Serum indoleamine 2,3-dioxygenase and kynurenine aminotransferase enzyme activity in patients with ischemic stroke.

We investigated the activation and pathophysiological roles of indoleamine 2,3-dioxygenase (IDO) and kynurenine aminotransferase (KAT) in patients with ischemic stroke. Patients were recruited from the acute stroke unit of a general hospital within 24 hours post-stroke. The immune transmission turbidity method was used to determine the concentration of serum high-sensitivity C-reactive protein (hsCRP), apolipoprotein A-1 and apolipoprotein B. The concentrations of triglyceride, cholesterol, high density lipoprotein (HDL), low density lipoprotein and non-esterified fatty acids were determined using an enzymatic method. Tryptophan (TRP), kynurenine (KYN) and kynurenine acid (KYNA) concentrations were determined by high performance liquid chromatography. The National Institutes of Health Stroke Scale (NIHSS) was used to assess the neurological deficits at admission and 3 weeks post-stroke. The IDO and KAT activity ratio were calculated by KYN/TRP and KYNA/KYN, respectively. The correlation between hsCRP and IDO, KAT and NIHSS score was also analyzed. A total of 81 patients with ischemic stroke and 35 normal controls were recruited. Lower TRP, KYNA, HDL and KAT activity ratio were found in the stroke group compared to the control group (p<0.05). The levels of hsCRP and IDO activity ratio were much higher in the stroke group than the control group (p<0.01). The IDO activity in patients with ischemic stroke showed a positive correlation with hsCRP (r=0.425, p=0.027). In addition, hsCRP and IDO levels were positively associated with the NIHSS score both at admission and 3 weeks post-stroke. These data suggest an inflammatory response characterized by up-regulated IDO activation in ischemic stroke, which might be closely relevant to its pathophysiology.

Carbonic anhydrases III and IV autoantibodies in rheumatoid arthritis, systemic lupus erythematosus, diabetes, hypertensive renal disease, and heart failure.

In the present study, the CA III and IV autoantibodies, CA activity, antioxidant enzymes and cytokines in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), diabetes, hypertensive renal disease, and heart failure were investigated. The anti-CA III antibody titers in patients with RA, SLE, and type 1 diabetes (T1D) were significantly higher than that in control groups (P < 0.05). The anti-CA IV antibody titers in patients with RA, SLE, type 1 diabetic nephropathy (T1DN), and heart failure were significantly higher than that in control groups (P < 0.05) while anti-CA IV antibody could suppress the total CA activity. The SOD and GPx levels in patients with RA, SLE, and T1DN were significantly lower than that in control groups (P < 0.05). IL-6, IL-17, IFN-γ, and TNF-α levels were significantly higher in SLE group compared with the control group (P < 0.05). Weak but significant correlations were found between anti-CA III antibodies and ESR in RA (r = 0.403, P = 0.013) and SLE patients (r = 0.397, P = 0.007). These results suggested that the generation of CA III and IV autoantibodies, antioxidant enzymes, and cytokines might influence each other and CA autoantibodies might affect the normal physiology function of CA.

[Simultaneous determination of kynurenine and kynurenic acid in serum by high performance liquid chromatography-fluorescence detection].

A method of high performance liquid chromatography-fluorescence detection (HPLC-FLD) for the simultaneous determination of kynurenine (Kyn) and kynurenic acid (KYNA) in serum was developed. A 20 microL of the supernatant of a serum sample deproteinized by 5% perchloric acid solution was separated on a Hypersil C8 column (300 mm x 6.0 mm, 10 microm) with an isocratic elution of 0.25 mol/L zinc acetate-50 mmol/L acetate containing 3% (v/v) acetonitrile. The fluorescence excitation and emission wavelengths were 365 nm and 480 nm at the beginning of the run; and 10 min later, the excitation and emission wavelengths were changed to 344 nm and 404 nm, respectively. The retention times of Kyn and KYNA were 8.1 min and 13.0 min, respectively. The linearities of the assay were from 98 to 19,600 nmol/L for Kyn and from 2.62 to 1047 nmol/L for KYNA; the detection limits were 50 nmol/L for Kyn and 0.11 nmol/L for KYNA. The average recoveries were 94.88% for Kyn, and 102.72% for KYNA. The within-day and between-day relative standard deviations (RSD) were 3.87% and 3.94% for Kyn; and 3.79% and 4.71% for KYNA, respectively. The results indicated phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), 5-hydroxytryptamine (5-HT), and creatinine (Cr) had no interfering effects to the determination of Kyn and KYNA. Kyn and KYNA concentrations in the sera of 71 health people were (1.40 +/- 0.34) micromol/L and (24.22 +/- 8.67) nmol/L, respectively. The method is simple, rapid, accurate, convenient, and suitable for routine analysis.

More rapid and sensitive method for simultaneous determination of tryptophan and kynurenic acid by HPLC.

OBJECTIVE: To describe a simple, rapid, and sensitive HPLC method for simultaneous determination of TRP and KYNA in human serum. DESIGN AND METHOD: Samples were deproteinized by perchloric acid. KYNA was detected at 344 nm of excitation wavelength and 404 nm of emission wavelength, TRP was detected at 254 nm and 404 nm, with a total run time of 13 min per sample. RESULTS: Standard curves of 0.49 micromol/L to 196 micromol/L of TRP were linear. Inter-day and intra-day coefficient of variations were 3.31% and 4.14%, respectively. Average recovery was 104.43%. Detection limit was 0.001 micromol/L. The linearity of the assay was maintained from 1.5 nmol/L to 2093 nmol/L of KYNA. Inter-day and intra-day CVS were 3.20% and 4.27%, respectively. Average recovery was 101.19%. Detection limit was 0.05 nmol/L. CONCLUSION: The developed HPLC method is simple, convenient and can be applied in the diagnosis of related diseases.

[Determination of kynurenine in serum using high performance liquid chromatography-fluorescence detection].

A sensitive method of high performance liquid chromatography-fluorescence detection (HPLC-FLD) for the determination of kynurenine (Kyn) in serum was developed. A 20 microL supernatant of a serum sample deproteinized by 5% perchloric acid solution was assayed and separated on a Hypersil C8 column (300 mm x 6.0 mm, 10 microm) with an isocratic elution of 0.25 mol/L zinc acetate-50 mmol/L acetate containing 3% (v/v) acetonitrile. The fluorescence excitation and emission wavelengths were 365 nm and 480 nm, respectively. The limit of detection was approximately 0.04 micromol/L (signal-to-noise ratio of 3) and the linearity of the assay was from 0.098 micromol/L to 19.6 micromol/L. The relative standard deviations of intraday and interday determinations were 3.8% and 4.6%, respectively. The results indicated tryptophan (Trp), 5-hydroxytryptamine (5-HT), kynurenic acid (KYNA), phenylalanine (Phe), tyrosine (Tyr) and creatinine (Cr) had no interfering effects to the determination of Kyn. The method is of high accuracy, easy operation, satisfactory recoveries and good reproducibility, and can be used for routine analysis.