Current status and future of clinical islet xenotransplantation.

ß-Cell replacement therapy, including allogeneic pancreas and islet transplantation, can normalize HbA1c levels in unstable type 1 diabetic (T1D) patients, but a donor shortage is a serious issue. To overcome this problem, xenotransplantation is an attractive option. In fact, islet transplantation from porcine pancreata was performed in the 1990s, which opened the door for islet xenotransplantation, but the possibility of porcine endogenous retrovirus (PERV) infection was raised, which has restricted progress in this field. The International Xenotransplantation Association published a consensus statement on conditions for undertaking clinical trials of porcine islet products in T1D to restart islet xenotransplantation safely. Clinical porcine islet xenotransplantation was restarted under comprehensive regulations in New Zealand. In addition, newly emerged gene-editing technologies have activated the xenotransplantation field. Islet xenotransplantation is becoming a clinical reality, with the results of recent studies showing promise to advance this field.

Effect of an omega-3 lipid emulsion in reducing oxidative stress in a rat model of intestinal ischemia-reperfusion injury.

OBJECTIVES: The usefulness of omega-3 lipid emulsions has been extensively studied. The objectives of the present study were to examine the effect of an omega-3 lipid emulsion in reducing oxidative stress in a rat model of intestinal ischemia-reperfusion injury and the underlying mechanism. METHODS: A total of 66 rats were divided into three dietary groups (lipid-free, soybean oil, and fish oil groups). Each animal was administered total parenteral nutrition for 3 days, followed by induction of intestinal ischemia for 100 min. Animals subjected to sham surgery served as the controls. Intestinal tissue and blood were harvested 6 and 12 h after the surgery, then, assessment of the histological damage score, plasma-related parameters, and statistical evaluation were performed. RESULTS: The histological damage score in the intestinal tissues was significantly lower in the fish oil group than in the soybean oil group (P = 0.0121). The late-phase urinary level of 8-hydroxy-2-deoxyguanosine was also significantly lower in the fish oil group as compared with that in the other groups (P = 0.0267). Furthermore, the plasma level of high-mobility group box 1 protein was also significantly lower in the fish oil group as compared with that in the lipid-free group (P = 0.0398). CONCLUSION: It appeared that intravenous administration of an omega-3 lipid emulsion prior to ischemia-reperfusion injury reduced the oxidative stress and severity of tissue damage. Modification of membrane fatty acids may serve as the mechanism underlying this reduction of tissue damage.

Determination of kynurenic acid levels in rat brain microdialysis samples and changes in kynurenic acid levels induced by N-acetylaspartic acid.

The levels of kynurenic acid, an endogenous antagonist of alpha(7) nicotinic acetylcholine and N-methyl-D-aspartate receptors, were measured in microdialysis samples obtained from the prefrontal cortices of rats using column-switching high-performance liquid chromatography with fluorescence detection. When the perfusate was constantly infused at a rate of 1.0 mu/min, the in vitro recovery of kynurenic acid through the dialysis membrane was approximately 20.4%, and the precision was within 1.31%. Endogenous kynurenic acid in the microdialysis sample was clearly detected using column-switching high-performance liquid chromatography. As an application study, N-acetyl-L-aspartic acid, an endogenous metabolite and precursor of N-acetyl-L-aspartyl-L-glutamic acid, which is an agonist of metabotropic glutamate receptors, was infused for 120 min through the microdialysis probe. The kynurenic acid level significantly increased during the infusion of N-acetyl-L-aspartic acid, suggesting that kynurenic acid might have some association with N-acetyl-L-aspartic acid in vivo.

Alteration of kynurenic acid concentration in rat plasma following optically pure kynurenine administration: a comparative study between enantiomers.

L-Kynurenine (KYN), a tryptophan metabolite, is metabolized to kynurenic acid (KYNA), which is an antagonist of N-methyl-D-aspartate and alpha7 nicotinic acetylcholine receptors, by kynurenine aminotransferase (KAT) I and KAT II. In this study, optically pure KYN, namely L-KYN or D-KYN, was administered intraperitoneally to male Sprague-Dawley rats (16.3 micromol kg(-1)), and the change in plasma KYNA was investigated by using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. Unexpectedly, no remarkable alteration in the plasma KYNA was observed when a natural isomer, L-KYN, was administered, whereas plasma KYNA concentration was unequivocally increased when an unnatural isomer, D-KYN, was administered. Serum protein bindings of L-KYN and D-KYN were also studied, and the protein binding of L-KYN (approximately 65%) in rat serum was larger than that of D-KYN (approximately 12%), suggesting that D-KYN may be easily incorporated and metabolized in tissues during blood circulation to generate KYNA in mammals. In addition, the increase in plasma KYNA by the administration of D-KYN was suppressed in rats pretreated with a selective inhibitor of D-amino acid oxidase (DAAO), 5-methylpyrazole-3-carboxylic acid (80 mg/kg). These results suggest that DAAO might be responsible for the production of KYNA from D-KYN in vivo.

Simultaneous determination of N-acetylaspartylglutamate and N-acetylaspartate in rat brain homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization.

Simultaneous determination method of N-acetyl-l-aspartyl-l-glutamate (NAAG), an endogenous agonist at type 3 metabotropic glutamate receptor, and its degradation product, N-acetyl-l-aspartate (NAA) was developed by using reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. The detection limits of NAAG and NAA were approximately 12 and 34 fmol on the column, respectively (signal to noise ratio 3). The proposed HPLC method was applied to determine NAAG and NAA simultaneously in the rat brain homogenate. Both concentrations of NAAG and NAA in the male rat cerebrum (13 weeks old) were 5.7+/-0.30 and 2.1 x 10(2)+/-9.2 nmol/mg protein, respectively (n=6), while those in the hippocampus were 6.8+/-0.48 and 1.9 x 10(2)+/-8.5 nmol/mg protein, respectively (n=5). Hippocampal NAA concentration was significantly increased in the ketamine-treated rats as compared to the control rats (p<0.01).

Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization.

N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84 +/-4.6 nmol/mg protein (n = 3) [corrected].

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization.

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 degrees C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 microL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and -10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4+/-0.32 micromol L(-1) (n=4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats--the animal model of schizophrenia.

Development of a column-switching high-performance liquid chromatography for kynurenine enantiomers and its application to a pharmacokinetic study in rat plasma.

Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N-methyl-d-aspartate receptor. In this study, an enantiomeric separation of d,l-KYN derivatized with the benzofurazan fluorescence reagent 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD-d,l-KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD-d,l-KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H(2)O/CH(3)CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d- and l-KYN enantiomers in 10 microL of rat plasma following the intraperitoneal administration of d,l-KYN to rats (10 mg kg(-1)). The result revealed that the concentration of l-KYN was higher than that of d-KYN, suggesting that d-KYN was eliminated faster than l-KYN.

Alterations in serum amino acid concentrations in male and female schizophrenic patients.

BACKGROUND: Since several studies have investigated gender-related differences in the onset of disease, response to drug therapy, etc. in schizophrenic patients, we examined the alterations in serum amino acids concentrations in male and female patients separately. METHODS: Serum amino acid concentrations in the normal (n=35; male 21 and female 14) and schizophrenic patients (n=32; male 19 and female 13) were determined by HPLC using a pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole. RESULTS: Serum glutamate and serine concentrations were significantly increased in the male schizophrenic patients (p=0.0161 and 0.0257, respectively), while the serum Pro concentration was significantly increased in female schizophrenic patients (p=0.0398). Serum Glu, Ser, and Pro concentrations in the patients did not significantly correlate with the age, age of onset of disease, duration of illness, and chlorpromazine equivalents. Among the amino acids, serum Orn concentrations in male and female schizophrenic patients positively correlated with the duration of illness (p<0.01, r=0.685 and 0.688, respectively). CONCLUSION: The present data suggest the existence of gender-related differences in the alterations in serum amino acid concentrations in schizophrenic patients; further, serum Orn concentration in both sexes might be influenced by medications.

Determination of rat brain kynurenic acid by column-switching HPLC with fluorescence detection.

Kynurenic acid (KYNA), one of the tryptophan metabolites, serves as an endogenous antagonist of N-methyl-d-aspartate and the alpha7 nicotinic receptors in mammalian brains. In the present study, the column-switching high-performance liquid chromatography (HPLC) method we developed for plasma KYNA was extended and validated for the determination of brain KYNA. Rat cerebrum, cerebellum and brainstem homogenates were deproteinized with acetone, and the extracts reconstituted with the mobile phase were injected onto the HPLC. In spite of the facile pretreatment, the fluorescence peak of KYNA in the cerebrum, cerebellum and brainstem was clearly observed with no interfering peaks. Intra- and inter-day precisions [relative standard deviation (%)] and accuracies [relative mean error (%)] were satisfactory (< +/-5.8%). The concentrations of KYNA in rat cerebrum, cerebellum, and brainstem were 224 +/- 65.8, 606 +/- 191, and 323 +/- 114 fmol/mg protein (n = 5), respectively. The proposed HPLC method will be a useful tool for pharmacokinetic and pharmacological researches on brain KYNA.

Determination of kynurenic acid in human serum and its correlation with the concentration of certain amino acids.

BACKGROUND: Kynurenic acid (KYNA)--a tryptophan metabolite--elicits antagonistic activity against glutaminergic and cholinergic receptors; it has been suggested to have some relationship with neurological disorders. Considering this, serum KYNA may be an important marker in clinical diagnosis. We determined serum KYNA concentration and elucidate its correlation with several amino acids in human serum. METHODS: KYNA and amino acids concentrations in human serum of healthy subjects [n=35 (21 males and 14 females)] were determined by HPLC with fluorescence detection; thus, the correlation between KYNA concentration and that of several amino acids was examined in these subjects. RESULTS: Of the amino acids examined in this study, a significant negative correlation was observed between KYNA and glutamine (Gln) concentrations (r=-0.452, p<0.01) in the healthy subjects, particularly males (r=-0.687, p<0.01), and age-related changes were not observed. In addition to Gln, Gly and Ala concentrations showed a significant negative correlation with KYNA concentration in the serum of male subjects (r=-0.440 and -0.456, respectively, p<0.05). CONCLUSION: The significant correlation between KYNA and Gln concentrations in vivo may support the previous finding that kynurenine aminotransferase I (KAT I), responsible for the biosynthesis of KYNA, was identical to Gln transaminase K (GTK), which catalyses the transamination of Gln to 2-oxoglutamic acid. Both KYNA and Gln concentrations in vivo might be influenced due to altered KAT I/GTK activity.

Alterations of plasma and cerebrospinal fluid glutamate levels in rats treated with the N-methyl-D-aspartate receptor antagonist, ketamine.

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia.