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Background: Lung subtraction iodine mapping (LSIM)-CT is a clinically useful technique that can visualize pulmonary mal-perfusion in patients with chronic thromboembolic pulmonary disease (CTEPD). However, little is known about the associations of LSIM images with hemodynamic parameters of patients with CTEPD. This study investigates a parameter of LSIM images associated with mean pulmonary arterial pressure (mPAP) and validates the association between pulmonary vascular resistance, right atrial pressure, cardiac index, and exercise capacity in patients with CTEPD., Methods: This single-center, prospective, observational study involved 30 patients diagnosed with CTEPD using lung perfusion scintigraphy. To examine the correlation of decreased pulmonary perfusion area (DPA) with mPAP, areas with 0-10, 0-15, 0-20, and 0-30 HU in lung subtraction images were adopted in statistical analysis. The DPA to total lung volume ratio (DPA ratio, %) was calculated as the ratio of each DPA volume to the total lung volume. To assess the correlation between DPA ratios of 0-10, 0-15, 0-20, and 0-30 HU and mPAP, Spearman's rank correlation coefficient was used., Results: The DPA ratio of 0-10 HU had the most preferable correlation with mPAP than DPA ratios of 0-15, 0-20, and 0-30 HU ( ρ  = 0.440, P  = 0.015). The DPA ratio of 0-10 HU significantly correlates with pulmonary vascular resistance ( ρ  = 0.445, P  = 0.015). The receiver operating characteristic curve analysis indicated that the best cutoff value of the DPA ratio of 0-10 HU for the prediction of an mPAP of ≥30 mmHg was 8.5% (AUC, 0.773; 95% CI, 0.572-0.974; sensitivity, 83.3%; specificity, 75.0%). Multivariate linear regression analysis, which was adjusted for the main pulmonary arterial to ascending aortic diameter ratio and right ventricular to left ventricular diameter ratio, indicated that the DPA ratio of 0-10 HU was independently and significantly associated with mPAP ( B  = 89.7; 95% CI, 46.3-133.1, P  < 0.001)., Conclusion: The DPA ratio calculated using LSIM-CT is possibly useful for estimating the hemodynamic status in patients with CTEPD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Yamaguchi, Ehara, Yoshida, Himoto, Izuta, Hayashi, Hayashi, Ogawa, Shibata, Yamazaki, Izumiya and Fukuda.)

Objectives: Cases in which the left circumflex coronary artery (LCX) runs close to the mitral annulus are considered high risk for LCX injury during mitral surgery. We investigated the anatomical relationship between the LCX and the mitral annulus using 3-dimensional (3D) computed tomography (CT)., Methods: We constructed 3D-CT images of the LCX and the mitral annulus before surgery in 122 patients with mitral regurgitation (MR). We classified coronary dominance by 3D-CT and MR aetiologies (degenerative, atrial functional MR, ventricular functional MR and Barlow's disease) using echocardiography. We detected the point on the mitral annulus closest to the LCX (X point) and measured the minimum distance from the LCX to the mitral annulus (mCAD). We judged whether atrioventricular disjunction existed using CT. We also investigated the factors affecting mCAD and examined how coronary dominance and MR aetiologies relate to the location of the X point., Results: The median mCAD was 4.2 mm (range 0.9-11.4 mm). Considering coronary dominance and MR aetiologies, mCAD was shorter in patients with left coronary dominance and Barlow's disease. The X point mostly existed on the lateral side of the posterior annulus, but it sometimes existed on the medial side. Multiple regression revealed left dominance and mitral annular disjunction as significant factors affecting mCAD (P = 0.01)., Conclusions: The anatomical relationship between the LCX and the mitral annulus can be recognized using superimposed 3D-CT images. This approach is useful to avoid LCX injury in mitral valve surgery since the X point varies between patients., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Multidetector row computed tomography (CT) scanners perform dynamic scanning and have a wide scan range. Time-resolved three-dimensional CT (i.e., 4D CT) has recently enabled visualization of flow in neurovascular vessels. We hypothesized that 4D CT technology would be a useful and non-invasive method for visualizing the flow dynamics of the portal circulation. The aim of this study was to evaluate the technical feasibility of 4D CT for visualizing flow dynamics in the portal circulation using 320-detector-row CT. 4D CT images of 18 consecutive patients with portal circulation including gastrorenal shunt were retrospectively evaluated for their ability to generate flow dynamics of the portal circulation. Flow dynamics could be visualized by 4D CT in 68 of the 72 vessels in the portal vein, splenic vein, superior mesenteric vein, and gastrorenal shunt. Flow direction could not be identified in four vessels, all of them being superior mesenteric veins. Flow direction was recognized on 4D CT in the 68 vessels of the portal circulation. A preliminary validation study revealed that flow direction of all 19 vessels in the portal circulation had concordance between 4D CT and color Doppler ultrasound. 4D CT could visualize flow dynamics of the portal circulation.

Ketamine has been used to treat chronic pain; however, it is still unknown as to what types of chronic pain is ketamine effective against. To identify the effect of administration of subanesthetic-dose ketamine in patients with chronic pain and to clarify the mechanism of the effect, we retrospectively investigated brain functional connectivity using resting-state functional magnetic resonance imaging (rs-fMRI). Patients were divided into responders (Group R: ≥50% improvement on Numerical Rating Scale) and non-responders (Group NR). We compared the differences in terms of brain functional connectivity by seed-to-voxel correlation analysis. Two-sample t-test revealed significant lower connectivity between the medial prefrontal cortex (mPFC) and precuneus in Group R. We also found a significant negative correlation between the improvement rate and functional connectivity strength between the mPFC and precuneus. These findings suggest that subanesthetic-dose ketamine is effective in patients with chronic pain whose brain functional connectivity between the mPFC and precuneus is low. We believe that the current study explored for the first time the correlation between brain functional connectivity and the effect of subanesthetic-dose ketamine for chronic pain and indicated the possibility of use of the predictive marker in pharmacological treatment of chronic pain.

Aim: To investigate the anesthetic management of peroral endoscopic myotomy (POEM) and its associated complications., Methods: This study was a single-center, retrospective, observational study comprising a case series of all patients who underwent POEM in our hospital from April 2015 to November 2016. We collected data regarding patient characteristics, anesthetic methods, surgical factors, and complications using an electronic chart., Results: There were 86 patients who underwent POEM in our hospital during the study period. Preoperatively, patients were maintained on a low residue diet for 48 h prior to the procedure. They were fasted of solids for 24 h before surgery. There was one case of aspiration (1.2%). During POEM, patients were positioned supine with the upper abdomen covered by a clear drape so that pneumoperitoneum could be timeously identified. In three cases, the peak airway pressure exceeded 35 cmH 2 O during volume controlled ventilation with tidal volumes of 6-8 mL/kg and subsequent impairment of ventilation. These cases had been diagnosed with spastic esophageal disorders (SEDs) and the length of the muscular incision on the esophageal side was longer than normal., Conclusion: In the anesthetic management of POEM, it is important to prevent aspiration during induction of anesthesia and to identify and treat complications associated with CO 2 insufflation., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest relevant to this article were reported.

In a search for compounds interacting with ergosterol resin, a new compound named dipyrimicin B was isolated from a rare actinomycete strain, Amycolatopsis sp. K16-0194. In addition, another analog, dipyrimicin A, which does not interact with the resin, was also discovered. The structures of the two dipyrimicins were established by comprehensive 1D and 2D NMR and MS analyses and found to contain a unique core structure, a 2,2'-bipyridine skeleton. Dipyrimicin A showed strong antimicrobial and cytotoxic activity, whereas dipyrimicin B displayed distinctly poor antimicrobial and cytotoxic activities.

ᅟ: We report a case of unexpected ventilatory impairment that occurred during per-oral endoscopic myotomy (POEM) under general anesthesia. A 73-year-old woman underwent POEM for Jackhammer esophagus. The patient developed hypercarbia, pneumoperitoneum, and severe subcutaneous emphysema during the operation. Although she was treated with abdominal paracentesis, it became difficult to ventilate her lungs a few minutes later. We recommended the surgeons to interrupt the procedure and proposed repeating the abdominal paracentesis. Simultaneously, we switched to manual ventilation and waited for the subcutaneous emphysema to subside. Thereafter, her respiratory status gradually improved and the surgeons were able to continue the operation. We considered that the main reason for our patient's severe ventilatory impairment was that the length of surgical dissection was longer than usual.

Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes. Mammalian cells immobilized on a poly(ethylene glycol)-lipid coated substrate were rapidly and efficiently fractured, with the sheer stress of laminar flow in microchannels, resulting in isolation of the bottom cell membrane sheets with exposed intact cytoplasmic faces. On these faces of the cell membrane sheets, both ligand-induced phosphorylation of receptor tyrosine kinases and selective enzymatic modification of a G-protein coupling receptor were directly observed. Thus, the present cell membrane sheet should serve as a unique platform for studies providing new insights into juxta-membrane molecular networks and drug discovery.

A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm -1 was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the signal activation of the probe was also detected on living cells by stimulated Raman scattering microscopy. The ring-A-opened alkyne steroid analogue, thus, provides a first chemically-activatable Raman probe as a promising tool for potentially unravelling the intracellular formation and trafficking of cholesterol-rich microdomains.

The many known eukaryotic DNA polymerases are classified into four families; A, B, X, and Y. Among them, DNA polymerase η, a Y family polymerase, is a low fidelity enzyme that contributes to translesional synthesis and somatic hypermutation. Although a high mutation frequency is observed in immunoglobulin genes, translesional synthesis occurs with a high accuracy. We determined whether the misincorporation rate of DNA polymerase η varies with ambient conditions. It has been reported that DNA polymerase η is unable to exclude water molecules from the active site. This finding suggests that some ions affect hydrogen bond formation at the active site. We focused on the effect of pH and evaluated the misincorporation rate of deoxyguanosine triphosphate (dGTP) opposite template T by DNA polymerase η at various pH levels with a synthetic template-primer. The misincorporation rate of dGTP by DNA polymerase η drastically increased at pH 8.0-9.0 compared with that at pH 6.5-7.5. Kinetic analysis revealed that the Km value for dGTP on the misincorporation opposite template T was markedly affected by pH. However, this drastic change was not seen with the low fidelity DNA polymerase α.

The effect of T-5224, a selective inhibitor of c-Fos/activator protein (AP)-1, on lipopolysaccharide (LPS) induced liver injury was examined in mice. Administration of LPS (10 mg kg(-1), i.p.) markedly increased serum levels of tumor necrosis factor-alpha (TNFα), high mobility group box 1 (HMGB1), alanine aminotransferase/aspartate aminotransferase (ALT/AST), liver tissue levels of macrophage-inflammatory protein-1 alpha (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1), as well as hepatic necrosis and inflammation, leading to 67 % lethality. Administration of T-5224 (300 mg kg(-1), p.o.) after intraperitoneal injection of LPS imparted appreciable protection against acute elevations in serum levels of TNFα, HMGB1, ALT/AST as well as in liver tissue levels of MIP-1α and MCP-1, and reduced the lethality (27 %). These data indicate that T-5224 ameliorates liver injury and improves survival through decreasing production of proinflammatory cytokines and chemokines in endotoxemic mice.

DNA polymerase eta is recently found as a responsible gene product of xeroderma pigmentosum variant. Differently from other eukaryotic DNA polymerases, such as alpha, beta or gamma, eta polymerase is categorized in Y family. Specific inhibitors for DNA polymerases are useful tools to study the exact role of enzyme in the living cells, however, the inhibitor for DNA polymerase eta has not been developed. We examined the inhibitory effects of several sugar-modified nucleotide analogs on DNA polymerase eta. The arabinonucleotides (araCTP), dideoxynucleotides (ddTTP) and 3'-modified nucleotides (3'-dCTP and 3'-azidothymidine tri-phosphate) did not show any inhibitory effect on DNA polymerase eta. On the other hand, the oxetanocin derivatives those have the oxetane ring as a sugar moiety, OXT-GTP and OXT-ATP, strongly inhibited this polymerase. These results suggest that DNA polymerase eta has a unique recognition mode against the sugar moiety of nucleotide compared with other eukaryotic nucleotide polymerases.

To clarify the molecular mechanism of mismatch formation by DNA polymerase eta, the pH-dependency of misincorporation of dNTP was studied with the synthetic template-primer. Incorporation of dNTP formed Watson-Crick type base pair, such as the incorporation of dATP opposite template T, was slightly affected by pH between 6.5 to 9.0. On the other hand, the misincorporation rate of dGTP opposite template T by DNA polymerase eta was drastically increased according to the increasing pH. Kinetical analysis revealed that this change might be due to the change of Km value for dGTP rather than that of Vmax value. This suggests that the affinity of dGTP on DNA polymerase eta during the mismatch formation with template T should be affected by pH.

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G(0)/G(1) by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21(WAF-1/CIP-1) and p27(KIP-1), followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21(WAF-1/CIP-1), and p27(KIP-1) were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G(1)/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G(1) to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser(502) that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.

The mechanism of action of the antitumor nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl)cytosine (4'-thio-FAC) was investigated. 4'-Thio-FAC inhibited cellular DNA synthesis, but not RNA and protein syntheses. We observed potent inhibitory action of the triphosphate of 4'-thio-FAC (4'-thio-FACTP) against DNA polymerase alpha, whereas it showed moderate inhibition of DNA polymerase beta and little inhibition of DNA polymerase gamma. The kinetic analysis showed that the inhibition mode of 4'-thio-FACTP towards DNA polymerase alpha was mixed type, implying a chain-terminating effect of 4'-thio-FACTP. The triphosphate of 2'-deoxy-2',2'-difluorocytidine (gemcitabine), a known antitumor nucleoside, did not show potent inhibition of these three DNA polymerases. Thus, the effect of the diphosphate of gemcitabine on ribonucleotide reductase was suggested to be more important for the antitumor action of gemcitabine. From these findings, the main target enzymes of 4'-thio-FAC and gemcitabine appear to be different. We found a synergistic effect of the two drugs in an in vitro model, which supports the above idea.

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10-100-fold the activity of DNA polymerase alpha from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase alpha more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase alpha. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase alpha antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase alpha. This result indicates that DNA polymerase alpha exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase alpha specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase alpha trapped to anti-DNA polymerase alpha antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.

1-O-Acetyl-2,5-di-O-p-chlorobenzoyl-3-deoxy-D-ribofuranose (1), derived from the antibiotic cordycepin was coupled with trimethylsilylated derivatives (2a-c) of N4-propionylcytosine, N4-p-toluoyl-5-fluorocytosine and 5-fluorouracil in the presence of trimethylsilyl trifluoromethanesulfonate (TMS-triflate) to give fully acylated nucleosides (3a-b and 3d, respectively). Selective removal of the N4-propionyl group of 3a by treatment with hydrazine hydrate gave 2',5'-di-O-p-chlorobenzoyl-3'-deoxycytidine (4). Deamination of 4 with sodium nitrite in trifluoroacetic acid afforded 2',5'-di-O-p-chlorobenzoyluridine (3c) in good yield. Compounds 3a-d were saponified to give free 3'-deoxycytidine (5a), 5-fluoro-3'-deoxycytidine (5b), 3'-deoxyuridine (5c), and 5-fluoro-3'-deoxyuridine (5d), respectively. These 3'-deoxyribonucleosides (5a-d) were then converted to corresponding 5'-monophosphate and further phosphorylated to the 5'-triphosphates by the phosphoroimidazolidate method. The nucleosides (5a-d) were examined for growth-inhibitory effects on mouse leukemic L5178Y cells, and their IC50 values (microgram/ml) were 1.8, 33, 6.5, and 18, respectively. On the other hand, the antiviral activities of these compounds on a rhabdovirus, infectious hematopoietic necrosis virus (IHNV), were moderate (IC50 = 100-500 micrograms/ml in CHSE-214 cells). The 5'-triphosphates showed remarkable inhibitory effects on DNA polymerase beta and DNA polymerase alpha-primase purified from testes of the cherry salmon, Oncorhynchus masou, but not on common DNA polymerase alpha from same source.

We examined the mechanism of the inhibition of DNA synthesis by a new platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato+ ++)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis-diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase alpha were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase beta was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.

Normal and vitamin B6-deficient rats received an intraperitoneal injection of 30 mg/100g of body wt, and the contents of metabolites in kidney or plasma and the related enzyme activities in kidney were determined. The contents of kynurenine and 3-hydroxykynurenine in B6-deficient rat plasma and kidney were much higher than those in normal rat. The changes of those contents in plasma were parallel to those in kidney, but not in liver. The contents of kynurenic acid and xanthurenic acid in B6-deficient liver, plasma, and kidney were also much higher than those in normal rats. However, the changes of those contents in plasma were parallel to those in liver, but not in kidney. Xanthurenic acid and kynurenic acid accumulated to a much greater extent in kidney than in plasma and liver. Kidney kynureninase activity was very low, but kynurenine aminotransferase activities were very high. These observations indicated that the production of xanthurenic acid after tryptophan injection was favorable in B6-deficient kidney with respect to enzyme activities and substrate concentrations, and suggested that kidney took up kynurenine or 3-hydroxykynurenine from blood and after conversion of them it excreted xanthurenic or kynurenic acid into urine.