Human Fluid Samples Research Articles

Use of cervicovaginal fluid for the identification of biomarkers for pathologies of the female genital tract

Cervicovaginal fluid has an important function in the homeostasis and immunity of the lower female genital tract. Analysis of the cervicovaginal fluid proteome may therefore yield important information about the pathogenesis of numerous gynecological pathologies. Additionally, cervicovaginal fluid has great potential as a source of biomarkers for these conditions.This review provides a detailed discussion about the human cervicovaginal proteome and the proteomics studies performed to characterize this biological fluid. Furthermore, infection-correlated pathological conditions of the female genital tract are discussed for which cervicovaginal fluid has been used in order to identify potential biomarkers. Recent years, numerous studies have analyzed cervicovaginal fluid samples utilizing antibody-based technologies, such as ELISA or Western blotting, to identify biomarkers for preterm birth, premature preterm rupture of membranes, bacterial vaginosis and cervical cancer. The present article will discuss the importance of proteomic technologies as alternative techniques to gain additional meaningful information about these conditions. In addition, the review focuses on recent proteomic studies on cervicovaginal fluid samples for the identification of potential biomarkers. We conclude that the use of proteomic technology for analysis of human cervicovaginal fluid samples is promising and may lead to the discovery of new biomarkers which can improve disease prevention and therapy development.

Grafting of poly[1-(N,N- bis-carboxymethyl)amino-3-allylglycerol- co-dimethylacrylamide] copolymer onto siliceous support for preconcentration and determination of lead (II) in human plasma and environmental samples

A method is reported for surface grafting of polymer containing a functional monomer for metal chelating, poly[1-(N,N- bis-carboxymethyl)amino-3-allylglycerol- co-dimethylacrylamide] (poly(AGE/IDA- co-DMAA) onto silica modified by silylation with 3-mercaptopropyltrimethoxysilane. Monomer 1-(N,N- bis-carboxymethyl)amino-3-allylglycerol (AGE/IDA) was synthesized by reaction of allyl glycidyl ether with iminodiacetic acid. The resulting sorbent has been characterized using FT-IR, elemental analysis, thermogravimetric analysis (TGA), FT-Raman and scanning electron microscopy (SEM) and evaluated for the preconcentration and determination of trace Pb(II) in human biological fluid and environmental water samples. The optimum pH value for sorption of the metal ion was 5.5. The sorption capacity of functionalized resin was 15.06 mg g −1. The chelating sorbent can be reused for 15 cycles of sorption–desorption without any significant change in sorption capacity. A recovery of 96.2% was obtained for the metal ion with 0.5 M nitric acid as eluting agent. The profile of lead uptake by the sorbent reflects good accessibility of the chelating sites in the poly(AGE/IDA- co-DMAA)-grafted silica gel. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. The equilibrium adsorption data of Pb(II) by modified resin were analyzed by Langmuir, Freundlich, Temkin and Redlich–Peterson models. On the basis of equilibrium adsorption data the Langmuir, Freundlich and Temkin constants were determined as 0.70, 1.35 and 2.7, respectively at pH 5.5 and 20 °C. Isotherms have also been used to obtain the thermodynamic parameters such as free energy, enthalpy and entropy of adsorption.

Application of Galactomannan Analysis and Protein Electrophoresis in the Diagnosis of Aspergillosis in Avian Species

Previous studies support the possible application of galactomannan, a major antigen of Aspergillus sp., to aspergillosis diagnosis in avian and other animal species. An assay is commercially available for use with human serum and bronchoalveolar lavage fluid samples. In the current study, galactomannan results from plasma samples were compared between birds with histologically confirmed aspergillosis and those that were clinically normal presumptively non-Aspergillus infected birds per submitting practitioners' responses to a questionnaire. It was observed that infected birds demonstrated a 2.6-fold increase in galactomannan over birds without evidence of aspergillosis. With the use of a galactomannan index of 0.5 as a cutoff, the sensitivity and specificity of the test were found to be 67% and 73%, respectively. In addition, plasma samples were analyzed for abnormalities in protein electrophoretic patterns. Infected birds had a higher incidence of increased beta and/or gamma globulin concentrations. Test sensitivity and specificity were 73% and 70%, respectively. If the 2 tests were used as a panel, then the sensitivity was 89% and specificity was 48%. These data indicate that both galactomannan and protein electrophoresis may be valuable tools in the diagnosis of avian aspergillosis.

An HPTLC Method for the Determination of Minocycline in Human Plasma, Saliva, and Gingival Fluid after Single Step Liquid Extraction

A rapid, sensitive and specific high-performance thin-layer chromatographic (HPTLC) method was developed and validated for determination of minocycline in human plasma, saliva, and gingival fluid samples. Densitometric analysis of minocycline was carried out at 345 nm after single step extraction with methanol. The method uses TLC aluminium plates pre-coated with silica gel 60F-254 as a stationary phase and methanol-acetonitrile-isopropyl alcohol-water (5:4:0.5:0.5, v/v/v/v) as mobile phase. In all the three matrices, the calibration curve was linear (r(2) >/= 0.9958) in the tested range of 100 - 1200 ng spot(-1) with a limit of quantification of 15.4 ng spot(-1). Drug recovery from plasma, saliva and gingival fluid averaged 97.7%. Intra- and inter-day accuracies, determined at three different concentrations, were 95.08 to 100.6% and the corresponding precision (% CV) values were < 4.61%. In all the three matrices, rapid degradation of drug occurred and the half-life of drug ranged from 9.9 to 16.1 h at 4 degrees C and from 6.3 to 11.5 h at 20 degrees C. Frozen at -20 degrees C, this drug was stable for at least 2 months and can tolerate two freeze-thaw cycles without losses higher than 10%. The method's ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.

Single-drop micro-extraction and diffuse reflectance Fourier transform infrared spectroscopic determination of chromium in biological fluids

The present paper deals with a new micro-extraction procedure for selective separation of Cr(VI) in the form of a metaloxy anionic species namely dichromate (Cr 2O 7 2−) with N 1-hydroxy- N 1, N 2-diphenylbenzamidine (HOA) in to dichloromethane and its subsequent and rapid diffuse reflectance Fourier transform infrared spectroscopic (DRS-FTIR) determination employing potassium bromide matrix. The diffuse reflectance Fourier transform infrared spectroscopy gives both qualitative and quantitative information about the dichromate. The determination of chromium is based on the analytical peak selection, among the various vibrational peaks, at 902 cm −1. The micro-extraction was based on the liquid–liquid solvent extraction (LLSE) principle. The dichromate binds with the nitrogen and oxygen atoms of N 1-hydroxy- N 1, N 2-diphenylbenzamidine (HOA) and forms 1:2, Cr(VI):HOA complex in 0.1 mol L −1 HCl medium. The formation of above complex, in the acidic medium, is confirmed by the appearance of chocolate-brown color in the micro-extract. The speciation studies of Cr(III) and Cr(VI) is done by conversion of Cr(III) into Cr(VI) utilizing H 2O 2 as an oxidizing agent. The chemistry of pure dichromate and that of its HOA complex is discussed. The limit of detection (LoD) and the limit of quantification (LoQ) of the method are found to be 0.01 μg g −1 Cr 2O 7 2− and 0.05 μg g −1 Cr 2O 7 2−, respectively. The standard deviation value and the relative standard value at a level of 10 μg Cr 2O 7 2−/0.1 g KBr for n = 10 is found to be 0.26 μg Cr 2O 7 2− and 2.6%, respectively. The relative standard deviation ( n = 8 and 6) for the determination of dichromate (Cr 2O 7 2−) in real human biological fluid samples is observed to be in the range 3.1–7.8%.

Evidence of in vitro glucuronidation and enzymatic transformation of paralytic shellfish toxins by healthy human liver microsomes fraction

Paralytic Shellfish Toxins (PST) are endemic components found in filter bivalves in Southern Chile. Post-mortems analysis of fluid and tissue samples has shown biotransformation of PST in humans. The Gonyautoxin 3 (GTX3) and Gonyautoxin 2 (GTX2) are the major PST components in the toxin profile found in Chilean shellfish extracts, being as much as 65% of the total content of PST in filter bivalves. Therefore, they are the major accountable components of the human intoxication by shellfish consumption. The aim of this study is to show in vitro glucuronidation and biotransformation of GTX3 and GTX2 when they are incubated with microsomal fraction isolated from healthy human livers. Microsomes fractions isolated from human livers were incubated with GTX3 and GTX2 purified from contaminated mussels. After different incubation times, incubated samples were extracted and analyzed by HPLC with fluorescent on line detection and HPLC-MS analysis. The results revealed that GTX3 and GTX2, only when they were incubated with microsomal fraction and appropriated cofactors, showed to be enzymatic transformed in vitro. The glucuronidation of GTX3 and GTX2 followed typical Michaelis–Menten kinetics, resulting in apparent kinetic parameters of K m = 39.4 ± 0.24 μM and V max = 6.0 × 10 −3 pmol/min/mg protein. In addition, the microsomes fraction also oxidized GTX3 and GTX2 into Gonyautoxin 4 (GTX 4) and Gonyautoxin 1 (GTX 1) resulting in 0.339 × 10 −3 pmol/min/mg protein. In conclusion, this study reports oxidation and glucuronidation of GTX3 and GTX2 when they are incubated with human liver microsomal fraction. The metabolism occurs via a glucuronidation reaction, the basis first step of biotransformation in human liver. Also it is showed that GTX4 and GTX1 came by biotransformation from GTX3 and GTX2 in humans. This data confirm human biotransformation found in human post-mortem fluid and tissue samples described previously. This data is the first evidence of in vitro glucuronidation of PST, given a metabolic pathway of detoxification and excretion of PST in human.

Multiplexed quantitation of endogenous estrogens and estrogen metabolites in human peritoneal fluid

Endogenous estrogens and estrogen metabolites (EM) in human peritoneal fluid may play an important role in health and disease, yet little is known regarding their types and levels present in human peritoneal fluid, primarily due to the lack of an analytical method that is capable of directly quantifying their absolute abundances. In this report, we describe the application of a capillary LC-MS/MS method for identifying and quantifying biologically active and total endogenous EM in human peritoneal fluid. The method requires only 50 muL of peritoneal fluid, yet can quantify 13 distinct EM. Calibration curves for each EM were linear over a 10(3)-fold concentration range and the lower LOQ was 50 fg on-column. For a charcoal stripped human peritoneal fluid sample containing 10 pg/mL of each EM, accuracy ranged from 83 to 118%, and intrabatch precision ranged from 0.2 to 4.4% RSD and interbatch precision ranged from 5.5 to 15.5% RSD. The analyses of human female peritoneal fluid shows that at least 10 biologically active and 11 total endogenous EM can be positively identified and quantitatively measured. Many of the biologically active forms are present in high abundance and possess distinct biological activities which warrant further study. Although micellar EKC gave baseline separation of a standard mixture of 10 EM, the LOQs using UV detection were not suitable for the assay of the low level estrogens in biological samples.

Matching of oligoclonal immunoglobulin transcriptomes and proteomes of cerebrospinal fluid in multiple sclerosis

We describe a method for correlating the immunoglobulin (Ig) proteomes with the B cell transcriptomes in human fluid and tissue samples, using multiple sclerosis as a paradigm. Oligoclonal Ig bands and elevated numbers of clonally expanded B cells in the cerebrospinal fluid (CSF) are diagnostic hallmarks of multiple sclerosis. Here we compared the Ig transcriptomes of B cells with the corresponding Ig proteomes in CSF samples from four subjects with multiple sclerosis. We created individual Ig transcriptome databases that contained the subject-specific mutations introduced by V(D)J recombination and somatic hypermutation and then searched the CSF for corresponding characteristic peptides by mass spectrometry. In each sample, the Ig transcriptomes and proteomes strongly overlapped, showing that CSF B cells indeed produce the oligoclonal Ig bands. This approach can be applied to other organ-specific diagnostic fluid or tissue samples to compare the Ig transcripts of local B cells with the corresponding antibody proteomes of individual subjects.

Rapid Antimicrobial Susceptibility Determination of Uropathogens in Clinical Urine Specimens by Use of ATP Bioluminescence

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

Joint fluid antioxidants are decreased in osteoarthritic joints compared to joints with macroscopically intact cartilage and subacute injury

Excess reactive oxygen species and oxidative damage have been associated with the pathogenesis of osteoarthritis (OA). Extracellular superoxide dismutase (EC-SOD or SOD3) scavenges superoxide is the major catalytic antioxidant in joint fluid and is decreased in OA cartilage. We studied human joint fluid samples to test whether there is an association between OA and EC-SOD or other low molecular antioxidants in the joint fluid. Joint fluid samples were obtained from 28 subjects with severe OA undergoing arthrocentesis or knee joint replacement and compared to joint fluid from 12 subjects undergoing knee arthroscopy for chronic knee pain, meniscal tears or anterior cruciate ligament reconstruction. EC-SOD protein was assayed by enzyme-linked immunosorbent assay (ELISA). Ascorbate and urate were measured with high performance liquid chromatography (HPLC) and total nitrates by the Greiss reaction. Glutathione (GSH) and oxidized glutathione were measured using a colorimetric method. Interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta) were both measured with ELISA. Human joint fluid contains significant amounts of the extracellular, catalytic antioxidant EC-SOD. Joint fluid from OA subjects is characterized by significantly decreased EC-SOD levels and significant decreases in GSH, and ascorbate compared to the reference group of knee joints with pain or subacute injury but macroscopically intact cartilage. GSH and ascorbate show only an age effect with no effect from disease state on regression modeling. Urate is present in joint fluid but does not show a significant difference between groups. IL-6 and TGF-beta both show non-significant trends to increases in the arthritic subjects. There was no correlation of EC-SOD levels with IL-6 as a marker of inflammation in either the comparison group or the OA group. EC-SOD, the major scavenger of reactive oxygen species (ROS) in extracellular spaces and fluids, is decreased in late stage OA joint fluid compared to fluid from injured/painful joints with intact cartilage. Injured joints may be able to increase or maintain secretion of EC-SOD but it appears that late stage OA joints fail to do so in spite of increased oxidative stress seen in the disease. Associated age related declines in GSH and ascorbate might also contribute to the development of severe OA. The net effect of these changes in joint fluid antioxidants is likely to accelerate the damaging oxidant effects on extracellular matrix stability in cartilage tissue.

Use of Electrochemical DNA Biosensors for Rapid Molecular Identification of Uropathogens in Clinical Urine Specimens

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

Electroanalytical Characteristics of Amisulpride and Voltammetric Determination of the Drug in Pharmaceuticals and Biological Media

AbstractThe electrochemical oxidation of antipsychotic drug amisulpride (AMS) has been studied in pH range 1.8–11.0 at a stationary glassy carbon electrode by cyclic, differential pulse and square‐wave voltammetry. Two oxidation processes were produced in different supporting electrolyte media. Both of the oxidation processes were irreversible and exhibited diffusion controlled. For analytical purposes, very resolved voltammetric peaks were obtained using differential pulse and square‐wave modes. The linear response was obtained in the range of 4×10−6 to 6×10−4 M for the first and second oxidation steps in Britton‐Robinson buffer at pH 7.0 and pH 3.0 (20% methanol v/v), respectively, using both techniques. These methods were used for the determination of AMS in tablets. The first oxidation process was chosen as indicative of the analysis of AMS in biological media. The methods were successfully applied to spiked human serum, urine and simulated gastric fluid samples.

Identification of trichloroethylene and its metabolites in human seminal fluid of workers exposed to trichloroethylene.

We have investigated the potential of the male reproductive tract to accumulate trichloroethylene (TCE) and its metabolites, including chloral, trichloroethanol (TCOH), trichloroacetic acid (TCA), and dichloroacetic acid (DCA). Human seminal fluid and urine samples from eight mechanics diagnosed with clinical infertility and exposed to TCE occupationally were analyzed. In in vivo experimental studies, TCE and its metabolites were determined in epididymis and testis of mice exposed to TCE (1000 ppm) by inhalation for 1 to 4 weeks. In other studies, incubations of monkey epididymal microsomes were performed in the presence of TCE and NADPH. Our results showed that seminal fluid from all eight subjects contained TCE, chloral, and TCOH. DCA was present in samples from two subjects, and only one contained TCA. TCA and/or TCOH were also identified in urine samples from only two subjects. TCE, chloral, and TCOH were detected in murine epididymis after inhalation exposure with TCE for 1 to 4 weeks. Levels of TCE and chloral were similar throughout the entire exposure period. TCOH levels were similar at 1 and 2 weeks but increased significantly after 4 weeks of TCE exposure. Chloral was identified in microsomal incubations with TCE in monkey epididymis. CYP2E1, a P450 that metabolizes TCE, was localized in human and monkey epididymal epithelium and testicular Leydig cells. These results indicated that TCE is metabolized in the reproductive tract of the mouse and monkey. Furthermore, TCE and its metabolites accumulated in seminal fluid, and suggested associations between production of TCE metabolites, reproductive toxicity, and impaired fertility.

Measurement of bisphenol A levels in human blood serum and ascitic fluid by HPLC using a fluorescent labeling reagent

A sensitive column-switching HPLC method with fluorescence detection was developed for the determination of bisphenol A (BPA) in human blood serum and ascitic fluid samples. 4-(4,5-Diphenyl-1 H-imidazol-2-yl)benzoyl chloride (DIB-Cl) was used as the fluorescent label, and the excess reagent was removed by a column-switching technique. Liquid–liquid extraction with chloroform was used for the pretreatment of serum and ascitic fluid samples. BPA in both the samples could be determined in the concentration range of 0.1–7.0 ppb with the detection limit of 0.04 ppb at a signal-to-noise ratio of 3. The recoveries of BPA spiked to serum and ascitic fluid were 78.6 and 77.7%, respectively. The mean concentrations of BPA ( n=9) in maternal and umbilical cord blood sera obtained from healthy pregnant women were 0.46±0.20 and 0.62±0.13 ppb, respectively. BPA levels ( n=21) in blood sera and ascitic fluid obtained from the patients with sterility were also determined to be 0.46±0.20 and 0.56±0.19 ppb, respectively. Relationships of BPA concentrations were observed between maternal and umbilical cord blood serum samples ( r=0.626), as well as blood serum and ascitic fluid samples ( r=0.785).

An ELISA technique for quantification of surfactant apoprotein (SP)-C in bronchoalveolar lavage fluid.

Pulmonary surfactant apoprotein C (SP-C) is a small, unique peptide that contributes to the reduction of alveolar surface tension. Due to the extreme hydrophobic nature of this peptide it was hitherto not possible to quantify SP-C in biological samples by immunological techniques. Using a newly developed polyclonal antibody raised against recombinant human SP-C in rabbits, we now describe an enzyme-linked immunosorbent assay (ELISA) to quantitate SP-C in bronchoalveolar lavage fluid (BALF). Solid phase binding of the hydrophobic SP-C was achieved by transfer of the standard or BALF samples (diluted in 80% isopropanol, pH 3.5) to polystyrene microtiter plates. Sequential treatment with trifluoroethanol and methanol (2x) was employed to improve antigen presentation and to minimize the influence of phospholipids. With this assay, SP-C from human, rabbit, porcine, and bovine surfactant was detectable. No cross-reactivity of the antibody to human SP-A and monomeric and dimeric SP-B was encountered. Total serum proteins did not affect ELISA signals, as evident from spiking experiments. The detection limit of the ELISA ranged below 3 ng/ml, and intra- and interassay coefficients of variation were 3.5% (n = 16) and 5.3% (n = 6), respectively. Serial dilutions of BALF showed good linearity, and excellent recovery rates were obtained upon spiking of human BALF. A mean value of 579.5 +/- 45.9 ng/ml (mean +/- SEM) SP-C was found in BALF samples of human healthy volunteers (n = 22), corresponding to 26.61 +/- 1.91 microg SP-C/mg total phospholipids (PL). SP-C levels were significantly lower in BALF of patients with acute respiratory distress syndrome (ARDS) (286.9 +/- 19.8 ng/ml [p < 0.001]; 13.92 +/- 1.93 microg SP-C/mg PL [p < 0.001], n = 48). We conclude that SP-C may be quantified with high specificity, reproducibility, and sensitivity in bronchoalveolar lavage samples by the presently described ELISA technique and that SP-C levels are significantly decreased in ARDS.

Instrumental detection limits in total reflection X-ray fluorescence: Comparison of cut-off and monochromatic excitation devices

“Cut-off” reflectors, which allow obtaining a reflected beam with almost no high energy radiation, as well as different monochromator devices that reflect a beam of monoenergetic and polarized radiation are the most widely employed artifacts to perform the excitation radiation improvement in analytical “non-expensive” TR spectrometers. The aim of this work consisted in testing several monochromator devices: a multilayer structured mirror for soft X-rays (0.7–12.5 nm), as well as different bent and flat crystals of wavelength dispersive spectrometers. The achieved instrumental detection limits for the selected monochromator device are compared with the values obtained using a Suprasil Cut-off reflector and a C-W multilayer (d∼5 nm). Several human corporal fluid samples were analyzed to compare the spectral characteristics when using the Cut-Off Reflector and the RAP flat crystal, respectively.

High resolution [formula omitted] NMR spectroscopic studies on dynamic biochemical processes in incubated human seminal fluid samples

High resolution 600 MHz 1 H NMR spectroscopy was used to investigate the changes in biochemical composition of whole human seminal fluid (SF) and an artificial mixture of prostatic (PF) and seminal vesicle fluid (SVF). A variety of time-related biochemical changes were monitored simultaneously and non-invasively in SF, including enzymatic hydrolysis of phosphorylcholine to choline and polypeptides to amino acids. The fastest NMR-observable reactions in SF were the conversion of phosphorylcholine to choline ( t 1/2≅9 min) and uridine-5′-monophosphate (UMP) to uridine ( t 1/2<2 min). UMP has not previously been detected in SF because of its rapid hydrolysis. Artificial mixtures of separately obtained prostatic and SVF showed very similar biochemical changes to those observed in whole SF. Addition of EDTA to SF incubated for 2 min post ejaculation strongly inhibited peptide hydrolysis. Zn 2+, present in whole SF was shown to be non EDTA-chelatable 2 min after ejaculation, whereas after 7 min, a singlet signal from the ethylenic protons of the Zn-EDTA 2− complex was clearly observed which remained constant after 7 min. This indicates that soon after ejaculation (<5 min) Zn 2+ is immobilised in a macromolecular complex which is rapidly broken down by proteolytic enzymes, the released Zn 2+ then being free to react with EDTA. Mg- and Ca-EDTA 2− complexes were observed at 2 min and remained constant (at 1.4 and 2.1 mM, respectively) throughout the entire time course of the experiment. These studies cast new light on the time-related biochemical changes occurring in the post-ejaculatory SF which may have an important role in reproductive function.

Proton MRS of human prostatic fluid: Correlations between citrate, spermine, andmyo-inositol levels and changes with disease

This is a study of the unusual small molecular components of human prostatic fluid using a non-destructive technique. Single pulse high resolution proton MRS of 38 human prostatic fluid samples (12 control, 10 with benign prostatic enlargement, 4 with prostatic cancer, 11 with vasal aplasia, and one with prostatodynia). Regression models for the metabolites measured were made and compared, and correlations were analyzed. A very strong correlation between the secretion of citrate and spermine (r = 0.94), two of the major components of prostatic fluid, was found. The molar ratio was 5:1 citrate: spermine. There was no difference seen between samples obtained by expression or ejaculation. The regression models suggest there is a significant difference (P < 0.02) in the citrate to spermine ratio in prostatic fluid from men with prostate cancer, with a relatively higher level of spermine. The authors speculate that citrate and spermine secretion is linked and may be forming a novel complex.

Human simple renal cyst fluid contains a cyst formation-promoting activity for Madin-Darby canine kidney cells cultured in collagen gel.

Simple renal cysts are commonly observed in the elderly, but few examinations have been performed to study the mechanism of this disease. We have previously shown that simple renal cyst fluid contains some growth factors, including epidermal growth factor (EGF; less than 54 pg mL-1) and insulin-like growth factor I (IGF-I; 30-2070 pg mL-1) (M. Taide et al. Clin Chim Acta, 1993; 217:199-203). In this report, the biological significance of growth factors or of human simple renal cyst fluid on the formation of renal epithelial cyst was determined using Madin-Darby canine kidney (MDCK) cells cultured in collagen gel. Both EGF (200 pg mL-1 or more) and IGF-I (100 pg mL-1 or more) were found to stimulate cyst formation in MDCK cells. Additionally, 12/21 human simple renal cyst fluid samples were found to be capable of stimulating cyst formation in MDCK cells. This activity was due to a heat- and acid-labile protein and not inhibited by the anti-IGF-I neutralizing antibody. These results indicate that in some instances simple renal cyst fluid would thus appear to contain specific activity for cyst formation, which may affect cyst formation in vivo.

PA·FPNS: its synthesis and use in spectrophotometric determination of magnesium

A new polymeric chromogenic reagent PA·FPNS has been synthesized by condensing polyallylamine (PA) with 3-(4-formylphenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic acid (FPNS) and its properties studied. In alkaline media, PA·FPNS reacts with magnesium to form a water-soluble blue complex, whose absorption maximum is at 604 nm. The molar absorptivity ( ɛ) of the complex is 5.2 × 10 4l mol −1 cm −1, which is four times that of the FPNS-Mg complex, and Beer's law is obeyed over the range 0–0.35 μg ml −1 magnesium. Compared to the corresponding low-molecular-weight FPNS and other chromogenic reagents, PA·FPNS offers considerably improved sensitivity and selectivity for magnesium, which may be attributed to incorporating FPNS into a water-soluble polymer and the effect of the polymeric chain on the reaction microenvironment. Also, a simple and sensitive spectrophotometric method for the determination of magnesium has been developed and applied to water and human fluid samples with satisfactory results.