Detection of proteins related to a salivary glycoprotein (EP-GP). Concentrations in human secretions (saliva, sweat, tears, nasal mucus, cerumen, seminal plasma).

With a highly specific monoclonal antibody against a previously isolated and characterized human salivary 19-20-kDa glycoprotein, designated as extra-parotid glycoprotein [Rathman et al. (1989) J. Biol. Buccale 17, 199-208], a common epitope was detected on proteins in several excretory human body fluids. With a quantitative ELISA the EP-GP epitope was measured in widely different concentrations in several secretory human body fluids in the descending order of seminal plasma much greater than tears approximately nasal mucus approximately sweat much greater than saliva. Crossreactivity was also observed in cerumen but not in milk, cerebrospinal fluid, blood plasma and urine. The relative amount of EP-GP in the positively reacting secretions was however, in the same order in each fluid per mg of protein on an average of 1% of the total protein amount. The EP-GP-epitope bearing proteins found in the various human secretions were further characterized by means of electrophoresis and immunoblotting. The molecular masses and the isoelectric points of the proteins in the different secretions display strong resemblance to values found for the salivary glycoprotein EP-GP (molecular masses 19 and 20 kDa; pI values between 4.8 and 5.4). All these findings point to the presence of proteins related to EP-GP in human secretions other than saliva.     

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400–1,500 μg protein from platelets, and 100–600 μg from PBMC. 30 μL plasma depleted of albumin and IgG provided 350–650 μg protein. A sample of morning urine provided 0.9–8.6 mg protein/dL, and a sample of saliva provided 70–950 μg protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Fully automated high-performance liquid chromatography : A new chromatograph for pharmacokinetic drug monitoring by direct injection of body fluids

A new fully automated high-performance liquid chromatograph is described which detects drugs from directly injected plasma (urine, saliva) without sample pretreatment. The apparatus consists of a programmable automatic sampling unit, which is connected via two alternating working pre-columns to an analytical column (“alternating pre-column sample enrichment”). The new device is able to operate with directly injected body fluids like an auto-analyzer and is especially useful for pharmacokinetic and clinical studies, where drug concentration have to be determined from plasma, urine or saliva.     

Human parotid and submandibular glands express and secrete surfactant proteins A,B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.     

A carbon column-based liquid chromatography electrochemical approach to routine 8-hydroxy-2'-deoxyguanosine measurements in urine and other biologic matrices: a one-year evaluation of methods.

8-Hydroxy-2'-deoxyguanosine (8OH2'dG) is a principal stable marker of hydroxyl radical damage to DNA. It has been related to a wide variety of disorders and environmental insults, and has been proposed as a useful systematic marker of oxidative stress. Analytic procedures for 8OH2'dG in DNA digests are well established; however, routine measurement of free 8OH2'dG in other body fluids such as urine or plasma has been problematic. This has hindered its evaluation as a general clinical, therapeutic monitoring, or environmental assessment tool. Therefore, we developed a liquid chromatography electrochemical column-switching system based on the use of the unique purine selectivity of porous carbon columns that allows routine accurate measurement of 8OH2'dG in a variety of biologic matrices. This paper describes the rationale of the system design and the protocols developed for 8OH2'dG in urine, plasma, cerebrospinal fluid, tissue, DNA, saliva, sweat, kidney dialysis fluid, foods, feces, culture matrix, and microdialysates. Concentrations in both human and animal body fluids and tissues are reported. The system performance is discussed in the context of a 1-year evaluation of the methods applied to approximately 3600 samples, using internal quality control and external blind testing to determine long-term accuracy. The methods are reliable and accurate, and therefore should prove useful in assessing the role and utility of oxidative DNA damage in aging and human illness.     

Neuraminic acid derivatives newly discovered in humans: N-acetyl-9-O-L-lactoylneuraminic acid, N,9-O-Diacetylneuraminic acid and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid.

The free and glycosidically bound acylneuraminic acids from human serum and saliva and the free acylneuraminic acids from human urine have been characterized by thin-layer chromatography and gas-liquid chromatography/mass spectrometry. Acylneuraminic acid mixtures obtained from serum and saliva contain mainly N-acetylneuraminic acid and N-acetyl-9-O-L-lactoylneuraminic acid, whereas small amounts of N,9-O-diacetylneuraminic acid are also present. No free N,O-diacylneuraminic acids could be detected in the urine samples. None of the investigated fluids contained N-glycoloylneuraminic acid. The unsaturated N-acetyl-2,3-dehydro-2-deoxyneuraminic acid is usually a component of the free acylneuraminic acid fractions of serum, saliva and urine. The body fluids of a patient with sialuria contain the same O-acylated and unsaturated N-acetyl neuraminic acid derivatives as mentioned above, but the total amounts of free acylneuraminic acids in these materials are significantly higher than found for normal persons.     

Estimation of plasma and saliva levels of coenzyme Q10 and influence of oral supplementation

Coenzyme Q10 (CoQ(10)) levels in human saliva were measured by HPLC with a highly sensitive electrochemical detector (ECD) and a special concentration column. This HPLC system showed satisfactory analytical results within the standard range of 0.78-50 ng/ml. We also found a significant correlation between CoQ(10) levels in plasma and in saliva from parotid glands, while this correlation was lacking between plasma CoQ10 and CoQ10 in whole saliva. Unlike in plasma, there are some fluctuations of saliva CoQ(10) levels throughout the day. A good correlation was obtained by collecting parotid gland saliva at times between meals. The mean saliva CoQ(10) level for 55 healthy volunteers was 17.0 ng/ml (S.D. 6.8 ng/ml); approximately one fiftieth of that in plasma. Regarding the influence of oral supplementation, CoQ(10) was analyzed in plasma and parotid gland saliva from 20 healthy volunteers supplemented daily with 100 mg of CoQ(10) for the first week and 200 mg for the second. The plasma CoQ(10) levels of all volunteers increased to different extents in accordance with the CoQ(10) daily intake and the corresponding change in saliva showed almost the same trend.     

Simple urinary sample preparation for proteomic analysis

Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.     

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.     

Quantitative analysis of oxepinac in human plasma,urine and saliva by gas chromatography—mass fragmentography

A sensitive and specific method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]oxepin-3-acetic acid (oxepinac) in human plasma, urine and saliva. Oxepinac and internal standard are extracted from acidified plasma, urine or saliva, converted to the corresponding n-propyl esters and analysed by gas chromatography—mass fragmentography using selected ion monitoring. The method is accurate and precise over the range 100 μg/ml to 1.0 ng/ml. The method has been applied to the analysis of plasma, urine and saliva from healthy volunteers receiving therapeutic doses of oxepinac.     

Evidence by gas chromatography-mass spectrometry of ex vivo nitrite and nitrate formation from air nitrogen oxides in human plasma, serum, and urine samples

Nitrite and nitrate in body fluids and tissues result from dietary source, endogenous nitric oxide (NO) production and from NO and its higher oxides (NO_x) present as pollutants in the atmosphere. Nitrite and nitrate in human blood serum and plasma or urine are commonly used as biomarkers and measures of endogenous NO synthesis. In addition to dietary intake of nitrite and nitrate, our study indicates that NO_x naturally present in the laboratory air may be an abundant source for nitrite and nitrate in human serum, plasma, and urine ex vivo. These artifacts can be effectively reduced by closing sample-containing vials during sample treatment.     

Trace elements in human parotid saliva

Although various proteins and some electrolytes have been measured in human saliva, little systematic data about the major and minor elemental components of this body fluid have been obtained. In order to obtain such data, concentrations of C, Na, P, Cl, K, Ca, Sc, Cr, Fe, Co, Zn, Se, Br, Rb, Sb, I, and Cs in human parotid saliva were measured by instrumental nuclear methods. The data obtained confirmed the relative lack of Zn in saliva of patients with hypogeusia (decreased taste acuity) and suggested that concentrations of Na, Cl, Br, and Ca followed the order: normals > hypogeusia > hyposmia (decreased smell acuity). To compare concentrations of elements in saliva with those in blood and urine, absolute concentrations were normalized to that of Na through the use of a concept called an enrichment factor. On this basis, parotid saliva is relatively depleted in Se, Zn, and Fe and enriched for most other elements relative to blood plasma indicating that the fluid is not simply a transudate of blood plasma. Using this same technique, saliva composition was found more similar to urine than blood plasma, being relatively depleted in Se, Cs, and Co, being enriched in I, Br, and Cr and having about the same relative concentrations of P, Cl, Zn, Fe, Ca, K, and Rb. As the total body concentrations of many of the enriched elements in saliva are extremely small, their enrichment in saliva suggests special roles for these elements in the oral cavity. Because of its accessibility, ease of collection, and interaction with some body constituents, saliva represents a useful, albeit neglected, tool in the diagnosis of some physiological and pathological changes in body function and in understanding important aspects of trace metal metabolism.     

Analysis of theaflavins in biological fluids using liquid chromatography–electrospray mass spectrometry

A HPLC–MS procedure for the sensitive and specific analysis of the black tea flavonoid theaflavin in human plasma and urine was developed. Levels were measured after enzymatic deconjugation, extraction into ethyl acetate, and separation by HPLC, using tandem mass spectrometry as a detecting system. Two healthy volunteers consumed 700 mg theaflavins, equivalent to about 30 cups of black tea. The maximum concentration detected in blood plasma was 1.0 μg l⁻¹ in a sample collected after 2 h. The concentration in urine also peaked after 2 h at 4.2 μg l⁻¹. Hence, only minute amounts of theaflavins can be detected in plasma and urine samples of healthy volunteers after ingestion.     

Determination of BAY 12-8039, a new 8-methoxyquinolone, in human body fluids by high-performance liquid chromatography with fluorescence detection using on-column focusing

A reversed-phase (RP) high-performance liquid chromatographic (HPLC) method with fluorescence detection allowing the sensitive and specific quantification of BAY 12-8039, a new antimicrobially active 8-methoxyquinolone, in biological fluids is described. The method is compared to a microbiological assay (bioassay) based on B. subtilis test strain with a limit of quantification of approximately 60 μg/l. Following dilution and centrifugation, plasma, saliva or urine supernatant is directly injected onto the HPLC system. Concentrations down to a limit of quantification of 2.5 μg/l can be quantified in plasma, saliva and urine. Data on recovery, accuracy and precision of the method throughout the whole working range as well as results on stability of the analyte are presented. The concentration data are correlated with results from the bioassay. BAY 12-8039 is stable in plasma after repeated freeze-thaw cycles and following storage at −20°C for at least 12 months. The results of HPLC measurements excellently agree with bioassay data indicating the relevance of the method as a tool in clinical development to answer pharmacokinetic questions related to antimicrobial activity. The method was applied to human plasma, saliva and urine from subjects after a single oral dose of 400 mg of BAY 12-8039.     

Ascorbic acid in nasal and tracheobronchial airway lining fluids

Ascorbic acid (AA) is thought to be an important antioxidant in the respiratory tract, whose regulation is yet to be fully characterized. We investigated whether AA in respiratory tract lining fluids (RTLFs) can be augmented by oral supplementation with AA. Plasma, nasal lavage fluids (NLFs), induced sputum (IS), and saliva were analyzed for AA immediately before and 2 h after ingestion of 2 g of AA in 13 healthy subjects. Concentrations of AA (median and range) were 52.5 (16.0-88.5), 2.4 (0.18-4.66), 2.4 (0.18-6.00), and 0.55 (0.18-18.90) micromol/l, respectively. Two hours after ingestion of AA, plasma AA increased 2-fold (p = .004), NLF AA increased 3-fold (p = .039), but IS and saliva AA did not increase. As AA concentrations in saliva and tracheobronchial secretions were low compared with other common extracellular components (such as urate), we evaluated the fate of AA in these fluids. Addition of AA to freshly obtained saliva or IS resulted in rapid depletion, which could be largely prevented or reversed by sodium azide or dithiothreitol. These findings suggest that oxidant-producing systems in saliva and airway secretions, such as heme peroxidases and other oxidizing substances, rapidly consume AA. Whereas oral supplementation resulted in detectable increases of AA in NLFs, its levels in tracheobronchial lining fluid, as measured by IS, were unaffected and remained relatively low, suggesting that AA may play a less significant antioxidant role in this compartment as compared with most other extracellular compartments.     

Human urine deoxyribonuclease II (DNase II) isoenzymes: a novel immunoaffinity purification, biochemical multiplicity, genetic heterogeneity and broad distribution among tissues and body fluids.

Deoxyribonuclease II (DNase II) was purified from the urine of a 48-year-old male (a single individual) using a column chromatography series, including concanavalin A-agarose and an immunoaffinity column utilizing anti-human spleen DNase II antibody, and was then characterized. Based on the catalytic properties of the purified enzyme, we have devised a technique of isoelectric focusing by thin-layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with a specific zymogram method, for investigating the possible molecular heterogeneity of human DNase II. DNase II in urine as well as the purified form was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 5-7. Since sialidase treatment of the urine sample induced simplification of the isoenzyme patterns with diminishment of anodal bands, it was clear that the multiplicity of the enzyme was in part due to differences in the sialic acid content. On screening of DNase II isoenzyme patterns in urine samples from more than 200 Japanese individuals, only the common isoenzyme pattern was observed and no electrophoretic variations were detected. However, genetic studies of urinary enzyme activity and comparative studies on the activity in urine, semen and leukocytes from the same individuals suggest that the enzyme activity level of DNase II may be under genetic control. The enzyme was widely distributed in human tissues and showed high activities in secretory body fluids such as breast milk, saliva, semen and urine, and leukocyte lysates.     

细胞外囊泡检测分析方法研究进展

细胞外囊泡是真核细胞和原核细胞共有的细胞间信号传递的重要媒介。细胞外囊泡可以传递蛋白质、脂质和核酸,影响供体细胞和受体细胞的生理学、病生理学功能。细胞外囊泡存在于多种体液中,当前已在血液、尿液、唾液、母乳、羊水、脑脊液、胆汁等体液中鉴定到细胞外囊泡的存在。这些体液很多是临床检测的样本,因此体液中含有的细胞外囊泡可能成为鉴定临床疾病的标志物,这引起了科研人员的极大兴趣。该综述重点关注了不同体液样品中细胞外囊泡的功能,并且针对临床样本和细胞外囊泡结构的特殊性,综述了样品收集、储存、检测等标准流程研究,为临床医生和科学家在细胞外囊泡研究中提供指引。     

A competitive enzyme-linked immunosorbent assay for quantification of tetrastatin in body fluids and tumor extracts

Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression.     

High Molecular Complexes of Insulin-Like Growth Factor-I in Human Saliva

Insulin-like growth factor-I is one of the most important growth factors involved in oral biology. It circulates in plasma in a form of complex with binding proteins—IGFBPs and acid labile subunit—ALS. It was decided to assess the content of IGF-I in human saliva in relation to other proteins, and the expression and content of its binding proteins and ALS of healthy people of different gender and age. Research material was mixed resting saliva obtained from 70 healthy volunteers, which were divided into seven groups, taking into account age and gender. For qualitative and quantitative evaluation of IGF-I complexes with IGFBP-5 and ALS there were used: western immunoblot and ELISA assay. It was shown that human saliva contained IGF-I mainly in the form of macromolecular complexes. Expression and content of IGFBP-5 and ALS were affected by gender and age.