YgfY Contributes to Stress Tolerance in Shewanella oneidensis Neither as an Antitoxin Nor as a Flavinylation Factor of Succinate Dehydrogenase.

YgfY(SdhE/CptB) is highly conserved while has controversial functions in bacteria. It works as an antitoxin and composes a type IV toxin-antitoxin system with YgfX(CptA) typically in Escherichia coli, while functions as an flavinylation factor of succinate dehydrogenase and fumarate reductase typically in Serratia sp. In this study, we report the contribution of YgfY in Shewanella oneidensis MR-1 to tolerance of low temperature and nitrite. YgfY deficiency causes several growth defects of S. oneidensis MR-1 at low temperature, while YgfX do not cause a growth defect or morphological change of S. oneidensis MR1-1 and E. coli. YgfY do not interact with FtsZ and MreB nor with YgfX examined by bacterial two-hybrid assay. YgfY effect on growth under low temperature is not attributed to succinate dehydrogenase (SDH) because a mutant without SDH grows comparably with the wild-type strain in the presence of succinate. The ygfY mutant shows impaired tolerance to nitrite. Transcription of nitrite reductase and most ribosome proteins is significantly decreased in the ygfY mutant, which is consistent with the phenotypes detected above. Effects of YgfY on growth and nitrite tolerance are closely related to the RGXXE motif in YgfY. In summary, this study demonstrates pleiotropic impacts of YgfY in S. oneidensis MR-1, and sheds a light on the physiological versatility of YgfY in bacteria.

SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth.

Renal cell carcinoma (RCC) cells have increased lipogenesis and cholesterol synthesis. Sterol regulatory element-binding protein-1 (SREBP1) is cleaved by site 1 protease (S1P) to release the transcriptionally active amino-terminal domain. PF-429242 is a potent and competitive S1P inhibitor. We here tested its activity in RCC cells. In established and primary human RCC cells, PF-429242 potently inhibited cell proliferation, migration, and invasion. The S1P inhibitor provoked apoptosis activation in RCC cells. Furthermore, shRNA-mediated S1P silencing or CRISPR/Cas9-induced S1P knockout led to RCC cell growth inhibition and apoptosis activation. Conversely, ectopic overexpression of SREBP1 or S1P augmented RCC cell proliferation and migration. Daily i.v. injection of a single dose of PF-429242 robustly inhibited RCC xenograft growth in severe combined immunodeficiency mice. Additionally, intratumoral injection of S1P shRNA lentivirus inhibited RCC xenograft growth in mice. SREBP1, S1P, and its target gene low density lipoprotein receptor (LDLR) were significantly elevated in human RCC tissues. These results suggest that targeting S1P by PF-429242 inhibited RCC cell growth in vitro and in vivo.

Pyruvate accelerates palladium reduction by regulating catabolism and electron transfer pathway in Shewanella oneidensis.

Shewanella oneidensis is a model strain of the electrochemical active bacteria (EAB) because of its strong capability of extracellular electron transfer (EET) and genetic tractability. In this study, we investigated the effect of carbon sources on EET in S. oneidensis by using reduction of palladium ions (Pd(II)) as a model and found that pyruvate greatly accelerated the Pd(II) reduction compared with lactate by resting cells. Both Mtr pathway and hydrogenases played a role in Pd(II) reduction when pyruvate was used as a carbon source. Furthermore, in comparison with lactate-feeding S. oneidensis, the transcriptional levels of formate dehydrogenases involving in pyruvate catabolism, Mtr pathway, and hydrogenases in pyruvate-feeding S. oneidensis were up-regulated. Mechanistically, the enhancement of electron generation from pyruvate catabolism and electron transfer to Pd(II) explains the pyruvate effect on Pd(II) reduction. Interestingly, a 2-h time window is required for pyruvate to regulate transcription of these genes and profoundly improve Pd(II) reduction capability, suggesting a hierarchical regulation for pyruvate sensing and response in S. oneidensis IMPORTANCE The unique respiration of EET is crucial for the biogeochemical cycling of metal elements and diverse applications of EAB. Although a carbon source is a determinant factor of bacterial metabolism, the research into the regulation of carbon source on EET is rare. In this work, we reported the pyruvate-specific regulation and improvement of EET in S. oneidensis and revealed the underlying mechanism, which suggests potential targets to engineer and improve the EET efficiency of this bacterium. This study sheds light on the regulatory role of carbon sources in anaerobic respiration in EAB, providing a way to regulate EET for diverse applications from a novel perspective.

Dose-dependent effects of neuropeptide Y on the regulation of preadipocyte proliferation and adipocyte lipid synthesis via the PPARγ pathways.

The objective of this study was to investigate the impact of neuropeptide Y (NPY) on preadipocyte proliferation and differentiation. Preadipocytes were incubated with a range of concentrations of NPY (10(-15)M - 10(-7)M). After NPY-induced differentiation, the extent of preadipocyte adipogenesis was evaluated. The expressions levels of related adipocyte markers such as PPARγ, C/EBPα and DLK-1 were examined by real-time PCR (RT-PCR) or western blot analysis. Furthermore, the mitogen-activated protein kinase (MAPK) signaling pathway proteins were also analyzed by western blot. Our results showed that low doses of NPY stimulated preadipocyte viability and proliferation, while high NPY doses inhibited cell viability. At high concentrations of NPY significantly promoted lipid accumulation and increased the size of lipid droplets. DLK-1 mRNA expression was inhibited, but the expression levels of PPARγ and C/EBPα were increased during differentiation with the presence of high concentration of NPY. High-dose NPY also suppressed the phosphorylation of the extracellular signal-regulated kinase (ERK) 1/2 protein. We conclude that NPY has a biphasic effect on preadipocyte proliferation. A high dose inhibits the proliferation of 3T3-L1 cell while promotes adipocyte differentiation, increasing lipid accumulation especially enlarged lipid droplets' size. NPY may lead to a better understanding for drug development to prevent hyperplastic obesity and hypertrophic obesity.

Serum pepsinogen reference intervals in apparently healthy Chinese population with latex enhanced turbidimetric immunoassay.

AIM: Serum pepsinogen (sPG) has been used to help in diagnosing atrophic corpus gastritis and in screening for gastric cancer non-invasively. There are as yet no reports on sPG reference intervals (RIs) with latex enhanced turbidimetric immunoassay (LIA). In this study, we established the RIs for sPG in a healthy Chinese population using LIA. METHODS: Serum PGI and PGII levels in a healthy population (aged 17-80 years) were measured simultaneously using LIA. RIs were determined following Clinical Laboratory and Standards Institute C28-A3 guidelines using a non-parametric method. RESULTS: 95% RIs in men (ng/mL) were: ≤40 years old, 25.53-100.76 for PGI and ≤24.42 for PGII; 41-50 years old, 26.62-124.74 for PGI and ≤26.81 for PGII; and 51-80 years old, 30.40-153.25 for PGI and ≤32.62 for PGII. Corresponding RIs for women (ng/mL) were: ≤40 years old, 21.20-87.44 for PGI and ≤25.53 for PGII; and 41-80 years, 26.40-127.46 for PGI and ≤30.18 for PGII. 95% RI for PGI/PGII in both men and women at any age was ≥2.51. CONCLUSIONS: We established the RIs for sPG using LIA in a healthy Chinese population, which can provide a reference for clinical and laboratory studies.

Reference intervals for serum creatinine levels in the healthy geriatric population.

BACKGROUND: There are few reliable reference values of enzymatically assayed serum creatinine (Scr) levels categorized within a small age interval in the healthy geriatric population. The aim of this study was to establish the reference intervals (RIs) for Scr in the elderly population. METHODS: Healthy, elderly Chinese Han ethnic individuals aged between 60 and 89 years old were recruited for this study. We stratified the reference individuals by gender and age (60-69, 70-79 and 80-89 years), and the Scr values were measured by an enzymatic method. The central 95 percentile RIs were determined using non-parametric statistical methods. RESULTS: The Scr values in the elderly population show a Gaussian distribution and age/sex related differences. The RIs for Scr in the reference population with respect to age (ranges of 60-69, 70-79 and 80-89 years) were 52.9-94.5, 57.3-106.2 and 59.0-110.8 µmol/L for males, respectively, and 44.3-75.4, 47.1-85.5 and 45.1-90.9 µmol/L for females, respectively. CONCLUSIONS: We have established the RIs for Scr measured with an enzymatic method in the healthy Chinese Han ethnic elderly population, which can provide a reference for both clinical and laboratory studies.

Simultaneous determination of phenylalanine and tyrosine in peripheral capillary blood by HPLC with ultraviolet detection.

OBJECTIVES: Phenylalanine (Phe) and tyrosine (Tyr) are the most reliable indicators for the diagnosis of phenylketonuria (PKU). The purpose of this study is to establish a simple and rapid method for the determination of Phe and Tyr in peripheral capillary blood from newborns and children by high performance liquid chromatography with ultraviolet detection (HPLC-UV). METHODS: Peripheral blood specimens were taken from newborns or children by heel stick or skin puncture on the fingers. Blood samples were deproteinized by equal volume of 5% perchloric acid. Amino acid separation was carried out on a Hypersil C8 column. The mobile phase containing 5% acetonitrile (v/v) was run at a flow rate of 1.5 mL/min. Phe and Tyr were determined in the ultraviolet detector at 210 nm. RESULTS: The determination was within 10.0 min. The linear range was from 6.0 to 1512.0 µmol/L for Phe and 5.5 to 1250.0 µmol/L for Tyr. Although Phe and Tyr levels from peripheral blood samples were relatively lower than those from serum or plasma, the data showed a good agreement between them. CONCLUSION: The method we developed is simple, rapid and convenient. It is useful for screening and diagnosis of PKU in newborns and children.

Utility of NT-proBNP for identifying LV failure in patients with acute exacerbation of chronic bronchitis.

BACKGROUND: NT-proBNP has been widely regarded as a useful tool for diagnosis or exclusion of heart failure (HF) in many settings. However, in patients with acute exacerbation of chronic bronchitis (AECB), its roles have not been well described. The objective of this study was to evaluate the diagnostic performance of NT-proBNP for identifying left ventricular (LV) failure in such patients. METHODS AND RESULTS: 311 AECB patients and 102 stable chronic bronchitis patients with no history of HF were enrolled. Plasma NT-proBNP concentrations were measured using Roche Elecsys. The European Society of Cardiology (ESC) diagnostic principles were adopted to identify HF and the diagnostic performance of NT-proBNP was evaluated by ROC. Our results showed, the median NT-proBNP level in patients with LV failure [4828.4 (2044.4-9203.6) ng/L] was significantly higher than that in those without LV failure [519.2 (179.1-1409.8) ng/L, p<0.001] and stable controls [207.5 (186.5-318.2) ng/L, p<0.001]. LV failure, renal function, atrial fibrillation and systolic pulmonary artery pressure were independent predictors of NT-proBNP levels (all p<0.05). The area under ROC curve (AUC) of NT-proBNP for identifying LV failure was 0.884, significantly superior to clinical judgment alone (AUC 0.835, p = 0.0294). At the optimal cutoff value of 935.0 ng/L, NT-proBNP yielded sensitivity 94.4%, specificity 68.2%, accuracy 74.3% and negative predictive value 97.6%. Adding the results of NT-proBNP to those of clinical judgment improved the diagnostic accuracy for LV failure. CONCLUSION: As a tool for diagnosis or exclusion of HF, NT-proBNP can help physicians identify LV failure in patients with AECB.

Aberrant expression of E-cadherin in lung tissues of patients with probable lung cancer.

INTRODUCTION: This study assessed the relationship of E-cadherin mRNA and protein expression with the diagnosis of lung cancer with the aim of providing an auxiliary diagnostic method. METHODS: Semi-quantitative nested RT-PCR and western blotting were applied to detect E-cadherin mRNA transcripts and protein, respectively, in 30 cases of diagnostic lung cancer, 30 cases of clinically suspected patients with lung cancer and 30 cases of other disease. Immunohistochemical staining was also used to detect E-cadherin. RESULTS: Remarkably decreased levels of relative E-cadherin mRNA value and increased E-cadherin protein negativity were observed in probable lung cancer, when compared with possible lung cancer and others. With a threshold of 1.45, relative E-cadherin mRNA value showed a sensitivity of 90% and a specificity of 83% for the diagnosis of lung cancer. The combination of decreased relative E-cadherin mRNA value and negative E-cadherin protein increased the specificity and sensitivity. CONCLUSION: These data suggest that Chinese patients with diagnostic lung cancer have similar decreased levels of relative E-cadherin mRNA and E-cadherin protein value in the lung cancer tissues as in lung cancer patients in other countries. Measurement of relative E-cadherin mRNA and protein values in lung cancer tissues has potential for lung cancer diagnosis.

Simultaneous determination of tyrosine, tryptophan and 5-hydroxytryptamine in serum of MDD patients by high performance liquid chromatography with fluorescence detection.

BACKGROUND: Tyrosine (Tyr), Tryptophan (Trp) and 5-hydroxytryptamine (5-HT) are important amino acids in vivo and have been hypothesized to be involved in many mental disorders. We developed a rapid and sensitive HPLC method for simultaneous measurement of serum Tyr, Trp and 5-HT and explored the clinical significances of Tyr, Trp and 5-HT and the 5-HT/Trp ratio for patients with major depressive disorder (MDD) disease. METHODS: Serum samples were deproteinized by 5% perchloric acid and separated on an Atlantis C18 column (4.6 × 150 mm, 5 µm) with the mobile phase consisting of 0.1 mol/l KH(2)PO(4) and methanol (85:15, V/V).The eluates were monitored by the fluorescence detection with programmed wavelength. RESULTS: Analysis was achieved in <12.0 min. The limits of quantification were 0.014, 0.005, and 0.024 µmol/l for Tyr, Trp and 5-HT, respectively. Reproducibility and recovery were satisfactory. Tyr, Trp and 5-HT and the 5-HT/Trp ratio were significantly decreased in patients with MDD. CONCLUSIONS: In diseases, like MDD, Tyr, Trp and 5-HT play an important role. This method can potentially be applied as prognostic or diagnostic tool or even to follow the evolution of the illness or of the treatment.

Clinical significance of simultaneous determination of serum tryptophan and tyrosine in patients with lung cancer.

BACKGROUND: To explore the clinical significance of serum tyrosine (Tyr) and tryptophan (Trp) in patients with lung cancer, we used a simple and efficient method of high-performance liquid chromatography with fluorescence detection (HPLC-FD) that simultaneously measured serum Trp and Tyr contents. METHODS: The concentrations of Tyr and Trp were measured simultaneously by HPLC-FD in the sera of 80 patients with lung cancer and 120 healthy controls. RESULTS: Trp concentrations were significantly lower in patients with lung cancer than in healthy controls (39.26±5.44 vs. 49.93±5.43 µmol/l, respectively; P<0.01), whereas in Tyr concentrations there were no differences with healthy controls (65.38±7.94 vs.66.40±8.55 µmol/l, respectively; P>0.05). In addition, patients in the adenocarcinoma group had significantly lower Trp and Tyr concentrations than those in squamous cell carcinoma group. There was no difference between the early stage and advanced stage of lung cancer. CONCLUSIONS: Determination of serum Trp and Tyr concentrations can be employed to assist the diagnosis of the histotypes of lung cancer and tumor stage. Tyr and Trp as indexes on the lung cancer diagnostic sensitivity, specificity were 54.9, 62.9% and 82.4, 92.1%, Trp is an important and special index for lung cancer diagnosis of which the specificity of diagnosis of lung cancer is more than 92%.

HPLC-FLD determination of serum aromatic amino acids: application in chronic kidney disease patients.

BACKGROUND: The aromatic amino acids (AAA) are very important amino acids in the body and have been suggested to be involved in many diseases. We describe the development and full validation of a high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for simultaneously quantitative determination of serum AAA. Furthermore, we aimed to explore the clinical significances of AAA for chronic kidney disease (CKD). METHODS: Serum samples were deproteinized by perchloric acid. Separation was carried out on a Megres C18 column (4.6 mm×250 mm i.d., 5 µm). The mobile phase consisted of 10% acetonitrile (v/v) in water and was run at a flow rate of 1.0 ml/min. The eluted AAA was monitored by measuring the fluorescence intensity using the programmed wavelength detection setting. RESULTS: AAA determination in a serum sample was achieved in <10 min. The linear ranges of the method were 0.55-275 µmol/l, 3.05-1220.0 µmol/l and 0.049-49 µmol/l for Tyr, Phe and Trp, respectively. The limit of detection (LOD) was 0.014 µmol/l for Tyr, 0.5 µmol/l for Phe, and 0.0049 µmol/l for Trp. Recovery and reproducibility were satisfactory. Compared with healthy control subjects, the CKD patients showed lower serum levels of AAA. It should probably available to apply for screening and monitoring of CKD. CONCLUSIONS: The method is simple, fast, accurate, and suitable for routine analysis.

Insulin receptor substrate 1 regulates the cellular differentiation and the matrix metallopeptidase expression of preosteoblastic cells.

Insulin receptor substrate 1 (IRS1) is an essential molecule for the intracellular signaling of IGF1 and insulin, which are potent anabolic regulators of bone metabolism. Osteoblastic IRS1 is essential for maintaining bone turnover; however, the mechanism underlying this regulation remains unclear. To clarify the role of IRS1 in bone metabolism, we employed RNA interference to inhibit IRS1 gene expression and observed the effects of silencing this gene on the proliferation and differentiation of and the expression of matrix metallopeptidase (MMP) and tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B) in MC3T3-E1 cells. Our results showed that IRS1 short hairpin RNAs can effectively suppress the expression of IRS1, and inhibit the phosphorylation of AKT in IRS1 pathway; reduce the expression of MMP2, MMP3, MMP13, and MMP14, decrease the expression of TNFRSF11B and RANKL (also known as tumor necrosis factor (ligand) superfamily, member 11), however increase the RANKL/TNFRSF11B ratio; decrease cell survival, proliferation, and mineralization, and impair the differentiation of MC3T3-E1 cells. The downregulation of IRS1 had no effect on the expression of MMP1. Our findings suggest that IRS1 not only promotes bone formation and mineralization but also might play roles in bone resorption partly via the regulation of MMPs and RANKL/TNFRSF11B ratio, thus regulates the bone turnover.

Simultaneous determination of serum tryptophan metabolites in patients with systemic lupus erythematosus by high performance liquid chromatography with fluorescence detection.

BACKGROUND: To provide a more comprehensive clinic marker of tryptophan (TRP) catabolism in patients with systemic lupus erythematosus (SLE), we developed a simple and efficient method that simultaneously measured serum TRP, kynurenine (KYN), and kynurenic acid (KYNA) using high performance liquid chromatography with fluorescence detection (HPLC-FD). METHODS: A simple and specific high performance liquid chromatography (HPLC) method was developed for simultaneously quantitative determination of TRP, KYN and KYNA with fluorescence detection (FD) using programmed wavelength and on-column fluorescence derivatization. Thirty patients with SLE and 80 healthy control subjects were analyzed for serum TRP metabolites using the assay we developed. The tryptophan breakdown index (TBI) and neuroprotective ratio (NPR) were calculated. RESULTS: The retention time of KYN, KYNA and TRP were 8.5 min, 13.7 min and 17.6 min, respectively. The linear range for TRP was 0.245-196 micromol/L, the limit of detection (LOD) was 0.001 micromol/L and average recovery was 103.71%. The linear range for KYN was 0.049-98 v/L, the LOD was 0.0245 micromol/L, and average recovery was 97.45%. The linear range for KYNA was 1.05-2093 nmol/L, the LOD was 0.05 nmol/L, and average recovery was 100.60%. Inter-day and intra-day relative standard deviations (SDs) were <5%. Phenylalanine, tyrosine, 5-hydroxytryptamine and creatinine did not interfere with the method. The results showed great differences in TRP, KYN and KYNA contents and TBI between patients with SLE and healthy controls, but little difference in NPR. CONCLUSIONS: The method is simple, fast, accurate, and meets the requirements for simultaneous determination of TRP, KYN and KYNA in serum.

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 platelet serotonin by high performance liquid chromatography with fluorometric detector].

OBJECTIVE: To establish a simple, rapid and precise method to determine platelet serotonin. METHODS: Serotonin (5-hydroxytryptamine; 5-HT) was determined in platelet pellet after simple deproteinization, following ion-pair high performance liquid chromatography (HPLC) on reverse-phase column: Nova-pak C18 column (150 mm x 4.6 mm, 4 microm), serotonin and internal standard (N-methylserotonin) were detected by fluorometry. RESULTS: The intra-day CV was 2.29% and inter-day CV was 3.88%. The mean recovery of serotonin in platelet was 97.5%, the linear range was 20 - 20000 nmol/ L, and the detection limit was 1 nmol/L. 5-hydroxyindole acetic acid (5-HIAA), Trp, Phe and Tyr had no interference with the chromatographic condition used in this trial. CONCLUSION: The metod described here is simple, convenient, rapid, sensitive, and accurate for clinical and scientific research.

[Determination of phenylalanine and tyrosine in serum by high-performance liquid chromatography with fluorescent detection].

OBJECTIVE: To determine phenylalanine and tyrosine in serum by high-performance liquid chromatography (HPLC) method. METHODS: Serum precipitated with perchloric acid was isocratically eluted using a base-deactivated C8 column with 10% acetonitrile in water as the mobile phase. The flow rate was 1.0 ml/min. Fluorescent detector worked at an excitation wavelength of 210 nm and emission wavelength of 303 nm. RESULTS: Good linearity was observed in the range of 12.2 approximately 1 220.0 micromol/L for phenylalanine, the intra- and inter-assay coefficients of variation (CV) were 4.07% and 7.17% respectively, and the average recovery was 102.5%. There also was a good linearity in the range of 5.5 approximately 550.0 micromoL/L for tyrosine. The intra- and inter-assay coefficients of variation were 2.99% and 5.23% respectively, and the average recovery was 102.2%. The total assay time for the serum sample was within 30 min. We found a good correlation compared with HPLC ultraviolet detection. CONCLUSION: This method is simple, rapid, accurate in measuring phenylalanine and tyrosine in serum quantitatively.

[Changes in fatty acid composition of serum cholesteryl esters in patients with chronic heart failure].

OBJECTIVE: To investigate the changes in fatty acid composition of serum cholesteryl esters in patients with chronic heart failure (CHF). METHODS: The levels of serum TG, TC, HDL, and LDL in 36 patients with CHF and 20 healthy controls were measured and fatty acid cholesteryl esters were determined by high performance liquid chromatography. RESULTS: In the patients with CHF, the level of linoleic acid was significantly higher than that in the control group (P < 0.05), but the level of arachidonic acid was significantly lower than that in the control group (P < 0.05). The levels of TC, TG, HDL, and LDL were not significantly different between the two groups. CONCLUSION: Changes in fatty acid composition of serum cholesteryl esters exist in the patients with CHF.

[Determination of serum tryptophan by high performance liquid chromatography with fluorescence detection].

OBJECTIVE: To develop a rapid assay for determining tryptophan in human serum. METHODS: The serum sample was injected into the high performance liquid chromatography (HPLC) system after the precipitation of protein with perchloric acid, and was monitored with a fluorometric detector with wavelength settings of 254 nm for excitation and 313 nm for emission. The mobile phase is 20% acetonitrile. RESULTS: Good linearity was observed in 1.22-400 mumol.L-1 range for tryptophan. The detection limit was 0.1 mumol.L-1. The intra- and inter-assay coefficients of variation (CV) were 2.57% and 3.66%, respectively. The average recovery was 97.13%. The detection time of serum tryptophan was within 5 min. Phenyalanine, tyrosine, serotonin, and 5-hydroxyindoleacetic acid didn't interfere in the assay. CONCLUSION: This method is simple, rapid, sensitive, accurate, and suitable for clinical application.