Proteomic Analysis of Urine from California Sea Lions ( Zalophus californianus): A Resource for Urinary Biomarker Discovery.

Urinary markers for the assessment of kidney diseases in wild animals are limited, in part, due to the lack of urinary proteome data, especially for marine mammals. One of the most prevalent kidney diseases in marine mammals is caused by Leptospira interrogans, which is the second most common etiology linked to stranding of California sea lions ( Zalophus californianus). Urine proteins from 11 sea lions with leptospirosis kidney disease and eight sea lions without leptospirosis or kidney disease were analyzed using shotgun proteomics. In total, 2694 protein groups were identified, and 316 were differentially abundant between groups. Major urine proteins in sea lions were similar to major urine proteins in dogs and humans except for the preponderance of resistin, lysozyme C, and PDZ domain containing 1, which appear to be over-represented. Previously reported urine protein markers of kidney injury in humans and animals were also identified. Notably, neutrophil gelatinase-associated lipocalin, osteopontin, and epidermal fatty acid binding protein were elevated over 20-fold in the leptospirosis-infected sea lions. Consistent with leptospirosis infection in rodents, urinary proteins associated with the renin-angiotensin system were depressed, including neprilysin. This study represents a foundation from which to explore the clinical use of urinary protein markers in California sea lions.

Fabrication and evaluation of molecularly imprinted magnetic nanoparticles for selective recognition and magnetic separation of lysozyme in human urine.

In this study, lysozyme-based magnetic molecularly imprinted polymers (Lyz-MMIPs) for selective recognition and magnetic separation of lysozyme in human urine were prepared via surface imprinting technology. The morphology and structural properties of the resultant Lyz-MMIPs were characterized by using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), thermogravimetry (TG), X-ray diffraction (XRD) and a vibrating sample magnetometer (VSM). The results showed that the Lyz-MMIPs exhibited a uniform core-shell structure and favorable magnetic properties with a saturation magnetization of 14.8 emu g-1. To obtain the best selectivity and binding performance, the pH value of adsorption solution was investigated in detail. Under the optimized conditions, the Lyz-MMIPs possessed high binding and specific recognition capacity, as well as fast binding kinetics and phase separation rates. Moreover, the saturation binding capacity of Lyz-MMIPs reached 124.3 mg g-1, which was nearly 3.2 times that of lysozyme-based magnetic non-imprinted polymers (Lyz-MNIPs). In addition, the selectivity and reusability experiments showed that the Lyz-MMIPs displayed significant selectivity and favorable reusability. Furthermore, the Lyz-MMIPs were successfully applied for the determination and separation of lysozyme in human urine with satisfactory recovery rates. Above all, the synthetic process was quite simple and this strategy may provide a versatile approach for the fabrication of well-defined molecularly imprinted polymers on magnetic nanoparticles for the analysis of complicated matrixes.

Lysozyme aptasensor based on a glassy carbon electrode modified with a nanocomposite consisting of multi-walled carbon nanotubes, poly(diallyl dimethyl ammonium chloride) and carbon quantum dots.

An aptamer-based method is described for electrochemical determination of lysozyme. A glassy carbon electrode was modified with a nanocomposite composed of multi-walled carbon nanotubes, poly(diallyl dimethyl ammonium chloride), and carbon quantum dots. The composition of the nanocomposite (MWCNT/PDDA/CQD) warrants good electrical conductivity and a high surface-to-volume ratio. The lysozyme-binding aptamers were immobilized on the nanocomposite via covalent coupling between the amino groups of the aptamer and the carboxy groups of the nanocomposite. The modified electrode was characterized by electrochemical impedance spectroscopy, cyclic voltammetry and differential pulse voltammetry. The use of this nanocomposite results in a considerable enhancement of the electrochemical signal and contributes to improving sensitivity. Hexacyanoferrate was used as an electrochemical probe to study the dependence of the peak current on lysozyme concentration. In the presence of lysozyme, the interaction of lysozyme with immobilized aptamer results in a decrease of the peak current, best measured at +0.15 V vs. Ag/AgCl. A plot of peak current changes versus the logarithm of the lysozyme concentration is linear in the 50 fmol L-1 to 10 nmol L-1 concentration range, with a 12.9 fmol L-1 detection limit (at an S/N ratio of 3). The method is highly reproducible, specific and sensitive, and the electrode has a rapid response. It was applied to the determination of lysozyme in egg white, serum, and urine. Graphical abstract Schematic of a nanocomposite composed of multi-walled carbon nanotubes (MWCNTs), poly(diallyldimethyl ammonium chloride) (PDDA), and carbon quantum dots (CQDs) for use in a lysozyme aptasensor. The aptamer was immobilized on the surface, and bovine serum albumin (BSA) was applied to block the surface. The changes of peak current for the electrochemical probe hexacyanoferrate (Fe(CN)63-/4-) in the presence and absence of lysozyme was traced.

Hydrophobically modified quaternized celluloses as new dynamic coatings in CE for basic protein separation.

Hydrophobically modified quaternized celluloses (HMQCs), containing the quaternary ammonium groups and hexadecyl groups, were homogeneously synthesized as novel dynamic coatings for CE. Compared with quaternized cellulose coating, HMQC coating is able to generate stronger reversed EOF (ca. 10% increase) and has better efficiency in suppressing protein adsorption. The effects of the polymer concentration, the degree of hydrophobic substitution, and the buffer pH on the EOF mobility as well as on the separation of basic proteins were investigated in detail. It was shown that the use of HMQC coating could obviously improve the separation performance of basic proteins within a broad pH range. Five basic proteins (lysozyme, ribonuclease A, cytochrome c, bovine pancreatic trypsin inhibitor, and chymotrypsinogen) could be completely separated at pH 8.0. The successful performance of HMQC was further demonstrated by the analyses of lysozyme in tear and urine samples.

New strategy for label-free and time-resolved luminescent assay of protein: conjugate Eu3+ complex and aptamer-wrapped carbon nanotubes.

We report here a carbon nanotube-based approach for label-free and time-resolved luminescent assay of lysozyme (LYS) by engineering an antilysozyme aptamer and luminescent europium(III) (Eu(3+)) complex. The sensing mechanism of the approach is based on the exceptional quenching capability of carbon nanotubes for the proximate luminescent Eu(3+) complex and different propensities of single-stranded DNA and the DNA/protein complex to adsorb on carbon nanotubes. The luminescence of a mixture of chlorosulfonylated tetradentate ß-diketone-Eu(3+) and the antilysozyme aptamer was efficiently quenched by single-walled carbon nanotubes (SWNTs) unless the aptamer interacted with LYS. Due to the highly specific recognition ability of the aptamer for the target and the powerful quenching property of SWNTs for luminescence regents, this proposed approach has a good selectivity and high sensitivity for LYS. In the optimum conditions described, >700-fold signal enhancement was achieved for micromolar LYS, and a limit of detection as low as 0.9 nM was obtained, which is about 60-fold lower than those of commonly used fluorescent aptamer sensors. Moreover, due to the much longer lifetime of the Eu(3+) luminescence than those of the ubiquitous endogenous fluorescent components, the time-resolved luminescence technique could be conveniently used for application in complicated biological samples. LYS concentrations in human urine were thus detected using time-resolved luminescence measurement with satisfactory recoveries of 95-98%.

Magnetoelastic Immunosensor via Antibody Immobilization for the Specific Detection of Lysozymes.

Lysozymes in human urine have crucial clinical significance as an indicator of renal tubular and glomerular diseases. Most lysozyme detection methods rely on the enzyme-linked immunosorbent assay (ELISA), which is usually a tedious procedure. Meanwhile, aptamer sensors and fluorescence-based techniques for lysozyme detection have emerged in recent studies. However, these methods are time-consuming and highly complex in operation, and some even require exorbitant reagents and instruments, which restricts real-time clinical monitoring as diagnostic approaches. Therefore, a rapid and low-cost lysozyme detection method with facile preparation is still in demand for modern precision medicine. Herein, we propose a magnetoelastic (ME) immunosensor for lysozyme detection by detecting changes in resonance frequency under a magnetostrictive effect. The detection system is composed of a magnetoelastic chip with an immobilized lysozyme antibody, a solenoid coil, and a vector network analyzer. Since the ME sensor is ultrasensitive to mass change, the frequency offset caused by mass change can be utilized to detect the content of lysozyme. The immunosensor is evaluated to possess superior sensitivity of 138 Hz/µg mL-1 in terms of the resonance frequency shift (RFS). In addition, our sensor displays an outstanding performance in specificity experiments and shows a relatively lower detection limit (1.26 ng/mL) than other conventional lysozyme detection methods (such as ELISA, chemiluminescence assay, fluorescence, and aptamer biosensors).

Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia.

Markedly increased quantities of lysozyme have been found in the serum and urine (ranging to 2.6 g per day) of ten consecutive cases of monocytic and monomyelocytic leukemia. The enzyme has been isolated from the urine of several cases and physicochemically and immunochemically characterized. It is apparently identical to the lysozyme of normal tears, saliva, leukocytes, and serum, but structurally different from the lysozyme of hen's egg white. The activity of the human enzyme assayed with M. lysodeikticus organisms is 3 to 12 times greater than egg white lysozyme at equivalent concentrations. An agar plate method has been developed for quantitating lysozyme activity in small samples (approximately 25 microl) of serum, urine, or other biological fluids. The range and reproducibility of this method were found to be superior to previously available lysozyme assay procedures. Present evidence indicates that lysozyme is the principal, if not the sole, product of the proliferating monocytes in monocytic and monomyelocytic leukemia, and quantitation of serum and urine lysozyme should be a useful diagnostic procedure for these leukemias.

High serum and urine lysozyme levels in a dog with acute myeloid leukemia.

A 2-year-old, female German Shepherd Dog with facial nerve paralysis was diagnosed with acute myelomonocytic leukemia based on clinical, cytologic, and immunologic findings. Proteinuria (urine proteincreatinine ratio = 1.5) occurred in the absence of renal failure. Qualitative assessment of proteinuria by sodium dodecyl sulfate-agarose gel electrophoresis revealed a broad band with a molecular weight of approximately 15 kDa that was compatible with lysozyme (LZM). A diagnosis of tubular proteinuria was made, and a chemical evaluation of LZM in serum and urine samples was performed using a turbidimetric assay. The LZM concentrations were 24.5 mg/l (reference interval: 2.5-8.0 mg/l) and 274.5 mg/l (reference interval: <2 mg/l) in serum and urine, respectively.

Cystatin C, Neutrophil Gelatinase-associated Lipocalin, and Lysozyme C: Urinary Biomarkers for Detection of Early Kidney Dysfunction in Children With Urolithiasis.

OBJECTIVE: To screen for the presence of biomarkers involved in tubular injury and kidney damage in children with urolithiasis (RS), and to validate these proteins by ELISA. METHODS: Prospective-controlled pilot study of children with urolithiasis and their age- and gender-matched controls (HC). Initial screening test was done by quantitative proteomic comparison of pooled urine from RS versus HC, using liquid chromatography-mass spectrometry. Proteins of interest were selected using the following criteria: (1) ≥5 spectral counts; (2) ≥2-fold difference in spectral counts; and (3) ≤.05 P value for the Fisher's Exact Test. Validation was performed by ELISA testing. Statistical analysis was performed by Student t-test and Mann-Whitney U test. RESULTS: Proteomic analysis identified 3 proteins of interest, Cystatin C (CYTC), neutrophil gelatinase-associated lipocalin (NGAL) and lysozyme C that were significantly over-represented in RS group versus HC. ELISA analysis revealed significantly increased urinary levels of CYTC and NGAL, and nearly significantly increased urinary levels of lysozyme C in RS group (N = 24) compared to controls (N = 13). Subgroup analysis showed significantly higher urinary levels of CYTC in both hypercalciuria (N = 14) and hypocitraturia (N = 10) versus HC (P <.05). CONCLUSION: Children with urolithiasis showed significant increase in urinary CYTC and NGAL irrespective of their normal serum creatinine. These biomarkers indicate tubular injury and early kidney damage and represent valid tools for early screening when traditional tests are normal.

Integrated microfluidic system for electrochemical sensing of urinary proteins.

This study reports a new microfluidic system integrated with a microfluidic control module and a micro electrochemical module for detection of urinary proteins. The integrated microsystem can automatically detect proteins in urine with a high sensitivity. The microfluidic control module consists of a new two-way, spiral-shape micropump which can transport the urine samples to the sensing regions. The net ionic charges of the protein samples can be detected while the samples flow through the sensing region of the micro electrochemical module. Two major urinary proteins including lysozyme and albumin are detected in a multiple-channel layout with little human intervention and are analyzed in a short period of time, while only consuming a 100-mul urine sample. The developed microfluidic system could lead to a convenient, yet crucial, platform for chemical and biological detection and diagnosis.

Crystallization of human lysozyme.

Lysozyme, isolated from the urine of patients with monocytic leu kemia, has been crystallized as the chlo ride at pH 4.5 and at pH 10.5. The crystal forms of the human enzyme show certain similarities as well as dis tinct dissimilarities compared with the crystal forms of lysozyme chloride from hen's egg white.

Mouse lysozyme production by a monocytoma: isolation and comparison with other lysozymes.

A transplantable mouse tumor, GPC-11, produces large amounts of lysozyme. The tumor is a reticulum cell sarcoma, type A, and is a neoplasm of monocytes. The lysozyme was purified from mouse urine in quantities sufficient for structural analysis. Comparison of mouse lysozyme with lysozymes from; chicken egg white and patients with monocytic leukemia reveals similarities in size and electrophoretic mobility and, with human lysozyme, in functional properties; but considerable differences are found in antigenic characteristics and amino acid composition.

Acute health effects on planters of conifer seedlings treated with insecticides.

OBJECTIVES: The aim of this study was to assess acute health effects on planters caused by planting conifer seedlings treated with two insecticides, with active ingredients imidacloprid and cypermethrin, in comparison with untreated seedlings. METHODS: The investigation was a double-blind crossover study, which included a follow-up of 19 planters over a 3-week period. During Week 1, the 19 planters handled untreated conifer seedlings while they planted imidacloprid- and cypermethrin-treated seedlings during study Week 2 and 3, respectively. Signs and symptoms of acute health effects were documented by a questionnaire, administered by the field staff, during these 3 weeks. Inflammation markers in the nasal mucous membrane were also measured as an objective test. Exposure to cypermethrin was further assessed by measuring 3-phenoxybenzoic acid (3-PBA) in urine. No validated biomarker was available to assess internal exposure to imidacloprid. RESULTS: No clear, acute adverse health effects could be found in planters during the week of exposure to conifer seedlings treated with imidacloprid (Merit Forest) or cypermethrin (Forester), as compared to during the week of planting untreated seedlings. During the week of cypermethrin exposure, the individuals had 3-PBA values that were 12-54% higher (P < 0.05), depending on the worker, than those observed during the untreated week. There were no statistically significant correlations between the raised levels of 3-PBA and self-reported health problems. These results have been obtained during planting in late summer/early autumn and with good use of protective clothing. CONCLUSIONS: No clear, acute adverse health effects could be found in planters after exposure to conifer seedlings treated with imidacloprid (Merit Forest) or cypermethrin (Forester), as compared with planting untreated seedlings. The metabolite, 3-PBA, was found in low levels in urine and was increased after exposure to cypermethrin. However, no clear relationships could be found between exposure and reported symptoms or between elevated 3-PBA levels and reported symptoms.

Lysozyme turnover in man.

Lysozyme turnover studies with (125)I-labeled human lysozyme were carried out on 22 patients, viz. nine control patients, seven nephrological patients with varying degrees of renal insufficiency, including three bilaterally nephrectomized patients, and six hematological patients with disturbed turnover of the neutrophilic granulocytes. It was found that plasma lysozyme has a rapid turnover with a fractional catabolic rate of 76%/hr of the plasma content. Lysozyme catabolism varied with the endogenous creatinine clearance; in addition however, extrarenal sites of catabolism were demonstrated since lysozyme could be broken down in the anephric patients, although only at a rate amounting to about 15% of the rate found in persons with intact kidneys. In the uremic patients the increased plasma lysozyme concentration was due to decreased rates of catabolism; in the hematological patients the increased plasma lysozyme level was due to increased rates of synthesis which supports the hypothesis that plasma lysozyme mainly stems from disintegrating neutrophilic granulocytes. Furthermore, it was shown that in the nonhematological patients examined, the rate of synthesis varied with the endogenous creatinine clearance.

Serum and urinary proteins, lysozyme (muramidase), and renal dysfunction in mono- and myelomonocytic leukemia.

Serum levels, urinary excretion, and clearances of several proteins of different molecular weights were studied in 18 patients with mono- and myelomonocytic leukemia. Nine patients had normal renal function (group A) and nine had impaired renal function with azotemia (group B). The majority of patients in both groups had increased concentration of immunoglobulins, particularly IgG, IgA, and IgM; IgD level was normal. Serum transferrin and alpha(2)-macroglobulin were frequently reduced while the level of ceruloplasmin was often increased, especially in patients with azotemia. The activity of lysozyme in the serum was high in all patients, but was considerably higher in group B. Proteinuria was found in most patients but was more prominent in group B. Almost invariably albumin constituted less than 25% of the total protein excreted. Qualitative analysis of various urinary proteins by immunochemical techniques and clearance studies suggested the presence of glomerular as well as tubular dysfunction. Determination of urinary lysozyme frequently showed no direct correlation between the serum level of the enzyme and its concentration in the urine or its clearance by the kidney. In addition to glomerular filtration, impaired tubular reabsorption may account for the high level of lysozyme in the urine. It is postulated that the very high level of lysozyme in the glomerular filtrate and possibly hypergammaglobulinemia may play a role in the induction of tubular damage. Renal impairment has been correlated with histological changes in the kidneys. From a comparative study of various leukemias, it seems that the combined glomerular-tubular dysfunction is a manifestation unique to mono- and myelomonocytic leukemia.

Tumor-associated macrophages as the primary source of lysozyme in the urine of mice bearing GPC-11, a transplantable reticulum cell sarcoma.

Large amounts of lysozyme accumulated in the serum and urine of (NZB X BALB/c)F1 mice with GPC-11, a transplantable reticulum cell sarcoma, type A. We separated GPC-11 cell suspensions on 20% Ludox HS gradients. (HS is one of the nine general grades of Ludox offered by du Pont de Nemours & Co., Wilmington, Del.) We did morphologic, functional, and biochemical experiments to detect oncogenic and enzymatic activity in each fraction. Oncogenic cells did not produce lysozyme. In contrast, macrophages associated with the solid tumor did produce lysozyme. The lysozyme purified from the GPC-11-associated macrophages resembled in size, electrophoretic mobility, and antigenicity the lysozyme purified from the urine of mice with the GPC-11 tumor.

Lysosomal enzymes in preeclamptic women in northern Nigeria.

BACKGROUND: The incidence of preeclampsia is high in northern Nigeria, as it is in many other developing countries, and preeclampsia is associated with significant maternal and fetal morbidity and mortality. We inquired if proteinuria or hypertension alone could account for the altered concentrations of urinary lysosomal hydrolases that have been reported in preeclamptic women and pregnant women without preeclampsia. METHODS: The activities of urinary beta-hexosaminidase and beta-galactosidase were determined fluorometrically in pregnant women assigned to one of four groups: Group I: 41 preeclamptic women; Group II: 31 hypertensive aproteinuric women; Group III: 44 normotensive proteinuric women; and Group IV: 52 healthy pregnant women (controls). RESULTS: The urinary beta-hexosaminidase concentrations were decreased in the preeclamptic women (P<0.005) and proteinuric women (P<0.001) when compared to the healthy pregnant controls. There was no significant difference in beta-hexosaminidase concentrations between the hypertensive women and the healthy pregnant controls. The urinary beta-galactosidase concentrations for preeclamptic, hypertensive, and proteinuric women did not differ significantly versus healthy pregnant controls. CONCLUSIONS: The reduced urinary excretion of beta-hexosaminidase in preeclamptic women is associated with proteinuria, but not hypertension. Measuring urinary concentrations of lysosomal hydrolases alone or in conjunction with urinary protein concentrations is not likely to be useful in predicting or monitoring the clinical course of preeclampsia; however, it might prove important in gaining a more complete understanding of the pathogenesis of renal tubular epithelial cell injury and proteinuria that occurs in preeclampsia.

Hypokalemia in nonblastic chronic myelogenous leukemia.

Hypokalemia has been noted as a frequent complication of acute leukemia. To our knowledge, an association of this electrolyte disturbance with chronic myelogenous leukemia (CML), not in blast crisis, has never been reported. We report a unique case of hypokalemia that complicated the clinical course of a patient with nonblastic CML. The pathogenesis of the hypokalemia was shown to be inappropriate hyperkaluresis, which appeared to be related to lysozymuria and a large tumor burden. We discuss the pathogenesis of hypokalemia in leukemia. We think that the unusual features associated with CML in this case make a large tumor burden their most likely underlying cause. This may help explain the still confusing and unresolved relationship between lysozymuria and potassium wasting in leukemia.

Measurement of lysozyme by an enzyme-linked immunosorbent assay.

In this study an enzyme-linked immunosorbent assay has been developed for the determination of lysozyme in saliva, serum and urine. The assay relies on the detection of specific protein rather than lytic activity, a property which has been shown to be most suitable for the quantitation of lysozyme in mucin containing substances. Our results indicate that no pretreatment is necessary for the immunochemical method. The assay is sensitive to concentrations as low as 1 microgram lysozyme/l. The intra-assay and inter-assay coefficients of variation were 5.9% and 15.8% respectively. The lysozyme level in whole saliva was 55.53 +/- 30.35 mg/l, in serum the level was 0.64 +/- 0.15 mg/l and in urine it was 0.17 +/- 0.22 mg/l. Comparisons between immunochemical determination and lytic assays showed a good correlation (serum, r = 0.79, P less than 0.01; saliva, r = 0.85, P less than 0.005; treated saliva, r = 0.96, P less than 0.001).

Immunochemical quantitation of human submandibular-sublingual lysozyme.

A method for quantitating lysozyme by using carbamylated antiserum (commercially available) to human lysozyme in a electroimmunodiffusion technique has been developed. The method measures specific protein not lytic activity as the lyso-plate method does. We have applied this method to tears, parotid saliva, submandibular-sublingual saliva, nasal secretions, serum, and minor salivary gland secretions. We specifically selected submandibular-sublingual saliva for a comparison test with the lyso-plate method because of the known mucin content of the submandibular-sublingual saliva. Our findings indicate that no pretreatment is necessary for the electroimmunodiffusion technique. The lyso-plate method, on the other hand, requires pretreatment with Tris-acetate pH 4.5 to dissociate the mucin-lysozyme complex and give accurate values.