Effect of glutamine on glutathione kinetics in vivo in dogs

To determine whether glutamine affects glutathione (GSH, gamma-glutamyl-cysteinyl-glycine) metabolism, seven healthy beagle dogs received 6-h infusions of [(15)N]glutamate and [(13)C]leucine after a 3-day fast. Isotope infusions were performed during oral feeding with an elemental regimen, supplemented with either l-glutamine or an isonitrogenous amino acid mixture, on two separate days and in randomized order. Timed blood samples were obtained, and a surgical duodenal biopsy was performed after 6 h of isotope infusion. GSH fractional synthesis rate (FSR) was assessed from [(15)N]glutamate incorporation into blood and gut GSH, and duodenal protein synthesis from [(13)C]leucine incorporation into gut protein. Glutamine supplementation failed to alter erythrocyte GSH concentration (2189+/-86 vs. 1994+/-102 micromol L(-1) for glutamine vs. control; ns) or FSR (64+/-17% vs. 74+/-20% day(-1); ns). In the duodenum, glutamine supplementation was associated with a 92% rise in reduced/oxidized GSH ratio (P=.024) and with a 44% decline in GSH FSR (96+/-15% day(-1) vs. 170+/-18% day(-1); P=.005), whereas total GSH concentration remained unchanged (808+/-154 vs. 740+/-127 micromol kg(-1); P=.779). We conclude that, in dogs receiving enteral nutrition after a 3-day fast: (1) glutamine availability does not affect blood GSH, and, (2) in contrast, in the duodenum, the preserved GSH pool, along with a decreased synthesis rate, suggests that glutamine may maintain GSH pool and intestinal redox status by acutely decreasing GSH utilization.     

Oral [13C]bicarbonate measurement of CO2 stores and dynamics in children and adults

During exercise, less additional CO2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO2. To examine this, tracer doses of [13C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of 13CO2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO2 stores. CO2 production (VCO2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 +/- 13% (SD)] and adults (71 +/- 9%). MRT was shorter in children (42 +/- 7 min) compared with adults (66 +/- 15 min, P less than 0.001). VCO2 per kilogram was higher in the children (5.4 +/- 0.9 ml.min-1.kg-1) compared with adults (3.1 +/- 0.5, P less than 0.0001). Tracer estimate of CO2 production was correlated to VCO2 (r = 0.86, P less than 0.0001) and when corrected for mean recovery accurately predicted the VCO2 to within 3 +/- 14%. There was no difference in the estimate of resting CO2 stores between children (222 +/- 52 ml CO2/kg) and adults (203 +/- 42 ml CO2/kg). We conclude that orally administered [13C]bicarbonate can be used to assess CO2 transport dynamics. The data do not support the hypothesis of lower CO2 stores under resting conditions in children.     

The effects of cellular glutathione elevation on the oxygen enhancement ratio

The radiation responses at various oxygen tensions were evaluated in V79 Chinese hamster cells under conditions where their nonprotein thiols, primarily glutathione (GSH), were elevated by 2-oxothiazolidine-4-carboxylate (OTZ). OTZ, when cleaved by intracellular oxoprolinase, provides the cell with cysteine which stimulates GSH synthesis. A 2-hr pretreatment with 10 mM OTZ elevated GSH to 200% of controls. This elevation in GSH offered no protection to aerated cells; however, for O2 tensions less than or equal to 40,000 ppm modest protection was observed as evidenced by an increase in oxygen enhancement ratio. GSH elevation afforded maximal protection between 1000 and 10,000 ppm O2; however, the extent of protection was relatively small (protection factor = 1.3).     

Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa

Many patients with X-linked retinitis pigmentosa (XLRP) have lower than normal blood levels of the long-chain polyunsaturated omega3 fatty acid docosahexaenoic acid (DHA; 22:6omega3). This clinical trial was designed to test whether down-regulation of DHA biosynthesis might be responsible for these reduced DHA levels. DHA biosynthesis was assessed in five severely affected patients with XLRP and in five age-matched controls by quantifying conversion of [U-(13)C]alpha-linolenic acid (alpha-LNA) to [(13)C]DHA. Following oral administration of [U-(13)C]alpha-LNA, blood samples were collected at designated intervals for 21 days and isotopic enrichment of all omega3 fatty acids was determined by gas chromatography/mass spectroscopy. Activity of each metabolic step in the conversion of alpha-LNA to DHA was determined by comparison of the ratios of the integrated concentration of (13)C-product to (13)C-precursor in plasma total lipid fractions. The ratio of [(13)C]DHA to [(13)C]18:3omega3 (the entire pathway) and that of [(13)C]20:5omega3 to [(13)C]20:4omega3 (Delta(5)-desaturase) were significantly lower in patients versus controls (P = 0.03 and 0.05, respectively). The estimated biosynthetic rates of [(13)C]20:5omega3, [(13)C]22:5omega3, [(13)C]24:5omega3, [(13)C]24:6omega3, and [(13)C]22:6omega3 were significantly lower in XLRP patients (42%, 43%, 31%, 18%, and 32% of control values, respectively; P < 0.04), supporting down-regulation of Delta(5)-desaturase in XLRP. The disappearance of (13)C-labeled fatty acids from plasma was not greater in XLRP patients compared with controls, suggesting that XLRP was not associated with increased rates of fatty acid oxidation or other routes of catabolism.Thus, despite individual variation among both patients and controls, the data are consistent with a lower rate of Delta(5)-desaturation, suggesting that decreased biosynthesis of DHA may contribute to lower blood levels of DHA in patients with XLRP.     

Corticosteroids increase glutamine utilization in human splanchnic bed

Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-h intravenous infusions of l-[1-(14)C]leucine and l-[(2)H(5)]glutamine, along with q. 20 min oral sips of tracer doses of l-[1-(13)C]glutamine in the fasting state, either 1) at baseline (control group; n = 6) or 2) after a 6-day course of 0.8 mg.kg(-1).day(-1) prednisone (prednisone group; n = 8). Leucine and glutamine appearance rates (Ra) were determined from plasma [1-(14)C]ketoisocaproate and [(2)H(5)]glutamine, respectively, and leucine and glutamine oxidation from breath (14)CO(2) and (13)CO(2), respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered [(13)C]glutamine that failed to appear into systemic blood. Prednisone treatment 1) did not affect leucine Ra or leucine oxidation; 2) increased plasma glutamine Ra, mostly owing to enhanced glutamine de novo synthesis (medians +/- interquartiles, 412 +/- 61 vs. 280 +/- 190 mumol.kg(-1).h(-1), P = 0.003); and 3) increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (means +/- SE 64 +/- 6 vs. 42 +/- 12%, P < 0.05), without any change in the fraction of glutamine oxidized (means +/- SE, 75 +/- 4 vs. 77 +/- 4%, not significant). We conclude that high-dose glucocorticosteroids increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remains to be determined.     

Physiological responses to food deprivation in the house sparrow, a species not adapted to prolonged fasting

Many wild birds fast during reproduction, molting, migration, or because of limited food availability. Species that are adapted to fasting sequentially oxidize endogenous fuels in three discrete phases. We hypothesized that species not adapted to long fasts have truncated, but otherwise similar, phases of fasting, sequential changes in fuel oxidization, and similar changes in blood metabolites to fasting-adapted species. We tested salient predictions in house sparrows (Passer domesticus biblicus), a subspecies that is unable to tolerate more than ～32 h of fasting. Our main hypothesis was that fasting sparrows sequentially oxidize substrates in the order carbohydrates, lipids, and protein. We dosed 24 house sparrows with [(13)C]glucose, palmitic acid, or glycine and measured (13)CO(2) in their breath while they fasted for 24 h. To ascertain whether blood metabolite levels reflect fasting-induced changes in metabolic fuels, we also measured glucose, triacylglycerides, and β-hydroxybutyrate in the birds' blood. The results of both breath (13)CO(2) and plasma metabolite analyses did not support our hypothesis; i.e., that sparrows have the same metabolic responses characteristic of fasting-adapted species, but on a shorter time scale. Contrary to our main prediction, we found that recently assimilated (13)C-tracers were oxidized continuously in different patterns with no definite peaks corresponding to the three phases of fasting and also that changes in plasma metabolite levels accurately tracked the changes found by breath analysis. Notably, the rate of recently assimilated [(13)C]glycine oxidization was significantly higher (P < 0.001) than that of the other metabolic tracers at all postdosing intervals. We conclude that the inability of house sparrows to fast for longer than 32 h is likely related to their inability to accrue large lipid stores, separately oxidize different fuels, and/or spare protein during fasting.     

Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation

Synthesis of [(11)C]celecoxib, a selective COX-2 inhibitor, and [(11)C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug transporter in biliary excretion was evaluated. The synthesis of [(11)C]celecoxib was achieved in one-pot by reacting [(11)C]methyl iodide with an excess of the corresponding pinacol borate precursor using Pd(2)(dba)(3), P(o-tolyl)(3), and K(2)CO(3) (1:4:9) in DMF. The radiochemical yield of [(11)C]celecoxib was 63±23% (decay-corrected, based on [(11)C]CH(3)I) (n=7) with a specific radioactivity of 83±23GBq/μmol (n=7). The average time of synthesis from end of bombardment including formulation was 30min with >99% radiochemical purity. [(11)C]SC-62807 was synthesized from [(11)C]celecoxib by further rapid oxidation in the presence of excess KMnO(4) with microwave irradiation. The radiochemical yield of [(11)C]SC-62807 was 55±9% (n=3) (decay-corrected, based on [(11)C]celecoxib) with a specific radioactivity of 39±4GBq/μmol (n=3). The average time of synthesis from [(11)C]celecoxib including formulation was 20min and the radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [(11)C]celecoxib and [(11)C]SC-62807 showed largely different excretion processes, and consequently, [(11)C]SC-62807 was rapidly excreted via hepatobiliary excretion without further metabolism. [(11)C]SC-62807 was shown to have a high potential as a PET probe for evaluating drug transporter function in biliary excretion.     

8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase

The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.     

13CO2 washout dynamics during intermittent exercise in children and adults.

To test the hypothesis that children store less CO2 than adults during exercise, we measured breath 13CO2 washout dynamics after oral bolus of [13C]bicarbonate in nine children [8 +/- 1 (SD) yr, 4 boys] and nine (28 +/- 6 yr, 5 males) adults. Gas exchange [O2 uptake and CO2 production (Vco2)] was measured breath by breath during rest and during light (80% of the anaerobic threshold) intermittent exercise. Breath samples were obtained for subsequent analysis of 13CO2 by isotope ratio mass spectrometry. The tracer estimate of Vco2 was highly correlated to Vco2 measured by gas exchange (r = 0.97, P < 0.0001). The mean residence time was shorter in children (50 +/- 5 min) compared with adults (69 +/- 7 min, P < 0.0001) at rest and during exercise (children, 35 +/- 7 min; adults, 50 +/- 11 min, P < 0.001). The estimate of stored CO2 (using mean Vco2 measured by gas exchange and mean residence time derived from tracer washout) was not statistically different at rest between children (254 +/- 36 ml/kg) and adults (232 +/- 37 ml/kg). During exercise, CO2 stores in the adults (304 +/- 46 ml/kg) were significantly increased over rest (P < 0.001), but there was no increase in children (mean exercise value, 254 +/- 38 ml/kg). These data support the hypothesis that CO2 distribution in response to exercise changes during the growth period.     

Spermatogenic cell-somatic cell interactions are required for maintenance of spermatogenic cell glutathione

Sertoli cells play a major role in the regulation of spermatogenic cell energy metabolism and differentiation. This study demonstrates that Sertoli cells are essential for the maintenance of spermatogenic cell glutathione (GSH), an important intracellular reductant and detoxicant. Primary spermatocytes and round spermatids isolated from Xenopus laevis contained 1.5 +/- 0.1 mM GSH, but sperm lacked detectable GSH. During a 5-day culture period, isolated spermatocytes and spermatids lost 80% of the initial GSH (t 1/2 = 55 h). The levels of GSH were unaffected by L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of GSH synthesis. Cultures of testicular lobules and spermatocysts (composed of germ cells and Sertoli cells) depleted of interstitial tissue lost only 30% of their initial GSH in 4.5 days; the GSH levels decreased during treatment with BSO. Spermatogenic cells in cultured testes maintained their GSH levels for 7 days by a BSO-sensitive mechanism. These results demonstrate that the intracellular GSH levels of spermatogenic cells are dependent upon germ cell-somatic cell interactions. Spermatogenic cells were shown to possess gamma-glutamyl transpeptidase, glutathione synthetase, 5-oxoprolinase, and gamma-glutamylcysteine synthetase activities. [35S] Cysteine incorporation and distribution as analyzed by high performance liquid chromatography (HPLC) showed that isolated spermatogenic cells are capable of GSH synthesis. The rate of GSH synthesis, however, was insufficient to compensate for GSH turnover. These results demonstrate that production of spermatogenic cell GSH is dependent upon Sertoli cells. To our knowledge, this is the first evidence that interactions between different cell types may be of significance in GSH metabolism.     

Intestinal absorption and postabsorptive metabolism of linoleic acid in rats with short-term bile duct ligation

We investigated in bile duct-ligated (BDL) and sham-operated control rats whether the frequent presence of essential fatty acid deficiency in cholestatic liver disease could be related to linoleic acid malabsorption, altered linoleic acid metabolism, or both. In plasma of BDL rats, the triene-to-tetraene ratio, a biochemical marker for essential fatty acid deficiency, was increased compared with controls (0.024 +/- 0.004 vs. 0.013 +/- 0.001; P < 0.05). Net and percentage of dietary linoleic acid absorbed were decreased in BDL rats compared with control rats (1.50 +/- 0.16 mmol/day and 81.3 +/- 3.3% vs. 2.08 +/- 0.07 mmol/day and 99.2 +/- 0.1%, respectively; each P < 0.001). At 24 h after [(13)C]linoleic acid administration, BDL rats had a similar ratio of plasma [(13)C]arachidonic acid to plasma [(13)C]linoleic acid concentration compared with control rats. Delta(6)-Desaturase activity was not significantly different in hepatic microsomes from control or BDL rats. At 3 h after [(13)C]linoleic acid administration, plasma appearance of [(13)C]linoleic acid and cumulative expiration of (13)CO(2) were decreased in BDL rats, compared with controls (by 54% and 80%, respectively). The present data indicate that the impaired linoleic acid status in cholestatic liver disease is mainly due to decreased net absorption and not to quantitative alterations in postabsorptive metabolism.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

Resistance exercise acutely increases MHC and mixed muscle protein synthesis rates in 78-84 and 23-32 yr olds

We determined whether short-term weight-lifting exercise increases the synthesis rate of the major contractile proteins, myosin heavy chain (MHC), actin, and mixed muscle proteins in nonfrail elders and younger women and men. Fractional synthesis rates of mixed, MHC, and actin proteins were determined in seven healthy sedentary 23- to 32-yr-old and seven healthy 78- to 84-yr-old participants in paired studies done before and at the end of a 2-wk weight-lifting program. The in vivo rate of incorporation of 1-[(13)C]leucine into vastus lateralis MHC, actin, and mixed proteins was determined using a 14-h constant intravenous infusion of 1-[(13)C]leucine. Before exercise, the mixed and MHC fractional synthetic rates were lower in the older than in the younger participants (P < or = 0.04). Baseline actin protein synthesis rates were similar in the two groups (P = not significant). Over a 2-wk period, participants completed ten 1- to 1. 5-h weight-lifting exercise sessions: 2-3 sets per day of 9 exercises, 8-12 repetitions per set, at 60-90% of maximum voluntary muscle strength. At the end of exercise, MHC and mixed protein synthetic rates increased in the younger (88 and 121%) and older participants (105 and 182%; P < 0.001 vs. baseline). These findings indicate that MHC and mixed protein synthesis rates are reduced more than actin in advanced age. Similar to that of 23-32 yr olds, the vastus lateralis muscle in 78-84 yr olds retains the capacity to increase MHC and mixed protein synthesis rates in response to short-term resistance exercise.     

Kinetics of L-[1-(13)C]leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein

In two groups of five adults, each adapted to two different dietary regimens for 6 days, the metabolic fate of dietary [1-(13)C]leucine was examined when ingested either together with a mixture of free amino acids simulating casein (extrinsically labeled; condition A), along with the intact casein (extrinsically labeled; condition B), or bound to casein (intrinsically labeled; condition C). Fed state leucine oxidation (Ox), nonoxidative leucine disposal (NOLD), protein breakdown, and splanchnic uptake have been compared using an 8-h oral [1-(13)C]leucine and intravenous [(2)H(3)]leucine tracer protocol while giving eight equal hourly mixed meals. Lower leucine Ox, increased NOLD, and net protein synthesis were found with condition C compared with condition A (19.3 vs. 24.9; 77 vs. 55.8; 18.9 vs. 12.3 micromol. kg(-1). 30 min(-1); P < 0.05). Ox and NOLD did not differ between conditions B and C. Splanchnic leucine uptake calculated from [1-(13)C]- and [(2)H(3)]leucine plasma enrichments was between 24 and 35%. These findings indicate that the form in which leucine is consumed affects its immediate metabolic fate and retention by the body; the implications of these findings for the tracer balance technique and estimation of amino acid requirements are discussed.     

Phenylalanine kinetics in healthy volunteers and liver cirrhotics: implications for the phenylalanine breath test

Expired 13CO2 recovery from an oral l-[1-13C]phenylalanine ([13C]Phe) dose has been used to quantify liver function. This parameter, however, does not depend solely on liver function but also on total CO2 production, Phe turnover, and initial tracer distribution. Therefore, we evaluated the impact of these factors on breath test values. Nine ethyl-toxic cirrhotic patients and nine control subjects received intravenously 2 mg/kg of [13C]Phe, and breath and blood samples were collected over 4 h. CO2 production was measured by indirect calorimetry. The exhaled 13CO2 enrichments were analyzed by isotope ratio mass spectrometry and the [13C]Phe and l-[1-13C]tyrosine enrichments by gas chromatography-mass spectrometry. The cumulative 13CO2 recovery was significantly lower in cirrhotic patients (7 vs. 12%; P < 0.01), in part due to lower total CO2 production rates. Phe turnover in cirrhotic patients was significantly lower (33 vs. 44 micro mol. kg(-1). h(-1); P < 0.05). When these extrahepatic factors were considered in the calculation of the Phe oxidation rate, the intergroup differences were even more pronounced (3 vs. 7 micro mol. kg(-1). h(-1)) than those for 13CO2 recovery data. Also, the Phe-to-Tyr conversion rate, another indicator of Phe oxidation, was significantly reduced (0.7 vs. 3.0 micro mol. kg(-1). h(-1)).     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

Synthesis of [(14)C]pyrroloquinoline quinone (PQQ) in E. coli using genes for PQQ synthesis from K. pneumoniae

Radiochemical forms of pyrroloquinoline quinone (PQQ) are of utility in studies to determine the metabolic role and fate of PQQ in biological systems. Accordingly, we have synthesized [(14)C]PQQ using a tyrosine auxotrophic strain of Escherichia coli (AT2471). A construct containing the six genes required for PQQ synthesis from Klebsiella pneumoniae was used to transform the auxotrophic strain of E. coli. E. coli were then grown in minimal M9 medium containing 3.7x10(9) Bq/mmol [(14)C]tyrosine. At confluence, the medium was collected and applied to a DEAE A-25 anionic exchange column; [(14)C]PQQ was eluted using a KCl gradient (0-2 M in 0.1 M potassium phosphate buffer, pH 7.0). Radioactivity co-eluting as PQQ was next pooled, acidified and passed through a C-18 column; [(14)C]PQQ was eluted with a phosphate buffer (0.1 M, pH 7.0). Reverse phase HPLC (C-18) using either the ion-pairing agent trifluoroacetic acid (0. 1%) and an acetonitrile gradient or phosphoric acid and a methanol gradient were used to isolate [(14)C]PQQ. Fractions were collected and analyzed by liquid scintillation counting. (14)C-labelled compounds isolated from the medium eluted corresponding to the elution of various tyrosine-derived products or PQQ. The radioactive compound corresponding to PQQ was also reacted with acetone to form 5-acetonyl-PQQ, which co-eluted with a 5-acetonyl-PQQ standard, as a validation of [(14)C]PQQ synthesis. The specific activity of synthesized [(14)C]PQQ was 3.7x10(9) Bq/mmol [(14)C]PQQ, equal to that of [U-(14)C]tyrosine initially added to the medium.     

Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective 18F-labeling of peptides and proteins

2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [(18)F]FDG as a building block for the synthesis of (18)F-labeled compounds. The present study describes the use of [(18)F]FDG as a building block for the synthesis of (18)F-labeled peptides and proteins. [(18)F]FDG was converted into [(18)F]FDG-maleimidehexyloxime ([(18)F]FDG-MHO), a novel [(18)F]FDG-based prosthetic group for the mild and thiol group-specific (18)F labeling of peptides and proteins. The reaction was performed at 100 degrees C for 15 min in a sealed vial containing [(18)F]FDG and N-(6-aminoxy-hexyl)maleimide in 80% ethanol. [(18)F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [(18)F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [(18)F]FDG-MHO to thiol groups was investigated by the reaction with the tripeptide glutathione (GSH) and the single cysteine containing protein annexin A5 (anxA5). Radiolabeled annexin A5 ([(18)F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [(18)F]FDG-MHO, n = 6), and [(18)F]FDG-MHO-anxA5 was used for a pilot small animal PET study to assess in vivo biodistribution and kinetics in a HT-29 murine xenograft model.     

Non-invasive Monitoring of L-2-Oxothiazolidine-4-Carboxylate Metabolism in the Rat Brain by In vivo 13C Magnetic Resonance Spectroscopy

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.     

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.