In order to extract Nystatin from urine and plasma samples, the SPE-CMN method developed in the present study was employed. Fresh tap water sample was collected from our laboratory (Khorramabad, Iran) and human urine samples were obtained from healthy female participants. Blood Transfusion Organization (Khorramabad, Iran) supplied the spiked tablet and plasma samples. Using distilled water, the specific urine and plasma samples were diluted tenfold in order to reduce the matrix effect. An extra preparation step was additionally conducted in order to remove protein from plasma in plasma samples; this was done by adding 75 ml of perchloric acid (0.5 M) to 50 ml of the diluted sample, followed by the centrifugation of the mixture to isolate the precipitated proteins. Also, to remove matrix effect in urine samples, the pH of urine samples was adjusted to 11 and centrifuged for 20 min until white lipidic solid was sedimented in the bottom of tube.
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immunoblotting (Fig. 8). The plasma levels of Lp(a) in these individuals were 0.3 and 0.2 mg/dl. Both individuals had apo(a) in the heparin-bound (lanes 6 and 10) and heparin-unbound (lanes 7 and 11) fractions. The samples were also subjected to size-fractionation on a nondenaturing gel (15) and in the hep- arin-unbound fraction, all of the full-length apo(a) comigrated with free apo(a). In contrast, the apo(a) in the heparin-bound fraction migrated a shorter distance in the gel, which is consis- tent with it being bound to apo-B100 or another heparin-bind- ing protein (data not shown). Although no apo-B100 could be detected in the plasma or in the heparin-unbound fraction of either individual (data not shown), the level of plasma apo- B100 may be below the detection limits of our immunoblotting method. Trace amounts of apo-B100 have been detected in the plasma of other individuals with abetalipoproteinemia (22). A series of smaller apo(a) fragments similar in size to those seen in the control subject (lane 3) was present in the heparin- unbound fraction. The proportion of apo(a) circulating as free apo(a) was 9 and 38% in these two individuals, which was within the same range as seen in normal individuals with very low plasma Lp(a) levels (see Fig. 5 B). Apo(a) fragments were also detected in the urine (lane 8), which was available from only one of the subjects. Based on these studies we concluded that apo-B100 was probably not required for the generation of the apo(a) fragments in humans and also that very low to ab- sent levels of plasma apo-B100 are not associated with an in- crease in the proportion of free apo(a).
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Table 4 summarizes the values of LR, RSD, LOD and LOQ of some analytical methods along with the proposed method for the extraction and determination of the selected analytes in different samples. The repeatability of the method is good and the RSDs for the proposed method are lower than or comparable with those of the mentioned methods. The LODs of the method are low and are comparable with that of the other methods. It should be noted that in some methods, a high sensitive detection system (mass spectrometry) has been used which is inherently more sensitive than FID. The wide LRs of the method are another advantage with respect to the others. These results reveal that the presented AALLME-GC-FID Table 3. Relative Recoveries Obtained by the Developed Method in Human Plasma and Urine
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New calibration graphs were constructed using spiked biological fluids as follows: 1 mL aliquots of human urine, plasma or breast milk samples were transferred into a series of 10-mL volumetric flasks, spiked with increas- ing concentrations of LOR and DSL and then completed to the mark with the mobile phase and mixed well (final concentration was in the range of 5.0–50.0 ng/mL for both analytes). The solution were then filtered through a 0.45-μm membrane filter and directly injected into the chromatographic system under the above described chromatographic conditions. The linear regression equa- tions relating the peak areas to the concentration (ng/ mL) were derived for each analyte.
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In the CE analysis the experimental parameters and con- ditions were selected carefully to prevent adsorption of large proteins like albumin onto the surface of the fused silica capillary. In order to accomplish this, polybrene, which is a pH-independent cationic polymer, was used to coat the inner wall of the silica capillary, and the pH of the analysis buffer ensured that albumin, which has an isoelectric point of 4.9 , was positively charged during the analysis. Fig. 1 shows a good peak shape for both bovine serum albumin (BSA) in the standard preparation and human serum albumin (HSA) in some of the precipitated plasma test solutions. The BSA peak migrated slightly earlier than HSA due to some slight struc- tural differences between the proteins. The humps preceding the albumin peak are interpreted as other proteins present in the plasma. The Lowry test, which is a general spectrophoto- metric protein test, was used to quantify total protein in the plasma samples. Although the plasma samples deproteinated by methanol and ethanol were 25 times more dilute than the samples treated by either acetone or acetonitrile, the albu- min peaks were still larger in the case of both methanol and ethanol. Albumin is the main protein in the plasma  and it is clear from the electropherograms in Fig. 1 that acetonitrile and acetone are much stronger deproteinisers than methanol and
Synthesis of the metabolites has been successfully car- ried out in our laboratory and structural confirmation has been performed. In addition, in this work we were concerned with the development and validation of two highly sensitive and selective chromatographic methods, HPTLC and HPLC–DAD methods, using developing sys- tems with the least hazardous solvents and the maximum chromatographic resolution. The developed methods were applied for determination of Flutamide in raw mate- rial and marketed tablets. Moreover, application of the methods was extended for determination of the drug and its metabolites in human plasma and urine samples. The developed HPTLC method is the first one reported for separation and quantitation of Flutamide and its metabo- lites, while the HPLC–DAD method has high selectivity, precision, and short analysis time (< 10 min). Moreover, the developed methods have advantages of lower cost comparing to previously reported LC–MS methods [4, 5]. Additionally, the facilities required for the methods developed in this article are mostly available in all labo- ratories, allowing them to be commonly applied for drug monitoring. The methods developed below are the only ones concerned with quantification of the drug along with its metabolites.
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Sample selection. A consecutive series of 860 archival aliquots of samples diagnostically tested for HSV-1, HSV-2, VZV, CMV, or EBV (EBV Virus Capsid Antigen-IgM analysis) at the Department of Medical Microbiology, Malmö University Hospital, Malmo ¨, Sweden, was retrieved. All archived aliquots had been stored at ⫺80°C or ⫺20°C (the EBV samples) for at least 18 months. For each positive sample, the negative sample, with the same type of biological material, submitted closest in calendar time was retrieved. Sample materials included bronchoalveolar lavage, conjunctival fluid, sore secretion, blister mate- rial, plasma, serum, and urine (Table 1). Thirty-two of the selected samples had been analyzed for three herpesviruses (HSV-1, HSV-2, and CMV [n ⫽ 3] or HSV-1, HSV-2, and VZV [n ⫽ 29]), 403 samples had been analyzed for two herpesviruses (HSV-1 and HSV-2), and 425 samples had been analyzed for one herpesvirus by clinical diagnostic testing. An additional 22 archived aliquots of serum samples that had tested positive for HHV8-specific antibodies, collected between 1996 and 2005 and stored at ⫺80°C, were obtained from the Depart- ment of Microbiology, Oncological Center, Aviano, Italy. Thus, a total of 1,349 herpesvirus analyses had been performed on a total of 882 samples (Table 1).
Determination of Caffeine in Biological Fluids: Aliquot volumes of human serum and urine samples were collected from the local hospital and stored in a refrigerator at 0-5 °C before using them to experiment. Both the samples were transferred into a small separating funnel, separately. Prior to analysis, a known amount of caffeine was spiked into the samples. Carbonate buffer (5 ml, pH 9.4) was added and mixed well with each sample. The caffeine extraction from both biological fluids was done three times using 5 ml of diethyl ether with handshaking the funnel for 20 min (liquid-liquid extraction). The ether extract (supernatant) was collected and then evaporated the ether using a fume hood at room temperature. The residue was
The concentration in serum of an antibody against a specific infectious disease agent can undergo oscillations over time. Such variations tend to be slow and are rarely established in a matter of days except in patients in the acute stage of the infection. When samples other than serum are used for diag- nosis of a chronic infection such as HIV infection, one problem that may arise is the presence of different HIV antibody con- centrations in the fluids used as alternatives to serum (16, 23, 31). The concentration may be influenced by the antibody lev- els that previously existed in the serum, by the quantity of serum filtrate that passes to the fluids, or by its final dilution, depending on the secretion volume of the alternative fluid. With this in mind it seems reasonable to venture that the an- tibody concentration in urine may suffer more circadian alter- ations than that in GCT. According to Mortimer and Parry (31), the immunoglobulin concentration in GCT is between four and five times lower than that in plasma. Slight fluctua- tions in RFVs for both GCT and urine throughout the day were observed in our study. At any rate, it should be indicated that just as when GCT and urine reactivities were observed, very significant quantitative reactivity differences were found between GCT and urine samples from the same individuals. The fact that the values for GCT samples were always higher than those for urine samples confirms what various investiga- tors have indicated, that more immunoglobulins are present in GCT than in urine (31). Nonetheless, we could observe how the titer of HIV antibodies found in urine paralleled the titer of HIV antibodies in the same individual’s GCT. It is therefore possible for the corresponding serum sample to be included in this correlation. Some studies that have observed the existence of a correlation between the antibody levels found in urine and the levels found in respective serum samples confirm this pos- sibility (10).
Then, the investigation was extended to plasma EEVs from 122 subjects (97 diseased and 25 healthy subjects) reporting TTV DNA detection in 34% of the samples at a mean level of 4.8 × 10 3 copies per ml, thus confirming that plasma EEVs are able to entrap the virus. Accumu- lating evidence demonstrates that many viruses hijack EVs pathways to ensure their survival and persistence, and that EVs have to be considered as important media- tors for virus infection-associated intercellular commu- nication and microenvironment alteration [37–40]. In this context, EVs can mediate virus egress from cells in the absence of lysis, facilitate infection of new suscep- tible and/or unsusceptible cells, and favor viral escape from the immune responses. The real significance of TTV and EEVs association is not yet known, but some hypotheses can be done starting from the findings of this study. First, TTV could utilize host EEVs as a vehicle for infecting naive healthy cells, thus increasing its potential of spreading, although the low prevalence of TTV de- tected in EEVs, seems to reveal that this is not the major mode with which TTV spreads in the host. The use of EEVs is well known for many viruses: for example, infec- tious particles of non-enveloped viruses such as HAV and HEV can be engulfed by host membranes that re- semble exosomes and as such are secreted from infected cells in a “quasi-enveloped” structure that permits a dif- ferent way of cell entry and a wider spread in the host . In this context, the acquisition of a “quasi-enve- loped” structure could facilitate the entry of TTV in otherwise non-permissive cells and permit also its diffu- sion in immunologically privileged site such as the cen- tral nervous system [3, 42]. Second, as reported for many viruses (i.e. HEV, HAV, and picornavirus), hiding within EVs for non-enveloped viruses is a barrier to neu- tralizing antibodies [37, 38, 40]. Thus, EEVs might shield TTV from neutralizing antibodies, acting as a possible mechanism of immune evasion. In fact, it’s well estab- lished that TTV infected hosts mount detectable anti- viral antibodies which fail to eradicate the virus, at least in the great majority of cases, and is also unsuccessful at protecting against superinfections sustained by heterol- ogous TTV types [2, 3]. Third, since no difference was seen between healthy subjects and diseased patients, TTV and EEVs association seems to be independent from the clinical status of analyzed individuals. However interestingly, TTV loads in EEVs from immunosup- pressed patients (i.e. HIV positive and transplant pa- tients) were higher to those in healthy people and other diseased patients, thus suggesting that the amount of TTV vehiculated by EEVs could be influenced by the status of host immunity. Thus, the carriage of TTV into EVVs could be a strategy that the virus uses to reduce Table 5 Late assembly domains in ORF1 gene of TTV genomes
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ion (RSD%), overy (ER), w ons to evalua ained results over a broad h correlation c Q, calculated b 10, were 0.5 e less than 5 cating an acc hod. The EF 7%, respectiv ER, and low he proposed m Urine sample a d to the pr orpyrifos in th uracy of the D h the target an 0, 200, and 1 the recommen wn in Table 2 overies ranged
The clinical performance of the JCV assay was demon- strated in a previously published study in patients at high-risk for JCV replication (8), as well as in low-risk healthy blood donors (10). In the former study, 27.2% of the urine samples from renal transplant recipients containing decoy cells, a result indicative of PyVAN, were positive for JCV DNA with our assay. In the patients with biopsy-proven PyVAN, 21.4% were positive with JCV DNA. In the latter study, 400 blood donors were screened for JCV DNA in urine and blood samples. FIG. 2. Alignment of the oligonucleotides of the JCV real-time PCR. The top sequence corresponds to the MAD-1 prototype strain. Each line corresponds to a representative sequence for a variant. The accession number and the position of the first nucleotide in the database entry are indicated. The number of sequences constituting a variant and the percentage of the total number of sequences are shown in the two columns on the right. Nucleotides identical to the query sequence are indicated as a dot (.); substitutions are indicated by the corresponding nucleotide one-letter code. Gaps are indicated by an underscore; insertions are indicated by a backslash (\). The binding sites of the oligonucleotides are underlined in the query sequence and shaded gray in the alignment. The corresponding sequences of BKV and SV40 are shown for comparison.
As another application of the studied method, recovery from human urine samples was carried out and treatment of drug with urine without any extraction step. Recovery studies were performed with the sample containing various amounts of pipazethate HCl. The results of recovery studies (Table 7.) revealed that, there was no interference from other constituents present in the urine in the method. The mean percent recovery obtained from five replicate measurements of urine containing pipazethate HCl ind indicate that the proposed method was effective for the determination of the drug in urine samples.
DNA was extracted and purified from the plasma and urine samples and concentrated on diatoms in the presence of guanidine thiocyanate (GuSCN). Briefly, 100 µl of plasma or urine were added to 900 µl of L6 buffer (GuSCN 120 g, 0.1 M Tris-HC1, pH 6.4, 100 ml, 0.2 M EDTA 22 ml, Triton X-100 2.6 ml) with 40 µl of diatom suspension (diatoms 10 g, distilled water 50 ml, 500 µl of HCl 36 YO w/v). The mixture was vortexed and incubated at room temperature for 10min and then centrifuged to spin down the complex of DNA-diatoms. After washing twice with L2 buffer (GuSCN 120 g, 0.1 M Tris-HCL 100 ml, pH 6.4), twice with ethanol 70% v/v, and once with acetone, the DNA-diatom complex was dried at 56°C for 10min and the DNA was eluted in the presence of proteinase IS 120 pg/ml solution at 56°C for 10 min. The proteinase K was subsequently inactivated by incubation at 100°C for 10 min (24).
fractions from macrophage infection models and CFP preparations. We found very few serum samples which produced highly elevated signals of the M. tuberculosis SOMAmers. In one patient from Peru with culture-positive, smear-negative TB and HIV coinfection, dozens of aptamer signals were 4- to 10-fold above the median signal observed in TB samples. These observations were conﬁrmed in separately acquired aliquots of both serum and plasma, although the M. tuberculosis proteins were not observed in afﬁnity capture eluates examined by LC-MS/MS. Based on standard curves using recombinant proteins run in the same experiments, even with greatly elevated signals well above the LODs for M. tuberculosis SOMAmers, the estimated antigen levels in this sample were only in the low picomolar range. Thus, conﬁrmation of the signals by MS may require a more advanced approach, such as targeted MRM-MS. It is not known whether this patient had an M. tuberculosis bloodstream infection that could explain such high levels of circulating antigens (58). In support of this hypothesis, peptides corresponding to the M. tuberculosis proteins antigen 85B, antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64, several of which were on our list of targets, had been previously identiﬁed by targeted MRM-MS assays of exosomes from TB patients (47, 59). That study also found a high variability in the identity of the corresponding targets and in the number of peptides detected, and some of the peptides (e.g., from MPT64) were also present in exosomes from LTBI. An additional possible explanation for the eleva- tion of SOMAmer signals in NTB samples compared to signals in control sample groups is aptamer cross-reactivity with homologous proteins from NTM or from other actino- bacteria.
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that might well be explained by Ab aggregation or oligo- merization, since there is clear evidence that aggregated Ab is cleared more slowly than non-aggregated forms. A less likely, but not impossible, scenario that could lead to elevated levels of Ab and p3-Alc species involves a formulation wherein SAD might, in some cases, be attri- butable to a defect in clearance of transmembrane (TM) domain-derived peptides. This formulation would dove- tail well with evidence that apolipoprotein E (APOE) iso- type modulates Ab clearance [19,20] and raises the possibility that APOE genotype may modulate clearance of many TM domain-derived peptides, including Ab and p3-Alc. In separate work still in progress, we are study- ing whether there exists any relationship between APOE genotype in the levels of plasma and CSF p3-Alc. In any event, there is substantial support for the possibility that alterations in Ab generation and/or clearance are con- sidered to be possible underpinnings for at least some cases of SAD. These alternative hypotheses are not mutually exclusive and could underlie different bio- chemical endophenotypes that all lead to clinical AD.
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Paired samples t-tests were used to determine the difference in mean salt intake measured from 24-h urine and spot urine samples. Estimates from spot urines were considered as slightly (< 1 g/day), moder- ately (1 to 2 g/day) or substantially (> 2 g/day) differ- ent to 24-h urine samples . A two-way, mixed- effects, single rater, consistency-of-agreement model was used to determine the intraclass correlation coef- ficient (ICC) of the two methods . Bland-Altman plots were used to determine the agreement between the methods, by plotting the difference in salt intake measured between 24-h urine and spot urine samples on the vertical axis against the mean of the two methods on the horizontal axis . Regression-based lines and the 95% limits of agreement were calculated and added to the Bland-Altman plots to better illus- trate the varying limits of agreement due to presence of proportional bias . Finally, the capacity of spot urine samples to classify population-level salt intake as above or below 5 g/day was examined. All these analyses were applied to the country-level datasets. For each country, the baseline and follow-up data points were combined. This was possible since the data were collected from different individuals at each time point and are therefore independent.
Spiked quality control plasma samples (LQC and HQC) were prepared in six replicates and different stability parameters were evaluated, like, bench top stability (48 h) and autosampler stability (20°C for 55.5 h), reinjection reproducibility (20°C for 27 h) The stability of spiked human plasma samples stored at room temperature (bench top stability) and freeze-thaw stability (at -30°C) and long term stability (71 days) was evaluated for 48 h.
irreversible 1:1 electron and proton process with diffusion character. MWCNTs showed electrocatalytic action for the reduction of LND, characterizing by the enhancement of the peak current and the reduction of the peak potential, which was probably due to the larger effective surface area of MWCNTs and PAN. This method was successfully used to determine LND in the pharmaceutical, human urine and serum samples. The proposed method offered the advantages of accuracy and time saving as well as simplicity of reagents and apparatus. In addition, the results obtained in the analysis of LND in spiked urine and serum samples demonstrated the applicability of the method for real sample analysis. Acknowledgement: I am very much thankful to UGC-BSR for providing financial assistance.
Urine is an adequate alternative biospecimen for monitoring HPV prevalence in female adolescents to determine the early effect of HPV vaccination on a population level. Strategies for recruitment should be optimized to avoid low response rates, sampling and HPV detection protocols should be detailed and standardized to ensure comparability, and importantly, care should be taken when extrapolating findings to the cervix. In males, urine samples do not seem to be optimal for moni- toring HPV prevalence due to a low human genomic DNA content compared to other urogenital sites. Although urine sampling has some advantages and is the only relevant option for sampling the general population in the youngest age groups, it also has several disadvantages, most importantly the fact that HPV prevalence in urine is only a distant measure of the main end point of vaccine impact, cervical cancer. In each situation the costs and benefits of HPV DNA detection in urine, compared to alternative monitoring options, should be carefully considered. 16,47
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