[Current issues in measurement and reporting of urinary albumin excretion]. / Données actuelles sur le dosage de l'excrétion urinaire de l'albumine.

Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the erro-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences has been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no referance materials for either analyte in urine. The recommanded reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethicity) nor the continuous increase in risk related to albumin excretion. Clinical needs have been identified for standardization of (a) urine collection methodes, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

Peak selection from MALDI-TOF mass spectra using ant colony optimization.

MOTIVATION: Due to the large number of peaks in mass spectra of low-molecular-weight (LMW) enriched sera, a systematic method is needed to select a parsimonious set of peaks to facilitate biomarker identification. We present computational methods for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectral data preprocessing and peak selection. In particular, we propose a novel method that combines ant colony optimization (ACO) with support vector machines (SVM) to select a small set of useful peaks. RESULTS: The proposed hybrid ACO-SVM algorithm selected a panel of eight peaks out of 228 candidate peaks from MALDI-TOF spectra of LMW enriched sera. An SVM classifier built with these peaks achieved 94% sensitivity and 100% specificity in distinguishing hepatocellular carcinoma from cirrhosis in a blind validation set of 69 samples. Area under the receiver operating characteristic (ROC) curve was 0.996. The classification capability of these peaks is compared with those selected by the SVM-recursive feature elimination method. AVAILABILITY: Supplementary material and MATLAB scripts to implement the methods described in this article are available at http://microarray.georgetown.edu/web/files/bioinf.htm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effect of substrate size on immunoinhibition of amylase activity.

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.

Macromolecular chromogenic substrates for measuring proteinase activity.

BACKGROUND: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates. METHODS: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and alpha(2)-macroglobulin-proteinase complexes was monitored spectrophotometrically. RESULTS: Macrosubstrates had hydrodynamic radii of approximately 20 A, similar to proteins with a molecular weight of 18,000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with alpha(2)-macroglobulin had approximately 10-fold lower activity vs macrosubstrates than small substrates. CONCLUSIONS: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as alpha(2)-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.

Detection of indinavir crystals in urine: dependence on method of analysis.

OBJECTIVES: To determine the frequency of crystalluria in patients treated with the human immunodeficiency virus protease inhibitor indinavir and to compare methods of detecting crystalluria. METHODS: A total of 308 freshly voided urine specimens from 168 patients treated with indinavir were evaluated by manual microscopy of sediment and microscopy with an automated workstation and by dipstick analysis. RESULTS: Crystals were detected in 22%, 31%, or 32% of specimens using, respectively, an automated workstation, manual microscopy, or both methods. Proteinuria or hemoglobinuria occurred significantly more often in specimens with (28%) than without (18%) crystals. Frequency of crystalluria was unrelated to specific gravity, but it increased at higher pH. Crystals were detected in 21% of specimens with pH less than 6 and 42% of specimens with pH of 6 or higher. CONCLUSIONS: Crystalluria occurs in more than 30% of urine specimens from patients treated with indinavir, but detection rates vary substantially with method of analysis. Manual microscopy detected crystalluria 41% more often than did an automated workstation.

Interferences with semen detection by an immunoassay for a seminal vesicle-specific antigen.

A commercial enzyme-linked immunosorbent assay (ELISA), the SEMA assay, for a seminal vesicle-specific antigen (SVSA) provides highly sensitive detection of semen. Here we show marked interference of proteins such as albumin, serum proteins, or mucin with the assay. This would substantially decrease the sensitivity for detecting semen mixed with other biological fluids such as blood or vaginal secretions.

Detection of azide in forensic samples by capillary electrophoresis.

Azide salts are highly toxic compounds that have been difficult to detect in forensic samples. Here, anion analysis by capillary electrophoresis with indirect spectrophotometric detection was applied to detect azide in forensic specimens from two suicide victims. Gastric specimens from the victims were shown to have high azide concentrations; azide represented one of the major anionic components and no corresponding component occurred in normal gastric juice. Samples of blood and bile had low concentrations of azide near the limits of detection. The method described for azide analysis used simple steps for sample preparation and analysis time was less than 10 min per sample. It offers a simple and reliable method for detecting azide in biological fluids.

Prostate-specific antigen in vaginal fluid as a biologic marker of condom failure.

Forty women participated in three clinic visits during which they were exposed to their partner's semen (10 microL, 100 microL, and 1 mL). At each visit they took vaginal fluid samples before exposure to their partner's semen, immediately after, and at 1, 24, and 48 h after exposure. PSA was measured with an enzyme-linked immunoassay. The mean PSA level for preexposure swabs ranged between 0.43 and 0.88 ng/mL. The mean PSA levels were 193 immediately after exposure to 10 microL, 472 after 100 microL, and 19,098 after 1 mL. The PSA levels declined within 1 h, and returned to background at 48 h. The findings confirm that our procedure is a sensitive and specific method for detecting recent semen exposure, and indicate that PSA levels depend on exposure intensity and time since exposure. Application of this method in condom efficacy studies provides objective evidence of condom failure that enhances the interpretation of self-report.

PIP: This article examines the efficacy of prostate-specific antigen (PSA) in vaginal fluid as a biologic marker of condom failure. The sample included 40 women who participated in three clinic visits during which they were exposed to their partner's semen (10 mcl, 100 mcl, and 1 ml). At every clinic visit, vaginal fluid samples were taken before exposure with their partner's semen, immediately after, and at 1, 24, and 48 hours after exposure; PSA was measured with an enzyme-linked immunoassay. A 0.43-0.88 ng/ml mean PSA level was found in preexposure swabs, while the mean PSA levels were 193 ng/ml immediately after exposure to 10 mcl, 472 ng/ml after exposure to 100 mcl, and 19,098 ng/ml after exposure to 1 ml. The PSA levels declined within 1 hour and returned to an original state at 48 hours. Findings show that the procedure is a sensitive and specific method for detecting recent exposure to semen and indicate that the signal intensity is a function of semen exposure and time since exposure. Application of this procedure in studies of condom efficacy provides objective evidence of condom failure, which improves the interpretation of self-reports.

Objective markers of condom failure.

BACKGROUND: Studies of condom efficacy rely on self-reported behavior. Objective markers of exposure to semen may provide a more valid assessment of condom failure and failure to use condoms. GOALS OF THIS STUDY: To compare three semen biomarkers: acid phosphatase (AP) activity, prostate specific antigen (PSA), and the human seminal plasma antigen (MHS-5). STUDY DESIGN: Twenty women were intravaginally inoculated with six measured, increasingly larger amounts of their partners' semen. Vaginal fluid was collected by the participant using swabs and tested. RESULTS: Background levels of PSA were low (0.00-1.25 ng/ml), background levels of AP were variable (0-350 U/l), and all preinoculation samples were negative for MHS-5. All postinoculation samples were positive for PSA, 64 of 117 (55%) for AP, and 14 of 120 (12%) for MHS-5. CONCLUSION: The PSA immunoassay was the best semen biomarker under these sampling and testing conditions.

PIP: Objective markers of exposure to semen provide a more valid assessment of condom failure and failure to use condoms than self-reports. The present study evaluated three of the assays commonly used in forensic medicine for detecting semen exposure: acid phosphatase (AP) activity, prostate specific antigen (PSA), and the human seminal plasma antigen (MHS-5). 20 US women were intravaginally inoculated with 6 measured, increasingly larger amounts of their partners' semen. Vaginal fluid was collected with swabs by study participants and tested for the three markers. Before semen inoculation, PSA levels were consistently low (median, 0.11 ng/ml; range, 0-1.25 ng/ml) while those of AP were highly variable (median, 13.4 U/l; range, 0-350 U/l); all preinoculation samples were negative for MHS-5. The median PSA concentration increased consistently with increasing volumes of semen, while median AP and MHS-5 levels showed an inconsistent pattern. All 120 swabs obtained after intravaginal inoculation with semen were positive for PSA, 64 (55%) were positive for AP, and 14 (12%) were positive for MHS-5. These findings indicate that self-sampling of vaginal secretions followed by the PSA immunoassay represents a simple, accurate marker of semen exposure. Because the PSA assay is available in most hospital laboratories for prostate cancer screening, the methodology used in the present study is suitable for widespread application.

Progress toward preparation of universal donor red cells.

Coupling of an inert polymer to the surface of red cells was examined as a potential means of covering blood group antigens and producing cells that could serve as universal donor cells for transfusion. Effective blockade of red cell antigens was achieved with N-hydroxysuccinimide-activated esters of polyethylene glycol. It was possible to block all antigens tested, but lower concentrations of reactants were required to block peptide-defined antigens than carbohydrate-defined antigens. Red cells remained intact after modification but were significantly damaged. Our results demonstrate the feasibility of antigenic blockade of red cells, but there is a need to reduce damage during coupling reactions to produce viable red cells.