Spatial and Temporal Brain Responses to Noxious Heat Thermal Stimuli in Burning Mouth Syndrome.

Burning mouth syndrome (BMS) is an idiopathic orofacial pain condition. Although the pathophysiology of BMS is not clearly understood, central and peripheral neuropathic mechanisms are thought to be involved. The authors compared brain response to noxious heat stimuli in 16 right-handed women with primary BMS and 15 sex- and age-matched right-handed healthy female controls. A thermal stimulus sequence of 32 °C to 40 °C to 32 °C to 49 °C was repeated 4 times in a cycle. Warm and noxious heat stimuli were delivered with a Peltier thermode placed on the right palm or right lower lip for 32 s each in a session. Functional magnetic resonance imaging data were obtained by recording echoplanar images with a block design. Statistical Parametric Mapping 8 software was used to analyze the data. Patients and controls both reported feeling more pain during palm stimulation than during lip stimulation. Repetition of noxious heat stimulus on the lower lip but not on the palm induced habituation in brain activity in the cingulate cortex without reduction in pain perception. Multiple regression analysis revealed a correlation between perceived pain intensity and suppression of brain activity in the anterior cingulate cortex when the repeated thermal sequence was applied at the lower lip. Furthermore, the response of the parahippocampal area differed in BMS patients and controls when the same repeated thermal sequence was applied at the palm. The authors' findings indicate that BMS patients show specific brain responses due to impaired function of the central and peripheral nervous systems (clinical trial registration: UMIN000015002).

Oxidation mechanism and overall removal rates of endocrine disrupting chemicals by aquatic plants.

The purpose of this study was to evaluate experimentally and theoretically the oxidation mechanisms and overall removal rates of phenolic endocrine disrupting chemicals (EDCs) by aquatic plants. EDCs used in this study were bisphenol-A (BPA), 2,4-dichlorophenol (2,4-DCP), 4-tert-octylphenol (4-t-OP), and pentachlorophenol (PCP). Referring to reported detection levels in aquatic environments and contaminated sites, the feed concentration of each EDC was set from 1 to 100µg/L. Experimental results showed that, except for PCP, phenolic EDCs were stably and concurrently removed by different types of aquatic plants over 70 days in long-term continuous treatments. Primal enzymes responsible for oxidation of BPA, 2,4-DCP, and 4-t-OP were peroxidases (POs). Moreover, enzymatic removal rates of BPA, 2,4-DCP, and 4-t-OP by POs were more than 2 orders of magnitude larger than those by aquatic plants. Assuming that overall removal rates of EDCs are controlled by mass transfer rates onto liquid films on the surface of aquatic plants, an electrochemical method based on the limiting current theory was developed to measure the mass transfer rates of EDCs. Because of extremely large removal rates of EDCs by POs, observed removal rates by aquatic plants were in reasonably good agreement with calculated results by a mathematical model developed based on an assumption that mass transfer limitation is a rate-limiting step.

Indometacin up-regulates TFF2 expression in gastric epithelial cells.

BACKGROUND: Trefoil factor family peptides are expressed in gastrointestinal epithelial cells and play a critical role in maintaining mucosal integrity. Although non-steroidal anti-inflammatory drugs (NSAIDs) are important causative agents of gastric mucosal lesions, few data are available about the effect of NSAIDs on trefoil family peptides in gastric mucosa. AIM: To examine whether indometacin, a widely used NSAID, affects trefoil factor family expression in gastric epithelial cells. METHODS: MKN45, a cell line derived from human gastric cancer, was used. TFF1, TFF2, and TFF3 mRNA expression was assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). TFF2 gene transcription was also examined by luciferase reporter gene assay. RESULTS: Relative expression level of TFF1, TFF2, TFF3 mRNA was 616: 12: 1 in unstimulated MKN45 cells. Although indometacin (1-250 micro mol/L) had no significant effect on the expression of TFF1 and TFF3 mRNA, it up-regulated TFF2 mRNA expression in a dose- and time-dependent manner. Luciferase reporter gene assay confirmed the up-regulation of TFF2 gene transcription by indometacin. Indometacin-induced up-regulation of TFF2 expression was not antagonized by externally applied prostaglandin E2. CONCLUSION: These results suggest that indometacin up-regulates gastric epithelial cell TFF2 expression through a COX-independent mechanism. Since TFF peptides play an important role in gastric mucosal protection, indometacin-induced TFF2 may reduce the degree of gastric mucosal damage induced by indometacin.

Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus.

A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutant's subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.

Effect of ticlopidine hydrochloride on erythropoietin-induced rise in blood pressure in patients on maintenance hemodialysis.

BACKGROUND/AIMS: A recent observation that antiplatelet-aggregation drugs, including ticlopidine hydrochloride, may prevent erythropoietin (EPO)-induced rise in blood pressure in hemodialysis (HD) patients remains a subject of particular interest. The aim of the present study was to determine the effect of ticlopidine hydrochloride on EPO-induced rise in blood pressure of HD patients with special reference to blood levels of vasoactive substances. METHODS: HD patients who showed hypertension or aggravation of preceding hypertension with EPO treatment were selected for this study. Ticlopidine hydrochloride was administered at a dose of 200 mg daily for 4 weeks. Blood pressure and serum levels of nitric oxide (NO), atrial natriuretic peptide (ANP) and endothelin (ET) were determined before and after drug administration. Patients were divided into two groups, one of which showed a drop in mean blood pressure (MBP) of >10 mm Hg (group I) and one which did not (group II), and a comparison was made between them with respect to the blood parameters. RESULTS: Five of 15 patients showed a drop of MBP of >10 mm Hg (group I), and 10 patients did not show any change in MBP (group II). In group I, there was a significant increase in blood NO levels compared to the concentrations before ticlopidine administration, while there was no change in group II. With respect to ANP and ET, there was no significant change in either of the groups. CONCLUSION: The findings suggest that the preventive effect of ticlopidine hydrochloride on EPO-induced rise in blood pressure may partly be related to the enhancement of NO production in patients on maintenance HD.

Renal cell carcinoma with skull base metastasis preceded by paraneoplastic signs in a chronic hemodialysis patient.

A 59-year-old man who had received chronic hemodialysis developed left occipital pain and hypoglossal nerve palsy. He was diagnosed as having skull base metastasis from renal cell carcinoma related to acquired cystic kidney. Retrospective analysis revealed the patient had had elevated serum C-reactive protein and alkaline phosphatase levels before the symptoms appeared. Radiotherapy to the skull base relieved the pain. Finally he died with generalized metastases. Serum interleukin-6 levels measured during admission had been elevated, and interleukin-6 mRNA was detected in the autopsy specimen of renal cell carcinoma. Interleukin-6 might be involved in the etiology of paraneoplastic signs.

Outcomes of hemodiafiltration based on Japanese dialysis patient registry.

Effectiveness of various therapeutic modalities was analyzed among 1,196 patients entered in the registry of the Japanese Society for Dialysis Therapy who were on hemopurification therapy as of the end of 1998 and developed dialysis-related amyloidosis during 1999. In the investigation, the effectiveness of various hemopurification modalities on the dialysis-related amyloidosis was ranked as exacerbation, unchanged, or alleviation, so as to analyze the possible relationship between the hemopurification modality and its effectiveness. The analysis was performed using a logistic regression approach, and the results were shown as "the risk of a worse therapeutic ranking" among the hemopurification modalities. The smaller "the risk of a worse therapeutic effect" was, the more effective the treatment modality. When the risk of a worse therapeutic effect for the hemodialysis patients treated by a regular membrane was put at 1.0, the risk for hemodialysis patients using high-flux membrane was 0.489, the off-line hemodiafiltration risk was 0.117, the on-line hemodiafiltration risk was 0.013, and the risk of push/pull hemodiafiltration was 0.017. For hemodialysis with a beta(2)-microglobulin adsorption column, a low risk of 0.054 was found. The results indicated that hemodiafiltration therapy and simultaneous hemodialysis with beta(2)-microglobulin adsorption therapy were more effective treatment for dialysis-related amyloidosis.

[Tubulointerstitial nephritis in the case of acute renal failure from sepsis after a cat bite].

Acute tubulointerstitial nephritis is associated with a variety of causes, such as drug interaction, and infectious or immunological mechanisms. We describe a patient who suffered from sepsis, septic shock, disseminated intravascular coagulation(DIC), hepatic failure and renal failure after receiving a bite from her house cat. The causes of her acute renal failure were initially thought to be due to circulatory failure with hypotensive shock, decrease in renal blood flow with fibrin formation by DIC, or microangiopathy such as hemolitic uremic syndrome. However, the renal biopsy on the 60th hospital day indicated tubulointerstitial nephritis, which was recognized by the presence of patchy and focal mononuclear small cell infiltration with invasion to the tubular epithelium. We concluded that prolonged renal failure was caused by tubulointerstitial nephritis. The cause of tubulointerstitial nephritis was not identified. Tubulointerstitial nephritis should be taken into consideration when the recovery from acute renal failure is slow.

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode.

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.

Effect of postural change on blood volume in long-term hemodialysis patients.

In healthy subjects, the blood volume (BV) increases rapidly after postural change from standing to the supine position. However, little is known about the effect of postural change on BV in long-term hemodialysis (HD) patients. Therefore, we have examined the BV change caused by adopting the supine position from standing by continuous hematocrit monitoring, using the CRIT-LINE instrument, in 8 anuric HD patients. The hematocrit was monitored for 25 min with the patient in the supine position just before HD. The percentage change in the BV (% Delta BV) was calculated from the hematocrit and approximated using the equation: % Delta BV = b - [1 - exp(-c x time (min)] -a x time (min). Coefficient a was the slope of the linear part in the % Delta BV, b was the magnitude of BV increase and c was the rate of BV increase. Then we examined the relationship between the coefficients (a, b and c) and clinical parameters. In all patients, % Delta BV increased quickly after adopting the supine position. The mean increases were 2.8 +/- 0.6% after 5 min and 4.8 +/- 0.5% after 25 min. There was a significant correlation between the value of % Delta BV calculated from the hematocrit and the value calculated using above equation (0.92 < r < 0.99, p < 0.001). Although coefficient a did not correlate with a clinical parameter, coefficient b showed a significant positive linear correlation with the serum albumin level (r = 0.816, p < 0.05) and coefficient c showed a significant positive linear correlation with the percentage change in interdialytic weight gain (r = 0.736, p < 0.05). Furthermore, based on the % Delta BV, we calculated the change in total BV, which had increased by 181.5 +/- 21.9 ml after 25 min in the supine position. In conclusion, the change in the BV with time by continuous hematocrit monitoring using the CRIT-LINE instrument can be approximated by a modified monoexponential equation. BV increased quickly in HD patients after postural change from standing to the supine position.

MS/NMR: a structure-based approach for discovering protein ligands and for drug design by coupling size exclusion chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy.

A protocol is described for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure-related information as part of a structure-based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, mass spectrometry, and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D 1H-15N HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of approximately 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.

Mechanism of inhibition of the class A beta -lactamases PC1 and TEM-1 by tazobactam. Observation of reaction products by electrospray ionization mass spectrometry.

The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A beta-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the beta-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58-82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125-141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A beta-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde.

The effect of EGF on electrolyte transport is mediated by tyrosine kinases in the rabbit cortical collecting duct.

Epidermal growth factor (EGF) inhibits amiloride-sensitive Na(+) conductance in the apical membrane of the isolated rabbit cortical collecting duct. However, there is no information on the relationship between electrolyte transport and tyrosine kinase. We examined the effect of EGF on transport of potassium and chloride as well as sodium and the roles of tyrosine kinases in the rabbit cortical collecting duct using in vitro isolated tubular microperfusion. Basolateral EGF depolarized the transepithelial voltage in a dose-dependent manner within a concentration range of 10(-10) in 10(-8) M. Basolateral ouabain and luminal amiloride completely abolished EGF-induced depolarization. However, luminal BaCl(2) did not abolish its depolarization. To confirm the mechanism, sodium, potassium, and chloride fluxes were measured in the presence of 10(-10) M EGF. EGF significantly decreased the lumen-to-bath isotope flux of sodium and chloride from 93.6+/-12.5 to 61.1+/-9.6 pmol/mm/min (n = 5, p<0.05) and from 86.6+/-10.0 to 54. 8+/-9.7 pmol/mm/min (n = 10, p<0.01), respectively. EGF also decreased net potassium secretion from -27.7+/-5.9 to -7.8+/-1.5 pmol/mm/min (n = 6, p<0.01). To examine whether EGF-induced depolarization is mediated by tyrosine kinase, tyrosine kinase inhibitors were applied from the basolateral side. Pretreatment with 1 microg/ml herbimycin A for 120 min completely abolished EGF-induced depolarization (90.9+/-5.4%, n = 4; NS). Herbimycin A itself also did not change the lumen-to-bath isotope flux of sodium and completely abolished the inhibition of Na(+) absorption on EGF action (control 65.4+/-6.8, herbimycin A 61.8+/-6.3, EGF with herbimycin A 60.0+/-4.4 pmol/min/mm, n = 5; NS). In conclusion, EGF depolarizes transepithelial voltage by inhibiting sodium transport primarily and potassium and chloride transport secondarily. These effects were blocked by nonspecific tyrosine kinase inhibitors.

Comparative mass spectrometric analyses of Photofrin oligomers by fast atom bombardment mass spectrometry, UV and IR matrix-assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry and laser desorption/jet-cooling photoionization mass spectrometry.

Photofrin (porfimer sodium) is a porphyrin derivative used in the treatment of a variety of cancers by photodynamic therapy. This oligomer complex and a variety of porphyrin monomers, dimers and trimers were analyzed with five different mass spectral ionization techniques: fast atom bombardment, UV and IR matrix-assisted laser desorption/ionization, electrospray ionization, and laser desorption/jet-cooling photoionization. All five approaches resulted in very similar oligomer distributions with an average oligomer length of 2.7 +/- 0.1 porphyrin units. In addition to the Photofrin analysis, this study provides a side-by-side comparison of the spectra for the five different mass spectrometric techniques.

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH.

Chronic hypokalemia increases the activity of proximal tubule apical membrane Na+/H+ antiporter NHE3. The present study examined the effect of the incubation of OKP cells (an opossum kidney, clone P cell line) in control medium (K+ concn ([K+]) = 5.4 mM) or low-K+ medium ([K+] = 2.7 mM) on NHE3. The activity of an ethylisopropyl amiloride-resistant Na+/H+ antiporter, whose characteristics were consistent with those of NHE3, was increased in low-K+ cells beginning at 8 h. NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%, respectively, at 24 h but not at 8 h. After incubation in low-K+ medium, intracellular pH (pHi) decreased by 0.27 pH units (maximum at 27 min) and then recovered to the control level. Intracellular acidosis induced by 5 mM sodium propionate increased Na+/H+ antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K+- and sodium propionate-induced activation of the Na+/H+ antiporter at 8 and 24 h. Our results demonstrate that low-K+ medium causes an early decrease in pHi, which leads to an increase in NHE3 activity via a tyrosine kinase pathway.

Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization - mass spectrometry.

A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.

Rapid methods for screening low molecular mass compounds non-covalently bound to proteins using size exclusion and mass spectrometry applied to inhibitors of human cytomegalovirus protease.

General and rapid methods were developed for determining the extent of non-covalent binding between small molecules and proteins, using the model system of human cytomegalovirus protease and several drug candidates which inhibit the protease by non-covalently binding to it. The assay was performed by off-line coupling of size-exclusion methods with mass spectrometry in the following manner. The protease and inhibitor were incubated together under native conditions and then subjected to separation based on size, by use of a spin column (gel permeation chromatography) and/or a microconcentrator (ultrafiltration). The spin column selectively passed the high molecular mass (M(r)) protease and trapped low M(r) molecules. Alternatively, the microconcentrator passed low M(r) molecules and retained the protease. If the inhibitor bound non-covalently to the protease, both the inhibitor and protease passed through the spin column (or were retained by the microconcentrator). Electrospray ionization mass spectrometry was used to assay the spin column eluate (or the microconcentrator retentate) and to characterize the amounts of protease and inhibitor based on known standards. An advantage of these techniques is that a mixture containing inhibitors can be analyzed in the presence of the protease, and inhibitors with the greatest binding affinity can be identified. Non-covalent binding specificity was demonstrated using spin columns by comparing the binding affinity of inhibitors using several mutants of cytomegalovirus protease. The techniques described are applicable to the rapid screening of compound libraries for selecting substances which bind non-covalently to a known protein.

Characterization of a recombinant fragment that contains a carbohydrate recognition domain of the filamentous hemagglutinin.

The filamentous hemagglutinin (FHA) of Bordetella pertussis plays an important role in establishing infection by attaching the bacteria to the ciliated respiratory epithelial cells. Expression of DNA encoding residues 1141 to 1279 of FHA in Escherichia coli yields a protein of 18,000 Da that exhibits some of the carbohydrate recognition properties of FHA (S. M. Prasad, Y. Yin, E. Rodzinski, E. I. Tuomanen, and H. R. Masure, Infect. Immun. 61:2780-2785, 1993). We have constructed an E. coli strain that expresses this protein, designated fragment A, in a soluble form at markedly elevated levels. Fragment A could be purified with high purity and yields and was immunogenic in mice. Both fragment A and anti-fragment A sera inhibited the binding of B. pertussis to asialo-GM2 and to rabbit ciliated cells. These observations demonstrate that this fragment of FHA contains a cellular binding domain capable of eliciting functional antibodies.

Cystatin C measurement and its practical use in patients with various renal diseases.

OBJECTIVE: To evaluate the clinical usefulness in terms of estimation for glomerular filtration rate (GFR), we determined the cystatin C levels in the serum and urine of 33 healthy volunteers as well as in the serum and urine of 35 patients with various renal diseases and compared them with those of creatinine. In addition, we evaluated this substance as an indicator of removal rate of low molecular weight protein with high flux membranes in 6 hemodialysis (HD) patients. METHODS: Serum and urinary cystatin C levels were measured by using an enzyme-linked immunosorbent assay (ELISA) method, 24-hour creatinine clearance was used as an indicator of GFR. RESULTS: Reference intervals with 95% ranges are 0.47-1.03 mg/l in the serum from healthy volunteers. There was a significant positive correlation between serum cystatin C and creatinine levels (r = 0.936, p < 0.001) in the patients with various renal diseases. Serum cystatin C and creatinine inversely and logarithmically correlated to creatinine clearance as shown in the following equations: log cystatin C = -0.564 x log creatinine clearance + 1.216 (r = -0.850), log creatinine = -0.678 x log creatinine clearance + 1.449 (r = -0.904). In these equations l/day is the unit used for creatinine clearance, mg/l is the unit used for serum cystatin C. The range for cystatin C is 0.67-6.15 mg/l, 0.66-7.23 mg/dl for creatinine and 8.9-186.3 l/day (6.2-129.4 ml/min) for creatinine clearance. Serum cystatin C levels started to increase over normal range when creatinine clearance fell below 135.9 l/day (94.4 ml/min), while serum creatinine remained within normal ranges. The daily urinary excretion of cystatin C was increased significantly in the group in which creatinine clearance was below 30 l/day (20.8 ml/min) compared to that in which creatinine clearance was higher than in 70 l/day (48.6 ml/min). Fractional clearance of cystatin C increased proportionally and markedly to the decrease of creatinine clearance. In a regular HD condition with high flux membrane, the cystatin C removal rate was 38.7 +/- 1.7%. CONCLUSIONS: These data suggest that combined measurement of cystatin C in the serum and urine is useful to estimate GFR, especially to detect the mild reduction of GFR. Cystatin C measurement can also be used as an indicator of removal rate of low molecular weight protein with different types of high flux membranes in hemodialysis.

Polysulfated carbohydrates analyzed as ion-paired complexes with basic peptides and proteins using electrospray negative ionization mass spectrometry.

Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.