High-throughput method to analyze tegafur and 5-fluorouracil in human tears and plasma using hydrophilic interaction liquid chromatography/tandem mass spectrometry.

RATIONALE: We developed a new high-throughput method to analyze tegafur (FT) and 5-fluorouracil (5-FU) in tear and plasma samples using hydrophilic interaction liquid chromatography (HILIC)/tandem mass spectrometry (MS/MS). METHODS: The tear samples (10 µL) spiked with FT, 5-FU, and 5-chlorouracil (internal standard) were diluted using 40 µL of 2 M ammonium acetate and 250 µL of acetonitrile with 2% formic acid; 20 µL of plasma spiked with the two drugs and internal standard was diluted with 80 µL of 2 M ammonium acetate and 500 µL of acetonitrile with 2% formic acid. After centrifugation, the clear supernatant extract (15 µL) was directly injected into the HILIC/MS/MS instrument, and each drug was separated on a Unison UK-Amino column (50 mm × 3 mm i.d., 3 µm particle size) with a linear gradient elution system composed of 10 mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.7 mL/min. We performed quantification by multiple reaction monitoring (MRM) with negative-ion atmospheric-pressure chemical ionization. RESULTS: Distinct peaks were observed for the drugs on each MRM channel within 2 min. The regression equations showed good linearity within the range 0.04-4.0 µg/mL for the tear and plasma samples with detection limits at 0.02-0.04 µg/mL. Recoveries for target analytes (FT and 5-FU) for the tear and plasma samples were in the 94-128% and 94-104% ranges, respectively. The intra- and inter-day coefficients of variation for the two drugs were lower than 10.8%. The accuracies of quantitation were 97-115% for both samples. CONCLUSIONS: We established a high-throughput, reproducible, and practical procedure for analyzing FT and 5-FU in human tear and plasma samples using HILIC/MS/MS analysis with an aminopropyl-bonded mixed-mode separation column. This method can be applied to the high-throughput routines used in clinical analyses.

Relationship between vascular endothelial growth factor and macular edema in retinal vein branch obstruction.

PURPOSE: Vascular endothelial growth factor (VEGF) plays an important role in branch retinal vein occlusion (BRVO) with cystoid macular edema (CME). Monitoring changes in VEGF is crucial for evaluating treatment but requires vitreous or aqueous humor sampling, which hampers its clinical application. We investigated the correlation between VEGF and protein concentration in the aqueous humor (flare) and whether this could be used to monitor treatment-related VEGF changes. DESIGN: This retrospective observational study involved 19 previously untreated patients with BRVO. Aqueous humor was obtained, and intravitreal ranibizumab (IVR) injection was administered to these patients. The correlation between VEGF and flare, central retinal thickness (CRT), and best-corrected visual acuity (BCVA) was investigated. Differences in these values were considered between pre-IVR and 1 week and 1-3 months post-IVR. Moreover, in patients with recurrence who received additional IVR, further changes in VEGF were examined. MAIN OUTCOME MEASURES: The end point of this study was BCVA, flare, and CRT at the fovea. RESULTS: Significant improvement was seen in BCVA and CRT at all time points and in Flare at 1 vs 3 months post-IVR; nevertheless, additional IVR was necessary in 94% of cases. In a patient with recurrence, CRT did not improve, even though VEGF decreased. CONCLUSION: Flare may be effective for estimating VEGF levels in aqueous humor pre-IVR. Inflammation-related molecules other than VEGF may be related to recurrence.

Measurement for canine IgE using canine recombinant high affinity IgE receptor α chain (FcεRIα).

To detect allergen-specific IgE in dogs with allergic diseases, we developed a recombinant canine high affinity IgE receptor α chain (FcεRIα)-based IgE detection system. Using the recombinant protein of canine FcεRIα expressed by an Escherichia coli expression system, we could detect house dust mite (Dermatophagoides farinae) allergen-specific IgE in sera from dogs naturally and experimentally sensitized to this allergen with ELISA and western blotting. The IgE binding activity of recombinant canine FcεRIα on ELISA was impaired by heat treatment of these sera. The specificity of this recombinant canine FcεRIα-based IgE detection system was confirmed by inhibition assays with canine IgE. The recombinant canine FcεRIα-based IgE detection system established in this study offers an alternative tool to measure allergen-specific IgE in dogs.

Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina induced by lipid hydroperoxide.

PURPOSE: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. METHODS: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by electrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. RESULTS: AP-1 binding activity was increased at 1-3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. CONCLUSIONS: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.

Anaemia, iron deficiency and vitamin A status among school-aged children in rural Kazakhstan.

OBJECTIVES: To investigate the prevalence of anaemia and iron deficiency and vitamin A status among school-aged children in rural Kazakhstan and identify factors associated with anaemia in this population. DESIGN: A cross-sectional design. SETTING: School-aged children in rural Kazakhstan. SUBJECTS: Socio-economic and anthropometric information was collected from 159 school-aged children living in the Kzyl-Orda region of Kazakhstan. Blood samples were collected and the concentrations of haemoglobin (Hb), serum iron, serum ferritin (SF), erythrocyte protoporphyrin (EP), serum retinol and beta-carotene, total iron binding capacity (TIBC), transferrin saturation (TS) and other haematological indices were measured. RESULTS: Among the 159 children, the prevalence of anaemia and iron deficiency defined by the multiple criteria model (SF, TS and EP) was 27% and 13%, respectively. Nine per cent had iron-deficiency anaemia and 21% had serum retinol value < 1.05 micromol l(-1). Mean SF and serum iron concentrations and TS were significantly lower in anaemic children than in their non-anaemic peers, while TIBC and EP were significantly higher in children with anaemia. Hb was significantly correlated with serum iron and retinol concentrations. Serum retinol and SF concentrations and mean corpuscular volume were significantly correlated with Hb by multiple regression analysis. CONCLUSIONS: Anaemia among school-aged children in rural Kazakhstan appears to be related to iron indices and vitamin A status.

Comparison of a small volume of hypertonic saline solution and dextran 40 on hemodynamic alternations in conscious calves.

The hemodynamic effects of rapid intravenous (IV) administration of 10% dextran 40 in saline solution (D40) and 7.2% hypertonic saline solution (HSS) in calves were compared. Calves received isotonic saline solution (ISS), HSS or D40 (3 calves/group) and were monitored of blood pressure, and cardiac output (CO) for 180 min. HSS and D40 infusions induced a significant increase in relative plasma volume reaching 134.9+/-2.8 and 125.0+/-1.9%, respectively at the end of fluid infusion. In the HSS group, CO, cardiac index (CI) and stroke volume (SV) remained constant at low levels after 90 minutes despite the maximal values of CO, CI and SV at the end of infusion, reaching 21.0+/-6.3 l/min (p<0.05), 177.8+/-14.2 ml/min/kg (p<0.001) and 0.20+/-0.03 l/beat (at t=10 min, p<0.001), respectively. In contrast, CI and SV in the D40 group showed significant increases to 14.7+/-2.9 l/min and 153.5+/-17.2 ml/min/kg, respectively, at the end of fluid infusion. And those values remained constant at higher levels than those of the before infusions values throughout the experimental periods. Positive effects for hemodynamic alternations of D40 in calf practice were milder and longer than those of HSS. Therefore, the D40 infusion should be explored as a possible treatment for dehydrated calves, since rapid infusion of D40 may be safe and more beneficial for rehydrating more than HSS treatment.

Production of a monoclonal antibody to canine thymus and activation-regulated chemokine (TARC) and detection of TARC in lesional skin from dogs with atopic dermatitis.

A monoclonal antibody to canine thymus and activation-regulated chemokine (TARC/CCL17) was developed to examine the association of TARC with the immunopathogenesis of canine atopic dermatitis (AD). Recombinant canine TARC was prepared using an E. coli expression system. Results of transwell chemotaxis assay demonstrated that the recombinant canine TARC showed chemotactic activity for canine lymphoid cells expressing CC chemokine receptor 4 (CCR4). Mice were then immunized with the recombinant canine TARC to obtain monoclonal antibodies. Among the monoclonal antibodies thereby obtained, one monoclonal antibody (CTA-1) was found to react with both recombinant and authentic canine TARC in ELISA and flowcytometric assays, respectively. Immunohistochemical analysis using the monoclonal antibody CTA-1 demonstrated that keratinocytes were major TARC producing cells in lesional skin of dogs with AD.

Isolation and expression of five-amino-acid-deleted variant of feline hepatocyte growth factor (HGF) cDNA.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and hematopoietic cells. We have cloned a different form of cDNA, with a deletion of 15 base pairs predicted to result in the loss of 5 amino acids from the first kringle domain. To investigate the biological activity, original and deleted variant of feline HGF cDNAs were transiently expressed in COS-7 cells. Both recombinant feline HGFs showed almost the same dose-response curves in the stimulation of the growth of BNL CL.2 cells (a mouse hepatocyte cell line) and scatter activity of Madin-Darby canine kidney (MDCK) cells. The findings reported here suggest that the deleted variant of feline HGF has almost the same biological activity as the original in terms of the proliferation and scatter activity.

Effects of intravenous infusion of hypotonic lactated Ringer's solution on calves with diarrhea.

The effects of intravenous infusion of hypotonic lactated Ringer's (LR: n=14) on plasma volume and venous blood gases were compared to those of hypotonic Ringer's solutions (RS: n=7) in diarrheic Japanese Black breed calves with metabolic acidosis. Venous blood samples were collected immediately before and after, and at 24 hr after the fluid infusion therapy. The LR and RS infusions increased relative plasma volume to 147.1 +/- 25.5% and 134.2 +/- 18.6%, respectively, just after the fluid therapy. The LR infusion induced an increase in the BE value (+5.1 +/- 4.8 mM) at 24 hr compared to that of RS. LR infusion should be explored as a treatment for dehydration and moderate metabolic acidemia caused by naturally occurring diarrhea in calves.

Canine hepatocyte growth factor: molecular cloning and characterization of the recombinant protein.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine originally identified and cloned as a potent mitogen for hepatocytes. The HGF receptor is the transmembrane tyrosine kinase encoded by c-MET proto-oncogene. Various lines of evidence suggest that the HGF/c-MET receptor system plays essential roles in monocyte-macrophage function, mammalian development, angiogenesis and organ regeneration. We have cloned canine HGF (CaHGF) cDNA from leukocytes by the methods of reverse transcription (RT)-polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). Canine HGF contains an open reading frame (ORF) of 2193 nucleotides, coding for 730 amino acids. The deduced amino acid sequence of canine HGF shows 97.5, 92.3, 92.1, and 92.0% homologies with those of feline, human, mouse, and rat, respectively. The possible glycosylation sites, cysteine residues linking the alpha and beta chains and the proteolytic processing site are conserved in all species. In addition, we have found a variant cDNA that deleted a sequence of 15 base pairs in the first kringle domain (K1) and resulted in the deletion of five amino acids. To confirm the biological activities of canine HGF cDNAs, both cDNAs were transiently expressed in COS-7 cells. The conditioned medium from the canine HGF-transfected COS-7 cells stimulated the growth of BNL CL.2 cells (a mouse hepatocyte cell) and scattering activity of Madin-Darby canine kidney (MDCK) cells. The materials reported here will be a crucial resource for further studies of canine HGF.

Cytokine profiles of peripheral blood mononuclear cells from dogs experimentally sensitized to Japanese cedar pollen.

Japanese cedar (Cryptomeria japonica, CJ) pollinosis is mediated by type-I hypersensitivity and induces seasonal rhinitis and conjunctivitis in humans. Previous studies showed that dogs could be experimentally sensitized with CJ pollen. In this study, we carried out quantitative analysis of mRNA levels of various cytokines in the peripheral blood mononuclear cells (PBMC) of 12 dogs experimentally sensitized to Japanese cedar pollen. Experimental sensitization was carried out by injection of crude CJ pollen extract with aluminium hydroxide gel. The expression levels of interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, interferon (IFN)-gamma, transforming growth factor (TGF)-beta(1), and tumor necrosis factor (TNF)-alpha mRNAs in the PBMC were quantified using a real-time sequence detection system. In the PBMC tested without culture, the expression levels of IL-8 and TNF-alpha mRNAs in experimentally sensitized dogs were significantly higher than those in control dogs. The expression level of IFN-gamma mRNA in the sensitized group was significantly lower than that in the control group. When the PBMCs were cultured in the presence of CJ pollen extract, the level of IL-4 mRNA expression was markedly increased in the PBMC from the experimentally sensitized dogs. In the PBMC stimulated with the CJ pollen extract, the expression level of IL-2 mRNA in the sensitized group was also significantly higher than that in the control group. Our data indicated that a Th2 response and proliferation of PBMC occur in response to the sensitizing antigen in dogs experimentally sensitized with CJ pollen, and revealed the presence of antigen-specific Th2 cells in this canine model. In addition, the expression levels of the mRNAs encoding proinflammatory cytokines were shown to be elevated after CJ pollen sensitization, indicating the activation of monocytes and macrophages.

Comparison of the effects of isotonic and hypertonic sodium bicarbonate solutions on acidemic calves experimentally induced by ammonium chloride administration.

The objectives of this study were to evaluate and compare the effects of intravenously (IV) administered infusion of isotonic solution (ISB) or hypertonic sodium bicarbonate solution (HSB) on acid-base equilibrium and the plasma osmolarity in acidemic calves experimentally induced by 5 M-NH(4)Cl, IV infusion (1.0 ml/kg, over 1 hr). The ISB and HSB infusion induced progressive and significant increases in their HCO(3)(-) and BE levels that persisted throughout the period of fluid administration. The plasma osmolarity in the ISB groups was significantly decreased. The plasma osmolarity in the HSB group was significantly higher than in the calves in the other groups (p<0.05). ISB solution might be safe and effective for treating and reviving conscious calves from experimentally induced metabolic acidosis.

Evaluation of isotonic sodium bicarbonate solution for alkalizing effects in conscious calves.

After intravenous (i.v.) infusion of various volumes of 1.35%-isotonic sodium bicarbonate solution (ISB), acid-base equilibrium, blood pressure, plasma volume and biochemical parameters in healthy Holstein calves were studied. Four calves each were randomly assigned to the low-dose (LD; i.v. infusion of 5 ml/kg ISB), middle-dose (MD; i.v. infusion of 10 ml/kg ISB) and the high-dose groups (HD; i.v. infusion of 15 ml/kg ISB). Administration volumes of ISB in the LD, MD and HD groups were decided based on the first half volumes of 5, 10 and 15 mEq of base requirement by the acceptable equation. Systemic, pulmonary artery and central venous pressures, cardiac output and plasma osmotic pressure were not changed by ISB infusion and remained constant throughout the experiment for all groups. There was good correlation (r(2) = 0.950) between relative changes in base excess and infused volume of bicarbonate (y=2.491x). The coefficient of distribution for bicarbonate ions was calculated to be 0.401 (=1/2.491). Therefore, it is suggested that a value of 0.4 would be most appropriate when calculating the base requirements in calves. Therefore, the first half volumes of ISB correcting base deficits of 5, 10 and 15 mEq in calves were estimated to be 6, 12 and 18 ml/kg, respectively. On the basis of the findings in this study, ISB may be used to correct metabolic acidosis without altering the plasma osmotic pressure, hemodynamic status and respiratory function in the calves.

Effect of isotonic and hypotonic lactated ringer's solutions with dextrose intravenously administered to dehydrated heifers.

To determine the effects of rapid infusion of essential fluids in a volume of hypotonic lactated Ringer's solution, the central venous pressure (CVP) and acid-base equilibrium were investigated in to mildly dehydrated heifers. Mild dehydration was induced in 9 Holstein heifers by withholding food and water until 7.0+/-5.7% of plasma volume had been lost. The heifers were randomly assigned to the ILG (lactated Ringer's + 5% dextrose), HLG (1/2 lactated Ringer's + 2.2% dextrose) or HRG (1/2 Ringer's + 2.5% dextrose) groups with 3 heifers in each group. Heifers received 30 ml/kg of one of the fluids, at a flow rate of 20 ml/kg/hr. The rapid intravenous (IV) infusions of HLG and HRG used in this study were found to be safe and effective in increasing plasma volume without increasing CVP, even though the infusion was given to the jugular vein at a dosage of 30 ml/kg. However, ILG infusion induced progressive increases in CVP, reaching 9.0+/-2.0 mmHg. No clinical signs, such as moist rales on auscultation, moist cough, jugular vein congestion, ophthalmoptosis, salivation or arrhythmia, were observed throughout the fluid infusion. The relative changes in base excess (rBE) for the ILG and HRG groups were significantly decreased until the end of fluid infusion. As for the HLG group, rBE slightly decreased until the end of the fluid infusion. Then the values significantly increased and exceeded the pre-infusion value at the end of the experiment. While IV infusion of HLG inhibited acidification caused by dilution, HRG infusion induced diluted acidification. It is suggested that HLG infusion should be examined as a treatment for cattle with dehydration and moderate metabolic acidosis, since rapid infusion of HLG may be more beneficial for rehydrating cattle with metabolic acidosis than current treatment.

Lipid hydroperoxide stimulates subretinal choroidal neovascularization in the rabbit.

The authors sought to evaluate the effect of linoleic acid hydroperoxide (18:2/LHP) in promoting choroidal neovascularization (CNV). Albino male rabbits received a subretinal injection of various amounts of LHP (ranging from 5 to 200 microg) dissolved in 50 microl of sodium borate buffer. Control eyes received the buffer only. Eyes were examined up to 4 weeks later with indirect ophthalmoscopy, fundus photography and fluorescein angiography. Animals were killed on days 3, 7, 14 or 28, and eyes examined by light and transmission electron microscopy. In eyes injected with LHP of 150-200 microg, exposed areas turned white as observed ophthalmoscopically and showed both severe retinal and choroidal atrophy histologically. Neither fluorescein leakage nor CNV was found in these eyes or in controls. In 33 eyes injected with LHP of 100 microg or less, prominent fluorescein leakage was seen in three (9%) and less prominent focal leakage in five (15%). In 11 (46%) of the 24 eyes injected with 12.5-50 microg LHP, CNV was found histologically. Subretinal injection of LHP is capable of inducing CNV.

Effect of intravenous infusion of isotonic sodium bicarbonate solution on acidemic calves with diarrhea.

The effect of 1.35% isotonic sodium bicarbonate solution (ISB) administered intravenously on acid-base equilibrium was examined in 18 acidemic Japanese black beef calves with spontaneous diarrhea. The infusion volumes of ISB were decided based on the first half volumes of base needed. In 72.2% (13/18) of calves, improvement of acidemia was detected. There was good correlation (r=0.693, p<0.01) between infused volume of ISB and changes in base excess (y=1.097x + 4.762). Infusion volumes of ISB were 7.5, 10.2, 12.9 and 15.7 ml/kg, respectively, enough to correcting the first half of 5, 10, 15 and 20 mEq/l of base deficit in acidemic calves. Our finding suggested that ISB could be used to correct metabolic acidosis without altering electrolyte concentrations in calves.