte rolling accompanied by an increase in the number of adherent leukocytes and transendothelial migration. Taurine given orally as a 4% solution significantly attenuated the LPS-induced leukocyte rolling and attenuated the number of adherent leukocytes as well as the increase in transendothelial cell migration.

Our hypothesis is that supplemental taurine in the drinking water increases the available taurine both systemically and at the site of inflammation. Leukocytes capable of generating hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride via the myeloperoxidase (MPO) pathway have intracellular concentrations of taurine of 20–50 mM. Moreover, in physiologic fluid extracellular taurine concentrations range from 50 to 100 mM after taurine supplementation [21]. Taurine reacts with HOCl to produce the less reactive and long-lived oxidant taurine chloramine (Tau-Cl). Thus, Tau-Cl, a stable oxidant, can be produced at the site of inflammation and down-regulate proinflammatory cytokine production leading to a significant reduction in the immune response. Taurine may provide a useful prophylactic approach to preventing tissue damage resulting from inflammation.

4 Taurine chloramine, the ‘active’ product of taurine, and the MPO pathway

Neutrophils and monocytes contain high levels of MPO, which, along with H2O2, catalyzes the formation of the potent oxidant, HOCl. Taurine, the most abundant free amino acid, scavenges HOCl to form the more stable and less toxic Tau-Cl [22,23].

Tau-Cl inhibits in a dose-dependent manner the production of both NO and TNF-α by activated RAW 264.7 cells, a macrophage-like cell line (Fig. 2) [24]. Tau-Cl (0.8 mM) inhibited secretion of TNF-α into the media and nitrite production from activated RAW 264.7 cells by 65% and 91%, respectively. To examine the mechanism(s) whereby Tau-Cl inhibits inflammatory cytokines, activated cell lysates in the presence or absence of Tau-Cl were analyzed for the inducible form of NO synthase (iNOS) by Western blot analysis, and TNF-α and iNOS mRNAs were assessed by Northern blot analysis [25]. Western blot analysis showed that iNOS protein was absent from cells activated with LPS and rIFN-γ in the presence of 0.8 mM Tau-Cl. Northern blot analysis demonstrated that Tau-Cl (0.8 mM) significantly inhibited iNOS mRNA at all time points examined (Fig. 3) demonstrating that Tau-Cl inhibits transcription of the iNOS gene. In the same experiments, Tau-Cl delayed the peak expression of TNF-α mRNA from 4 h to 8 h, with continuing expression of high TNF-α transcripts after 24 h of activation. TNF-α secreted into the medium was inhibited by the same doses of Tau-Cl used in the Northern blot experiments, indicating that although TNF-α mRNA is present, translation of this message is impaired. The effects of Tau-Cl are not a result of either changes in viability (data not shown) or a generalized effect on gene transcription because α-actin mRNA was intact with treatment of Tau-Cl (see Fig. 3 for increase in TNF-α message).

Figure 2

Tau-Cl inhibits the amount of NO2 and TNF-α recovered in the media of LPS- and IFN-γ-activated RAW 264.7 cells. Conditioned medium was collected 16 h after activation and assayed as described in the text. Values represent the mean±S.D. of triplicate samples. Asterisks indicate a significant difference from control values (P<0.05). Similar results were obtained in six to eight independent experiments. Reprinted from Park et al. [25], ©1995 The American Association of Immunologists, Inc.

Figure 3

Kinetics of iNOS, TNF-α and α-actin mRNA expression in RAW 264.7 cells. Cells were obtained 4, 8, 16, and 24 h after activation. Total RNA fractions from cells unactivated in the presence of either 0.8 mM taurine (lane 3) or 0.8 mM Tau-Cl (lane 4) are shown. Similar results were obtained in two to three additional independent experiments. Reprinted from Park et al. [25], ©1995 The American Association of Immunologists, Inc.

Studies on Tau-Cl have been performed using macrophage cell lines and activated murine and rat macrophages. Recent studies have demonstrated that Tau-Cl suppressed superoxide anion, IL-6 and IL-8 production in activated human peripheral blood PMNs [26]. In addition, using both adherent and non-adherent leukocytes, many proinflammatory mediators were significantly decreased by Tau-Cl [27]. Choray et al. have confirmed and extended these finding using LPS-stimulated peripheral blood monocytes from humans [28].

Early administration of Tau-Cl resulted in the delay of the onset of collagen-induced arthritis (CIA) in DBA1/J mice [29]. This is the first study to use Tau-Cl in vivo for immune intervention. An analysis of genes involved in the inflammatory process of joints in DBA1/J mice with CIA was performed using microarrays [30]. Of the 11,000 genes assayed, 223 increased four-fold or more. Nine genes mapped to the chromosome contributing to susceptibility to CIA, including the taurine transporter gene.

Kontny et al. [31] have shown that Tau-Cl inhibits the production of proinflammatory cytokines (IL-6 and IL-8) by fibroblast-like synoviocytes isolated from rheumatoid arthritis patients. In these studies Tau-Cl diminished the activity of NF-κB and to a lesser extent, that of AP-1 transcription factor. This possible mechanism for down-regulation of proinflammatory cytokines was also demonstrated by Barua et al. (see Section 5) [32].

Marcinkiewicz et al. found that treatment of T-cells with Tau-Cl prior to activation inhibited IL-2 release in response to both mitogen and antigenic stimulation [33]. In addition, this group found exposure of dendritic cells to Tau-Cl affected their ability to stimulate T-cell responses. The authors suggest that Tau-Cl may favor the development of a Th1 rather than a Th2 response.

5 Recent molecular studies on the function of taurine and its chloramine

Taurine has thus far not been found as a component of a protein or nucleic acid and its precise biochemical mechanism(s) are unclear. Exciting studies from Suzuki et al. [34] demonstrate the first reported evidence taurine is a constituent of biologic macromolecules, which is a significant new insight into the function of taurine. They identified two novel taurine-containing modified uridines (5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine) in human and bovine mitochondrial tRNAs. These nucleotides are synthesized by a direct incorporation of taurine supplied to the medium. They found an absence of taurine modified mitochondrial uridine in the cells from the mitochondrial diseases MELAS and MERRF. These findings will hopefully lead not only to development of therapies for these diseases but to clues for understanding an important biochemical function of taurine.

Barua et al. [32] have demonstrated that Tau-Cl depressed NF-κB migration into the nucleus of activated NR8383 cells, a cloned cell line derived from rat alveolar macrophages, and caused a more sustained presence of IκB in the cytoplasm. In additional experiments, Tau-Cl did not directly inhibit IκB kinase (IKK) activity suggesting that Tau-Cl exerts its effects at some level upstream of IKK in the signaling pathway.

Kanayama et al. [35] report Tau-Cl-induced inhibition of NF-κB activation by the oxidation of IκB-α. Deletion experiments showed that the Tau-Cl modification site causing the band shift is Met45, indicating that Met45 oxidation is a molecular mechanism underlying the Tau-Cl-induced inhibition of NF-κB.

6 Genetic studies on CSD, taurine transporter, and CDO

CSD was first identified in the liver as a rate-limiting enzyme in the biosynthesis of taurine. Reymond et al. [36] demonstrated that in addition to liver and kidney, rat brain expressed CSD mRNA. Brain CSD was strictly localized in glial cells, especially astrocytes, introducing a possible role of taurine in astrocyte–neuron interaction. Reymond et al. [36] and Kaisaki et al. [37] reported a sequenced CSD cDNA in the rat (GenBank accession numbers: X94152 and M64755, respectively). Human CSD has been registered in the GenBank (accession number: AF116548). Since the mouse is a good animal model for studies on the role of taurine in the immune system, we cloned murine CSD cDNA and examined the expression of CSD mRNA in various murine tissues including leukocytes. The cDNA sequence of murine CSD, which is a polypeptide of 493 amino acids (Fig. 4) [38], has 98% and 90% sequence homology of amino acids with rat and human CSD, respectively, indicating that it is a true ortholog of CSD. Northern blot analysis revealed that CSD mRNA is expressed in kidney and liver, and was not detected in lymphoid tissues and lung. These data suggest that lymphoid tissue may rely on transport of taurine and may not synthesize taurine directly.

Figure 4

Amino acid sequence alignment of CSD from mouse, human and rat. An asterisk underneath represents an amino acid conserved in all species whereas a dot represents an amino acid conserved only in mouse and rat. Mouse and human CSDs share 90% amino acid homology whereas mouse and rat CSDs share 98%. Reprinted from Park et al. [38] with permission from Elsevier Science.

Another key regulatory enzyme for cysteine metabolism is cysteine dioxygenase (CDO, EC 1.13.11.20) cloned from rat liver [39]. The levels of CDO activity changed by dietary protein level, in addition to cysteine availability, are key factors in determining the flux of cysteine between cysteine catabolism/taurine synthesis and glutathione synthesis [40]. Excess sulfur amino acids or protein increase CDO activity and CDO protein but not the levels of mRNA CDO. This suggests that CDO regulation may be posttranslational and possibly involve a decrease in the rate of CDO degradation.

To maintain adequate level of taurine in the tissues, taurine is tightly regulated by excretion and reabsorption by the kidney [41]. The taurine transporter in proximal tubule brush border membranes appears to be the primary target for adaptive regulation by dietary availability of taurine. The genes encoding the taurine transporter (TauT) for various species and tissues share a high degree of homology. TauT gene is located on the central region of mouse chromosome 6 and on human chromosome 3p21–25, where a conserved linkage group of genes has been found between mouse and man [42]. In patients with 3p syndrome, deletion of 3p25–pter is associated with profound growth failure, characteristic facial features, retinal changes and mental retardation, suggesting that deletion of TauT might contribute to some phenotypic features of the 3p syndrome [43].

Heller-Stilb et al. [44] have developed a mouse model with a disrupted gene encoding the taurine transporter (trans−/− mice). These mice show markedly decreased taurine levels in a variety of tissues, reduced fertility, and loss of vision due to severe retinal degeneration. A decrease of taurine concentration by 74% was observed in plasma, kidney, liver, and the eye. In skeletal muscle and heart, taurine levels were decreased by >95%. No data were reported for cells or organs of the immune system. The retinal involvement identifies the taurine transporter as an important factor for the development and maintenance of normal retinal functions and morphology. This progressive retinal degeneration was found to be caused by apoptosis. Han et al. [45] have shown that the taurine transporter gene is a transcriptional target of p53, which functions as a cell cycle checkpoint or may trigger apoptosis in cells with defective genomes. Of particular interest are the findings of Englert et al. [46] which show that amino chloramines induced apoptosis. Using B-cell lymphoma cells, Englert et al. have shown that long-lived aminoacyl chloramines (Tau-Cl being the most abundant) mediate HOCl-induced apoptosis. Since Tau-Cl is formed at the site of inflammation, neutrophil cell death and neutrophil-induced death at the inflammatory site would likely be apoptotic. Apoptotic cell death, in contrast to necrotic cell death, is a physiologic advantage in that cells are cleared by phagocytosis lessening tissue damage. Tau-C1 may promote apoptotic cell death and thereby decrease the detrimental effects of inflammation.

7 Taurine research: new insights

The schematic (Fig. 5) incorporates our findings as well as those of others on the possible mechanism(s) of action of taurine as an immunomodulator and as a component of RNA. Taurine has been shown to be tissue-protective in many models of oxidant-induced injury. Early events in inflammations include migration of leukocytes to the site of injury. These inflammatory cells produce high levels of HOCl via the MPO pathway and the abundance of taurine assures the production of Tau-Cl. Data show that Tau-Cl can be actively transported into leukocytes and can down-regulate the production of inflammatory mediators. New areas of research should extend these studies to include applications to clinical problems such as autoimmune diseases and inflammation. Two such areas include genetic manipulation of CSD, CDO and TauT which provide an approach to the fundamental roles of taurine in the immune system, CNS, reproduction and osmoregulation, as well as studies on the two novel taurine-containing modified uridines in human and bovine mitochondrial tRNAs.

Figure 5

Schematic representation of formation of Tau-C1 during inflammation, mechanism(s) utilized by Tau-Cl to inhibit production of inflammatory mediators by immune-responsive cells and possible catabolic flow and biosynthetic pathway for mitochondrial taurine. Adapted in part from Suzuki et al. [34] and Quinn and Schuller-Levis [47].

Acknowledgements

We thank Ms. Vanessa DeBello for secretarial help and William R. Levis, MD for reviewing the manuscript. The Office of Mental Retardation and Developmental Disabilities and USPHS Grant HL-49942 provided funding for this work. We apologize to those whose papers were not cited because of the publisher's space limitations.

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