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BACKGROUND Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from Mycobacterium tuberculosis (Mtb). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories. METHODS In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) rrs genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTMST). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium gordonae) and Mtb. RESULTS Using the fluorescent BiDz-NTMST assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye. CONCLUSIONS The BiDz-NTMST assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.

The recent outbreak of a novel coronavirus SARS-CoV-2 (also known as 2019-nCoV) threatens global health, given serious cause for concern. SARS-CoV-2 is a human-to-human pathogen that caused fever, severe respiratory disease and pneumonia (known as COVID-19). By press time, more than 70,000 infected people had been confirmed worldwide. SARS-CoV-2 is very similar to the severe acute respiratory syndrome (SARS) coronavirus broke out 17 years ago. However, it has increased transmissibility as compared with the SARS-CoV, e.g. very often infected individuals without any symptoms could still transfer the virus to others. It is thus urgent to develop a rapid, accurate and onsite diagnosis methods in order to effectively identify these early infects, treat them on time and control the disease spreading. Here we developed an isothermal LAMP based method-iLACO (isothermal LAMP based method for COVID-19) to amplify a fragment of the ORF1ab gene using 6 primers. We assured the species-specificity of iLACO by comparing the sequences of 11 related viruses by BLAST (including 7 similar coronaviruses, 2 influenza viruses and 2 normal coronaviruses). The sensitivity is comparable to Taqman based qPCR detection method, detecting synthesized RNA equivalent to 10 copies of 2019-nCoV virus. Reaction time varied from 15-40 minutes, depending on the loading of virus in the collected samples. The accuracy, simplicity and versatility of the new developed method suggests that iLACO assays can be conveniently applied with for 2019-nCoV threat control, even in those cases where specialized molecular biology equipment is not available.

Battery-free, wireless microfluidic/electronic system for multiparameter sweat analysis., Wearable sweat sensors rely either on electronics for electrochemical detection or on colorimetry for visual readout. Non-ideal form factors represent disadvantages of the former, while semiquantitative operation and narrow scope of measurable biomarkers characterize the latter. Here, we introduce a battery-free, wireless electronic sensing platform inspired by biofuel cells that integrates chronometric microfluidic platforms with embedded colorimetric assays. The resulting sensors combine advantages of electronic and microfluidic functionality in a platform that is significantly lighter, cheaper, and smaller than alternatives. A demonstration device simultaneously monitors sweat rate/loss, pH, lactate, glucose, and chloride. Systematic studies of the electronics, microfluidics, and integration schemes establish the key design considerations and performance attributes. Two-day human trials that compare concentrations of glucose and lactate in sweat and blood suggest a potential basis for noninvasive, semi-quantitative tracking of physiological status.

There is a need for rapid nucleic acid amplification and detection technologies suitable for point-of-care settings. PCR-based molecular diagnostics, portable thermocyclers, and rapid microfluidic techniques are emerging technologies (1), but the use of isothermal nucleic acid amplification would make less demanding instrumentation feasible. Most isothermal methods reported to date(2)(3)(4)(5)(6)(7) require reaction times >30 min. In this study, we describe very rapid isothermal DNA amplification through EXPAR (exponential amplification reaction)(8)(9) coupled with visual colorimetric detection facilitated by aggregation of DNA-functionalized gold nanospheres(10)(11)(12) applied to a genomic sequence derived from herpes simplex virus (HSV)1. Rapid detection of HSV infections is important in HIV-positive and immunocompromised individuals (13)(14), pregnant women, and newborns(15). PCR-based assays are superior to viral cultures and immunoassays for the diagnosis of HSV infections from cerebrospinal fluid(16) and genital, oral, and topical swabs(17)(18)(19)(20). New rapid, low-cost molecular diagnostic tools can enable broad-based testing of at-risk populations, providing effective patient treatment and preventing further disease spread. EXPAR (8)(9) isothermally amplifies a short oligonucleotide trigger 106- to 109-fold in

Background: Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADP-ribosylation by DT.Methods: Solid phase–bound his-tag eEF2 is ADP-ribosylated by toxins using biotinylated NAD+ as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti–his-tag antibody using an LAS-1000 System.Results: The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase.Conclusions: The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

BACKGROUND More than 70 million couples worldwide are affected by infertility, with male-factor infertility accounting for about half of the cases. Semen analysis is critical for determining male fertility potential, but conventional testing is costly and complex. Here, we demonstrate a paper-based microfluidic approach to quantify male fertility potential, simultaneously measuring 3 critical semen parameters in 10 min: live and motile sperm concentrations and sperm motility. METHODS The device measures the colorimetric change of yellow tetrazolium dye to purple formazan by the diaphorase flavoprotein enzyme present in metabolically active human sperm to quantify live and motile sperm concentration. Sperm motility was determined as the ratio of motile to live sperm. We assessed the performance of the device by use of clinical semen samples, in parallel with standard clinical approaches. RESULTS Detection limits of 8.46 and 15.18 million/mL were achieved for live and motile sperm concentrations, respectively. The live and motile sperm concentrations and motility values from our device correlated with those of the standard clinical approaches (R2 ≥ 0.84). In all cases, our device provided 100% agreement in terms of clinical outcome. The device was also robust and could tolerate conditions of high absolute humidity (22.8 g/m3) up to 16 weeks when packaged with desiccant. CONCLUSIONS Our device outperforms existing commercial paper-based assays by quantitatively measuring live and motile sperm concentrations and motility, in only 10 min. This approach is applicable to current clinical practices as well as self-diagnostic applications.

Consumers are increasingly interested in knowing more about their own health. One of the most familiar direct-to-consumer diagnostics tests has been the home pregnancy test. In this issue of Clinical Chemistry , Nosrati et al. (1) report their clever and practical approach toward developing a new point-of-care diagnostic test for another reproductive health need—the assessment of male fertility. The authors developed the test using paper, a familiar substrate used in pregnancy tests and the subject of a flurry of recent improvements in microfluidics (2). The authors placed at the bottom one laminate layer, and on top, paper layers patterned with wax (3). With the paper test they aimed to quantify sperm concentration via a dye that changes color in the presence of an enzyme (diaphorase flavoprotein) that resides within metabolically active human sperm. In one spot on the paper, the sample reacts directly with the dried dye. In a second spot, sperm must swim vertically through a highly viscous …

Potential usefulness of inflammatory markers to monitor respiratory functional impairment in sarcoidosis.Rothkrantz-Kos S, van Dieijen-Visser MP, Mulder PG, Drent M.Department of Clinical Chemistry, Sarcoidosis Management Center, University Hospital Maastricht, and Nutrition and Toxicology Research Institute Maastricht (NUTRIM), University Maastricht, The Netherlands.BACKGROUND: Sarcoidosis is a multiorgan inflammatory granulomatous disorder of unknown origin for which adequate markers to monitor disease severity are lacking. The aim of this study was to evaluate the potential clinical usefulness of serologic markers of inflammation [high-sensitivity C-reactive protein (hs-CRP) and serum amyloid A (SAA)], T-cell activation [soluble interleukin-2 receptor (sIL2R)], and granuloma formation [angiotensin-converting enzyme (ACE)] for monitoring of sarcoidosis. METHODS: Of the 185 sarcoidosis patients who visited the Sarcoidosis Management Center between 1999 and 2002, we selected 144 nonsmoking patients: 73 untreated (group I) and 71 treated (group II). Subgroups of the untreated patients [group Ia (nonchronic group with time since diagnosis < or = 2 years) and group Ib (chronic group with time since diagnosis >2 years)] were evaluated separately. ROC curves and logistic regression analyses were used to compare the diagnostic accuracy of different markers to assess disease severity. Pulmonary disease severity was defined by lung function test results. RESULTS: In untreated subgroup Ia and the total untreated group (group I), sIL2R had the largest areas under the curves (AUCs; 0.891 and 0.799, respectively) and the highest sensitivity (82% and 64%), specificity (94% and 88%), and positive (82% and 70%) and negative (94% and 88%) predictive values among the evaluated markers in both untreated groups. Nevertheless, the confidence intervals for sIL2R AUC, sensitivity, and specificity were broad and partly overlapped those of ACE, hs-CRP, and SAA. In the treated group (group II), all four markers appeared to have comparable AUCs, ranging from 0.645 for SAA to 0.711 for sIL2R. CONCLUSION: sIL2R appears to be useful for monitoring respiratory disease severity in sarcoidosis. We recommend sIL2R measurement in the follow-up of patients with sarcoidosis

Total iron-binding capacity (TIBC) indicates the maximum amount of iron needed to saturate plasma or serum transferrin (TRF), which is the primary iron-transport protein (1). Theoretically, 1 mol of TRF [average molecular mass, 79 570 Da (2)] can bind 2 mol of iron (55.8 Da) at two high-affinity binding sites for ferric iron (3). Therefore, TIBC correlates well with TRF concentration, and the theoretical ratio of TIBC (in μmol/L) to TRF (in g/L) is 25.1: TIBC (μmol/L) = 25.1 × TRF (g/L) (4)(5). Measurements of TIBC, serum iron, and the percentage of iron saturation of TRF are useful for the clinical diagnosis of iron-deficiency anemia and chronic inflammatory disorders (6)(7) and as screening tests for other clinical conditions (8). TIBC is routinely determined (9)(10)(11)(12) by saturation of TRF with an excess predetermined amount of iron, removal of the unbound iron, and measurement of the iron that is dissociated from TRF. For removal of the unbound iron, magnesium carbonate (9), ion-exchange resin (10), alumina columns (11), or magnetic particles(12) are used. Most direct TIBC measurement methods require manual procedures that involve centrifugation or pretreatment of serum samples. As an alternative to direct measurement methods, TIBC values are also calculated from the sum of serum iron and unsaturated iron-binding capacity (UIBC), both of which are determined by colorimetric methods (calculation method). We developed a direct and fully automated TIBC (DTIBC) assay for use with an automated multipurpose analyzer (13)(14). A fully automated TIBC measurement method is also commercially available (15). In our previous study (14), TIBC values obtained by DTIBC assay correlated strongly with serum TRF concentrations ( r = 0.984; n = 59), and the slope of the regression line was consistent with the theoretical TIBC/TRF ratio. We …

Background: Urinary iodine is a good biochemical marker for control of iodine deficiency disorders. Our aim was to develop and validate a simple, rapid, and quantitative method based on the Sandell–Kolthoff reaction, incorporating both the reaction and the digestion process into a microplate format. Methods: Using a specially designed sealing cassette to prevent loss of vapor and cross-contamination among wells, ammonium persulfate digestion was performed in a microplate in an oven at 110 °C for 60 min. After the digestion mixture was transferred to a transparent microplate and the Sandell–Kolthoff reaction was performed at 25 °C for 30 min, urinary iodine was measured by a microplate reader at 405 nm. Results: The mean recovery of iodine added to urine was 98% (range, 89–109%). The theoretical detection limit, defined as 2 SD from the zero calibrator, was 0.11 μmol/L (14 μg/L iodine). The mean intra- and interassay CVs for samples with iodine concentrations of 0.30–3.15 μmol/L were ≤10%. The new method agreed well with the conventional chloric acid digestion method (n = 70; r = 0.991; y = 0.944x + 0.04; Sy|x = 0.10) and with the inductively coupled plasma mass spectrometry method (n = 61; r = 0.979; y = 0.962x + 0.03; Sy|x = 0.20). The agreement was confirmed by difference plots. The distributions of iodine concentrations for samples from endemic areas of iodine deficiency diseases showed similar patterns among the above three methods. Conclusions: Our new method, incorporating the whole process into a microplate format, is readily applicable and allows rapid monitoring of urinary iodine.

Background: It would be useful to develop a simple kit method for determination of thiocyanate in urine, which could be used to monitor cyanide overload in cassava-consuming populations. Methods: The method was based on the quantitative oxidation of thiocyanate in acid permanganate at room temperature in a closed vial with liberation of HCN, which reacted with a picrate paper. For semiquantitative analysis in the field, the colored picrate paper was matched with a color chart prepared using known amounts of KSCN. In the laboratory, a more accurate result was obtained by elution of the colored complex in water and measurement of the absorbance at 510 nm. Over the range 0–100 mg/L, there was a linear relationship given by the equation: thiocyanate content (mg/L) = 78 × absorbance. Results: The picrate thiocyanate method gave no interference with urine samples containing protein at less than 7 g/L, 21 amino acids, histamine, glucose, NaCl, urea, blood, and linamarin. For 53 urine samples analyzed by an accurate column method and the thiocyanate picrate method, a regression line gave very good agreement (r2 = 1.000). Quantitative recoveries of thiocyanate added to urine samples were obtained with the picrate method. Conclusions: A simple picrate kit for determination of thiocyanate in urine was developed and is available free of charge for workers in developing countries.

We sought to determine if serum total iron-binding capacity (TIBC) is equivalent to serum transferrin (TRF) so that a low-cost colorimetric chemical assay for unsaturated iron-binding capacity (UIBC) could be substituted for a high-cost immunologic assay for TRF. Our study design included independent and blinded measurements of UIBC, serum iron, and TRF concentrations in human serum samples. Data from five independent correlation studies carried out at three different Quest Diagnostics laboratories were combined into one data set containing 570 paired results for TIBC and TRF. r2 was 0.941 when three outliers were eliminated from the 570-sample data set. Scatter about the regression line was fully accounted for by the CVs for the TIBC and TRF assays. When each test is measured precisely and without bias, the ratio of TIBC (μmol/L) to TRF (g/L) in SI units is close to the theoretically expected value of 25.0.

A fast competitive enzyme immunoassay (EIA) for measuring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcholinesterase as enzyme label. Enzyme detection was performed by an easy colorimetric assay. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 μmol/L, a CV

In this study, alpha-globin mRNA was captured on plastic plates to which alpha-globin-specific oligonucleotides had been covalently immobilized; the captured mRNA was immediately reverse-transcribed into cDNA in the presence of biotinylated dUTP. Addition of alkaline phosphatase-conjugated streptavidin to react with the biotin was followed by addition of substrate (p-nitrophenyl phosphate), and the resulting colorimetric development was measured at 405 nm. Because multiple biotin molecules are incorporated into cDNA during reverse transcription, and because synthesized cDNA is more stable than mRNA, we could successfully quantify by conventional colorimetry the amount of alpha-globin mRNA on the plate within a linear range of 100 pg to 10 ng. This is more sensitive than conventional Northern blotting. Therefore, the present method may be applicable to measurements of specific mRNAs in various test materials.

Liposomes entrapping the dye sulforhodamine B were used to develop an assay for anticardiolipin antibodies (ACAs). In the presence of magnesium ions, IgG ACAs induced liposomal lysis and the resulting absorbance changes were dependent on the amount of ACAs present. The liposomal assay (y) showed similar intraassay imprecision and detection limit to an ELISA for IgG ACAs (x), with a correlation coefficient of 0.90 (y = 1.05x - 1.61). No correlation with ELISA IgM ACA measurements was observed. Although ELISA remains the method of choice, particularly in examining different ACA classes, the liposomal assay offers advantages of speed and potential for processing large numbers of samples for IgG ACAs. This may facilitate study of the significance of increased IgG ACAs in the groups of conditions with which they are associated, and perhaps enable more laboratories to perform the test.

Patients with vascular disease have significantly higher concentrations of plasma total cysteine (tCYS) than do healthy individuals (1)(2)(3)(4)(5)(6)(7). The present method is a new, rapid, and sensitive enzymatic colorimetric assay for tCYS in plasma samples and is homogeneous in that it avoids separation methods. The tCYS assay uses only the recombinant enzymes methionine α,γ-lyase (rMETase) and S-adenosylhomocysteine hydrolase (rSAHH) cloned from Pseudomonas putida and Trichomonas vaginalis , respectively. We have also developed enzymatic assay methods for total homocysteine (tHCY) (8)(9) and vitamin B6 (10) in plasma that use the same analyte, H2S, that is used for tCYS in the present report. Simultaneous assay of tHCY, vitamin B6, and tCYS may be relevant to the study for the occurrence and prevalence of cardiovascular disease (11). The instrumentation included a Hitachi U-2000 Spectrophotometer (Hitachi, Ltd.) and Fl-1000 fluorescence spectrophotometer and a Hitachi HPLC (L-6200A Intelligent Pump) equipped with a Supelcosil LC-18DB column [25 cm × 4.8 mm (i.d.); particle size, 5 μm; Supelco]. The chemicals used were l-cysteine, d,l-homocysteine, l-methionine, adenosine (ADO), l-dithiothreitol, Triton X-100, pyridoxal 5-phosphate, and potassium ferricyanide and were purchased from Sigma Chemical Co. N,N -Dibutylphenylenediamine (DBPDA) was synthesized in our laboratory (8) …

p -Aminohippuric acid (PAH), a derivative of aminobenzoic acid, is almost completely extracted from the blood after a single passage by the kidney through a combination of glomerular filtration and proximal tubular secretion. On the basis of these properties, the renal clearance of intravenously administered PAH has been used as a measure of renal plasma flow (RPF) (1). PAH remains the “gold standard” for the noninvasive measurement of RPF in patients and study participants and may be useful for assessing the effect of disease states or pharmacologic agents on renal function. The standard method for PAH measurement is a colorimetric assay of a diazotation reaction that is labor-intensive (2)(3). Because of the complexities of the standard colorimetric PAH assay, its measurement has often been confined to research or used only in specialized laboratories. Measurement of PAH in urine and plasma by HPLC has been described (4)(5)(6)(7), but it requires relatively large sample volumes and time-consuming extraction procedures. We recently found that capillary electrophoresis (CE) is an efficient, inexpensive, and reliable method for measurement of the nonradiolabeled iothalamate in urine and plasma samples for the assessment of glomerular filtration rate (8)(9). Analysis by CE is analytically faster than standard HPLC or colorimetric assays, requires less reagent preparation and smaller sample size, minimizes drug interferences, and improves test turnaround time. With the development of a method for PAH on CE, assays for both RPF and glomerular filtration rate could also be obtained with a single methodology. This report describes a new quantitative CE assay for PAH in urine and plasma and compares it with the standard colorimetric assay. PAH was measured by CE on a Beckman Pace instrument using Beckman System Gold software, 50 mmol/L borate buffer (pH 10.2), ultraviolet detection at …

In solid-phase minisequencing, a defined point mutation is detected in microtiter plate-immobilized DNA by a single nucleotide primer extension reaction. We have here developed the method into a colorimetric assay and applied it to the detection of the Z mutation of the alpha 1-antitrypsin gene. We used novel nucleoside triphosphates modified with dinitrophenyl (DNP) hapten, permitting detection by anti-DNP-alkaline phosphatase conjugate, with p-nitrophenyl phosphate as substrate. The Z mutation is detected in two reactions: DNP-labeled dCTP is incorporated when the template is normal, DNP-dUTP when the Z mutation is present. Both modified nucleotides were incorporated with high specificity and with an efficiency similar to that of unmodified nucleotides. The test results are measured by spectrophotometry, yielding quantitative absorbance values. Calculation of the ratio of C to U signal permitted unambiguous distinction of normal homozygous, ZZ homozygous, and ZM heterozygous genotypes. The colorimetric minisequencing assay is rapid, standardized, and automatable, and thus provides an accurate and simple alternative for the analysis of known point mutations.

We report the evaluation of a new commercial kit for the determination of pancreatic lipase activity. The kit is based on the use of a 1,2-diglyceride as substrate and a specific monoglyceride lipase. The detection step is the continuous colorimetric measurement of hydrogen peroxide produced from glycerol by glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase reactions. The procedure appears to be precise (between-day CV < 9%) and the results show good correlation with those obtained by alternative procedures (vs turbidimetry, r = 0.965; vs ultraviolet absorbance-enzymatic method, r = 0.995; vs Ektachem, r = 0.976; vs immunometry, r = 0.970). However, the method is susceptible to interference by increased concentrations (> 4.5 mmol/L) of serum triglycerides. We estimated the reference interval for healthy adults to be 8-44 U/L. When we evaluated clinical efficacy by using receiver-operating characteristic curves and the overlap index, no significant differences were found between the commercial kit and a common turbidimetric assay for diagnosing patients with acute pancreatitis; both methods performed satisfactorily.

Acetone, a metabolite of fat catabolism, is produced in excessive amounts in subjects on restricted-calorie weight-loss programs. Breath acetone measurements are useful as a motivational tool during dieting and for monitoring the effectiveness of weight-loss programs. We have developed a simple, easy-to-read method that quantifies the amount of acetone in a defined volume of exhaled breath after trapping the sample in a gas-analyzer column. The concentration of acetone, as measured by the length of a blue color zone in the analyzer column, correlates with results obtained by gas chromatography. Using the breath acetone analyzer to quantify breath acetone concentrations of dieting subjects, we established a correlation between breath acetone concentration and rate of fat loss (slope 52.2 nmol/L per gram per day, intercept 15.3 nmol/L, n = 78, r = 0.81). We also discussed the possibility of using breath acetone in diabetes management.

In 1969, McCully (1) observed that increased plasma homocysteine (Hcy) was linked with vascular disease. Subsequent studies demonstrated that even mild hyperhomocysteinemia is an independent risk factor for cardiovascular, cerebrovascular, and peripheral vascular disease (2)(3)(4)(5)(6)(7)(8). Increased plasma Hcy is associated with a thermolabile variant of 5,10-methylenetetrahydrofolate reductase, smoking, lack of exercise, and excessive use of alcohol and coffee. Plasma or serum total Hcy (tHcy) concentrations are most commonly measured by HPLC (9), which is time- consuming and expensive, and by immunochemical (10)(11)(12) or enzymatic (13) methods, which may not be applicable to all colorimetric-based clinical chemistry analyzers. The thiol group of Hcy allows it to form a disulfide bond with other thiol-containing molecules, such as Hcy itself, cysteine, and the cysteine residue of plasma proteins. Biologic fluids may often contain both reduced and oxidized species of Hcy, and the sum of all the forms of Hcy is usually called total Hcy (tHcy) (14). Most clinical studies concerning Hcy have relied on the measurement of tHcy. An initial chemical reduction step of the sample is inevitable in the tHcy assay. Because reducing agents can interfere with the oxidation of redox indicators, such as Trinder’s reagents and derivatives of methylene blue generally used in diagnostic reagents, methods for tHcy that use these indicators have not been developed. The present method is a new enzymatic colorimetric assay for tHcy in biologic samples. The principle is as follows. In the first step, samples are reduced by dithiothreitol …

Increased serum cholesterol is a risk factor for atherosclerosis and coronary artery disease (CAD) (1)(2). Reduction of serum cholesterol has, therefore, been targeted for the prevention and management of CAD with cholesterol screening and is the primary tool for initial risk assessment (3)(4). Cholesterol, however, can also be quantified in skin. Skin reflects the vascular changes associated with age and aortic atherosclerosis. A simple, noninvasive procedure for the estimation of skin cholesterol, the “three-drop” test, has been proposed as an alternative screening method (5). The test, which uses three different concentrations of a digitonin–copolymer–horseradish peroxidase (HRP) conjugate and visual scoring, is capable of discriminating among healthy individuals, those at risk of developing atherosclerosis, and those with overt disease. Cholesterol 1,2,3TM is a refinement of this procedure. We describe here some properties of this test and report on our initial evaluation in a prospective clinical trial. Cholesterol 1,2,3 uses quantitative interpretation and a single concentration of detector. Briefly, the detector and the positive control each are added to die-cut wells in a foam template affixed to the palm. After a 1-min incubation, the reagents are removed by blotting and the HRP substrate is added to these wells and to a third well, which serves as negative control. After an additional 2-min incubation, color development in the center test well is measured by reflectance with a hand-held instrument (MD22 Spectrophotometer; X-Rite, Inc.) interfaced with a computer, and the result is reported numerically as the hue. Assay validity is assessed by visual interpretation of control wells. The specificities of digitonin conjugates have been demonstrated previously with several model systems [crystalline cholesterol, cholesterol-rich tissue sections, and antibody-immobilized LDL-cholesterol (LDL-C)], and the amount of skin-bound conjugate has been shown to correlate with epidermal cholesterol content (5). We extended the specificity …

Background: 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. Content: 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 error-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 have been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no reference materials for either analyte in urine. The recommended 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 ethnicity) nor the continuous increase in risk related to albumin excretion. Discussion: Clinical needs have been identified for standardization of (a) urine collection methods, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

Background: Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte concentrations as dialysis progressed. We observed that salivary nitrite (NO2−) and uric acid (UA) concentrations consistently decreased as dialysis proceeded. Methods: Solution-based colorimetric-detection chemistries for NO2− and UA were converted to a test strip format to produce a simple method for semiquantitatively measuring NO2− and UA concentrations in the clinic or at the patient’s home. We assessed the test strips with saliva samples collected from both ESRD patients undergoing dialysis and healthy control volunteers to qualitatively monitor the effect of dialysis on salivary NO2− and UA. We used computer software to analyze digital images of the resulting test strip color intensities. Results: Test strip measurements showed that mean salivary concentrations of NO2− and UA were decreased in ESRD patients by 86% and 39%, respectively, compared with 15% and 9% for time-matched controls. Comparison of test strip results with calibrated solution-based assays suggests that the test strips can semiquantitatively measure salivary concentrations of NO2− and UA. Conclusions: The colorimetric test strips monitored changes in salivary NO2− and UA concentrations that occurred in ESRD patients during dialysis. The test strips may prove useful for noninvasively evaluating dialysis progress and may also be useful for monitoring renal disease status.

We describe a new chromogenic substrate and assay for determining N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) activity in normal and pathological human urine. The novel substrate, ammonium 5-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3- methoxyphenylmethylene]-2-thioxothiazolidin-4-one-3-ethan oate (VRA-GlcNAc), is prepared by condensation of vanillyl N-acetyl-beta-D-glucosaminide and rhodanine-3-ethanoic acid. The phenol released by enzyme action has an intense absorption peak at 492 nm (epsilon = 37,000 L.mol-1.cm-1). The Km for NAG in urine was 0.5 mmol/L at pH 4.5. The substrate solution may be stored at 4-7 degrees C for one week without any increase in the substrate blank. For optimal assay conditions, we used a substrate concentration of 3 mmol/L and a 15-fold sample dilution at pH 4.5-5.0, with measurement at 505 nm, not significantly different from that at 492 nm. The sensitivity of the assay with VRA-GlcNAc as substrate is twice that of the MNP-GlcNAc [2-methoxy-4-(2'-nitrovinyl)-phenyl GlcNAc] procedure (Clin Chim Acta 1982;124:195-204) and can detect low amounts of minor NAG isoenzymes. This assay is an improvement on previously available colorimetric methods and could be easily incorporated into kits based on either an enzyme calibrant or the molar absorptivity of the phenol.

We developed a method for the selective determination of 4-aminobenzoic acid by use of 4-aminobenzoate hydroxylase (EC 1.14.13.27) to convert 4-aminobenzoic acid to 4-hydroxyaniline. The subsequent conversion of 4-hydroxyaniline to indophenol dye was quantified colorimetrically. The method is reproducible, and the assay response is linear up to 40 mumol/L. Selectivity exceeding that of the current colorimetric assays was demonstrated for these enzymatic determinations of 4-aminobenzoic acid concentrations in urine samples from patients undergoing tests of pancreatic function with bentiromide. This method effectively minimizes interferences from drugs and diet, a problem in current colorimetric methods.

We describe a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for quantifying ferritin in human and rat biological fluids. We used chlorophenol red beta-D-galactopyranoside as the colorimetric substrate of beta-galactosidase (EC 3.2.1.23), which is coupled to specific antibodies to either human or rat liver ferritin. The assay is sensitive (detection limit for human assay = 0.58 micrograms/L and for rat assay = 0.37 micrograms/L), accurate (average recovery for human assay = 93% and for rat assay = 92%), and precise (total CVs for human assay = 2.3-12.2% and for rat assay = 5.6-11.3%). The results correlated well with those of an established immunoradiometric technique (r = 0.99691). This assay has a prolonged shelf-life, is inexpensive, and utilizes a stable colorimetric substrate that requires relatively short incubation.

Background: Radioactive reagents are used in most assays for measurement of 1,25-dihydroxyvitamin D [1,25(OH)2D]. We evaluated a 1,25(OH)2D enzyme immunoassay (EIA) from IDS Ltd. that uses solid-phase immunoextraction and colorimetric detection and compared results to those of the thymus radioreceptor assay (RRA) for 1,25(OH)2D.Methods: We collected serum samples (n = 145) representing an even distribution (0–200 pmol/L) of 1,25(OH)2D concentrations and Vitamin D External Quality Assessment Scheme (DEQAS) proficiency survey samples from 2004 surveys (n = 15) and stored them at −20 °C. We analyzed all samples with both EIA and RRA methods. We calculated imprecision using 5 QC samples in quadruplicate in each run (n = 6), including both pooled patient material used for QC with the RRA and QC material included in the EIA reagent set. We evaluated calibration stability by analyzing calibrators from different lots on the same plate and determining if calculated sample values drifted significantly.Results: Deming linear regression between IDS EIA and RRA methods yielded slope 1.25 (95% CI 1.13–1.37), y-intercept −3 (95% CI −18 to 12), R2 = 0.74. DEQAS proficiency survey samples for 2004 were all within 30% of the all-methods-trimmed mean. Imprecision CVs were 12%–16% within-run and 15%–20% between-run.Conclusions: We find no evidence of inferiority to the classic calf-thymus receptor assay for 1,25(OH)2D and no disadvantage in the results generated by the IDS EIA using samples from the major proficiency survey for 1,25(OH)2D. According to the product insert, however, the IDS EIA underestimates 1,25(OH)2D2 compared with the D3 form.

Measurements of the activity of both lipase (triacylglycerolacylhydrolase, EC 3.1.1.3) and α-amylase (EC 3.2.1.1) in serum are commonly used as aids in the diagnosis and follow-up of acute and recurrent pancreatitis (1); 19% of pancreatitic patients present with normal serum amylase (2), thus leaving lipase as the main hematochemical marker of this disease in a substantial number of patients. Lipase and amylase are increased also in conditions other than pancreatitis, such as several nonmalignant hepatobiliary and gastrointestinal diseases, sepsis, renal failure, pulmonary failure, and subdural bleeding (1)(2)(3)(4). In 1987, Stein et al. (5) reported on a non-Hodgkin’s lymphoma patient with major lipase activity (up to seven times the upper reference value), with no parallel increase in amylase; in this case, the authors demonstrated a specific immunoglobulin G with high affinity for lipase as the cause of increased serum lipase activity. More recently, Donnelly et al. (6) have reported a chronic increase in serum lipase, again with no associated hyperamylasemia or clinical evidence of pancreatitis, which the authors suggested to be tumor-derived; furthermore, Munoz-Perez et al. (7), after observing chronic increased serum lipase in another patient with massive abdominal metastases from a suspected pancreatic adenocarcinoma, suggested that the biochemical profile characterized by increased lipase and normal amylase could be an early finding of a malignant neoplasm. We report on four cases in which serum lipase increase first anticipated an otherwise undetectable tumor relapse or progression. Serum lipase activity was measured with a commercially available kinetic colorimetric co-lipase method (Lipase KC, Bayer Italia S.p.A., Divisione Diagnostici). Briefly, in the assay, pancreatic lipase catalyzes the hydrolysis of the 1,2-diglyceride sulfate to produce fatty acids and 2-monoglyceride. The latter, in the presence of specific auxiliary enzyme, release glycerol, which is then converted, by glycerokinase, into glycerol-3-phosphate and …

Components of blood collection tubes have been implicated as interfering substances in various assays, especially immunoassays (1)(2)(3)(4)(5). Possible interfering components for blood collection tubes include the lubricants, clot activators, surfactants, and barrier gels. In 1 study, a surfactant was identified as the interferent (1). To our knowledge, no problems have been reported with general chemistry assays. Here we describe an apparent contamination of Terumo tubes with magnesium. In an evaluation of collection tubes, we collected samples from volunteers via a Vacutainer system into 3 tubes (Greiner, BD, or Terumo). The samples were obtained with no conscious order of draw, and the 3 types of tubes were treated identically. The fill volume of drawn blood was 5–7 mL for all tubes. The samples were stored at room temperature (∼21 °C) for

Many molecular methods have been developed for the direct identification and susceptibility testing of mycobacteria (1)(2)(3). Only 2 US Food and Drug Administration–cleared methods are available for direct detection of Mycobacterium tuberculosis , the enhanced Direct Test (Gen-Probe) and the Amplicor (Roche). The appearance of multiple-drug–resistant strains and elucidation of the rifampicin resistance mechanism in M. tuberculosis have prompted the development of new molecular techniques such as the INNO-LiPA-Rif-TB (Innogenetics), that can simultaneously detect M. tuberculosis and sensitivity/resistance to rifampicin (4). However, most molecular tests for M. tuberculosis detection are expensive and unsuitable for routine use. Therefore, we sought to develop an inexpensive molecular method for the fast and sensitive detection of M. tuberculosis . Our colorimetric method uses gold nanoparticles for rapid and sensitive direct detection of M. tuberculosis in clinical samples with high efficiency after an initial round of PCR. Gold-nanoparticle–based methods have been used extensively for detection of specific DNA and RNA sequences (5)(6)(7). We obtained 73 clinical specimens from patients with suspected pulmonary and extrapulmonary tuberculosis with …

Recent studies have suggested that abnormal hepatocellular function is associated with obesity, insulin resistance, and type 2 diabetes (1)(2)(3). Nonancoholic fatty liver disease (NAFLD) is the preferred term to describe a spectrum of liver damage, ranging from steatosis to steatohepatitis, liver fibrosis, and cirrhosis, that can be found in insulin-resistant (obese and type 2 diabetic) and hyperlipidemic patients (4). Insulin sensitivity and liver function exhibit a bidirectional relationship: a normally functioning liver determines, in part, the normal body response to insulin, whereas abnormal insulin sensitivity causes liver damage (5)(6). The insulin-resistant state is characterized by a failure to suppress hepatic glucose production and glycogenolysis (5), usually accompanied by fat accumulation in hepatocytes as a result of increased hepatic uptake and synthesis of triglycerides. The latter is known to impair liver function and, consequently, to worsen insulin resistance (5)(6). Aminotransferases are considered indicators of hepatocellular health. Alanine aminotransferase (ALT) is found primarily in the liver, whereas aspartate aminotransferase (AST) and γ-glutamyltranspeptidase (γGT) are also found in other tissues. NAFLD is characterized by mild-to-moderate increases in ALT and AST (7). Recent prospective studies have shown that increased ALT and γGT are predictors of the development of type 2 diabetes (1)(2). CD14, a 55-kDa glycoprotein, is a multifunctional receptor constitutively expressed in considerable amounts on the surface of mature monocytes, macrophages, and neutrophils (8). CD14 has specificity for lipopolysaccharide (LPS) and other bacteria-wall-derived components (9). LPS signaling triggers a cascade that leads to cytokine production and shedding of the extracellular domain of CD14 (sCD14) (10)(11)(12)(13). The buffering of LPS is crucial not only during acute inflammatory and infectious processes; in healthy humans, triglyceride-rich lipoproteins contain detectable amounts of endogenous LPS that are presumably scavenged in …

International groups recommend the following median urinary iodine concentration as the best single indicator of iodine nutrition in populations: severe deficiency, 0–0.15 μmol/L (0–19 μg/L); moderate deficiency, 0.16–0.38 μmol/L (20–49 μg/L); mild deficiency, 0.40–0.78 μmol/L (50–99 μg/L); optimal iodine nutrition, 0.79–1.56 μmol/L (100–199 μg/L); more than adequate iodine intake, 1.57–2.36 μmol/L (200–299 μg/L); and excessive iodine intake, ≥2.37 μmol/L (≥300 μg/L) (1). The range in which the median falls is more important than the precise number (2)(3). Many methods for assessing urinary iodine exist (3)(4)(5)(6)(7)(8), most based on the Sandell–Kolthoff reaction (9), in which iodide catalyzes the reduction of ceric ammonium sulfate (yellow) to the colorless cerous form in the presence of arsenious acid. Although iodide is the chemical form for both the catalytic reaction and in urine, some preliminary treatment is needed to rid urine of impurities, most commonly by acid digestion (3)(5). We have extended previous approaches (5)(6)(10) with improved conditions and here present a new method (“Fast B”) that is rapid, inexpensive, reliable, and flexible. The equipment required for the Fast B method includes a heating block, Pyrex test tubes (13 × 100 mm), two fixed-volume pipettes (0.5 mL and 1.0 mL), one adjustable pipette (0–200 μL), and a multipet (Eppendorf) for quick reagent volume additions of 0.125 and 0.1 mL. The basic chemicals used are potassium iodate, arsenic trioxide, ammonium persulfate, ammonium cerium(IV) sulfate dihydrate, sodium chloride, ferroine, and sulfuric acid. The solutions used in the assay are as follows

Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines.We added 250 ng of d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 degree C water bath, neutralized, and prepared by solid-phase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column.Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of 10-5000 microg/L. Interassay CVs were 5.7-8.6% at mean concentrations of 90-4854 microg/L for MN and NMN. The detection limit was 10 microg/L. Recovery of MN and NMN (144-2300 microg/L) added to urine was 91-114%. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y = 0.81x - 0.006 (r = 0.822; n = 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y = 1.09x + 0.05 (r = 0.998; n = 40).The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography-mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time.

Quantitative immunohistochemical (IHC) assays currently lack optimal reference quality-control material for cellular protein targets. To address this problem, we identified peptides that mimic the site on the native analyte to which the primary (monoclonal) antibody binds and used them as surrogate peptide controls.We identified peptide candidates from a combinatorial peptide phage-display library that mimic the epitope for the 1D5 estrogen receptor (ER) monoclonal antibody (mAb). The peptide inserts of the phage clones were sequenced. Several phage-encoded peptides were then synthesized and analyzed for affinity and specificity.We identified phage clones that specifically bound to the ER 1D5 mAb. The binding was specific, in that the phage clones did not bind to two other isotype-matched mAbs. Their ability to bind the ER 1D5 mAb was related to the presence of a consensus sequence. Binding analysis revealed a K(d) of 8.3 x 10(-8) mol/L. The peptide was not recognized by any of 15 other mAbs commonly used for clinical IHC testing. Moreover, the peptide was able to inhibit the binding of ER 1D5 mAb to native ER, indicating that the peptide bound to ER 1D5 mAb at or close to the antigen-binding site.Surrogate peptide controls behave like the native analyte in terms of affinity and specificity. This technology may be especially useful when the native analyte is in short supply.

Genetic variants of human transferrin (TF) have been described, but little is known about their functional differences. We studied iron status according to TF phenotype in a healthy Zimbabwean population and in subjects at risk of African iron overload.The study population consisted of 483 nondrinkers, 31 drinking spouse pairs, and 5 family pedigrees (n = 88) with index cases of iron overload. TF phenotypes were determined using starch gel electrophoresis. To evaluate iron status, serum iron, total iron-binding capacity (TIBC), ferritin, and soluble TF receptors were measured, and the percentage of saturation and the serum iron:TF ratio were calculated. The binding of the TF variants was studied by equilibrium dialysis.The reference population was characterized by a high TF D allele frequency (0.050) and a complete absence of homozygous TF DD individuals. Similar allele frequencies were observed in subjects at risk of African iron overload. In the reference population, male TF CD heterozygotes had significantly lower (P0.01) values for serum iron, TIBC, TF saturation, and serum iron:TF ratio than the TF CC homozygotes; in females, only TIBC was significantly different. Overall red blood cell indices did not differ according to TF phenotype. In the population at risk of African iron overload, only serum iron:TF ratio was consistently significantly lower in TF CD phenotypes (P0.05). After equilibrium dialysis, the amount of iron bound by TF was significantly lower (P0.01) in TF CD individuals.The present data demonstrate a functional difference between TF phenotypes in blacks.

Background: Automated electrophoresis combined with enzymatic cholesterol staining might improve routine assessment of LDL- and HDL-cholesterol (LDLC and HDLC), as an alternative to the Friedewald equation and precipitation. A new method (Hydrasys; SEBIA) that adapts the cholesterol esterase/cholesterol oxidase reaction within urea-free gels was evaluated.Methods: Fresh sera from 725 subjects (512 dyslipidemics) were analyzed by electrophoresis, in parallel with sequential ultracentrifugation, β-quantification, calculation, and precipitation.Results: Electrophoresis was linear up to 4 g/L cholesterol, with a detection limit of 0.042 g/L cholesterol/band. Within-run, between-run, between-batch, and between-operator imprecision (CVs) were 1.6%, 2.0%, 1.5%, and 2.7% for LDLC, and 3.9%, 4.3%, 5.5%, and 4.9% for HDLC, and remained unchanged up to 6.3 g/L plasma triglycerides (TGs). Precision decreased with very low HDLC (4 g/L (r = 0.91). Bias (2.88% ± 12%) and total error (7.84%) were unchanged at TG concentrations up to 18.5 g/L. Electrophoresis predicted National Cholesterol Education Program cut-points with 1.5 g/L) the percentage of subjects underestimated by calculation. One-half of the patients with TGs >4 g/L had LDLC >1.30 g/L. For HDLC, correlation was better with precipitation (r = 0.87) than ultracentrifugation (r = 0.76). Error (−0.10% ± 26%) increased when HDLC decreased (Conclusions: Electrophoresis provides reliable quantification of LDLC, improving precision, accuracy, and concordance over calculation, particularly with increasing plasma TGs. Implementation of methods to detect low cholesterol concentrations could extend the applications for HDLC assessment.

Monochromatic measurement of albumin by the bromcresol green method leads to overestimation of albumin in the presence of heparin. The interference seems to be caused by fibrinogen, which, in the presence of heparin, produces an almost constant increase of the measuring signal over the whole range of suitable wavelengths (550 to 700 nm). The albumin overestimation can be eliminated by using a bichromatic modification. An application of this procedure for the automated analyzer RAXT, RA-1000 (Bayer Diagnostik, Technicon) is presented. We recommend using only bichromatic methods for albumin determination with bromcresol green to avoid unexpected analytical artifacts.

Background: Various methods are available to measure serum cholesterol concentrations. Of these, the cholesterol ester hydrolase (CEH)-cholesterol oxidase-peroxidase chromogenic method is widely used. However, this method has the disadvantage of interference by reducing substances. We developed and evaluated an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method.Methods: Cholesterol esters are first hydrolyzed to free cholesterol by CEH. The free cholesterol is then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of β-NADH from β-NAD+. At equilibrium, the CDH reaction gives incomplete conversion of cholesterol to cholest-4-ene-3-one. To overcome this disadvantage, we added hydrazine monohydrate to the reaction mixture to remove cholest-4-ene-3-one, which allowed the reaction to proceed to completion and gave stoichiometric production of β-NADH from the reaction of β-NAD+ with cholesterol.Results: We tested whether the amount of cholesterol added was equivalent to the absorbance change of NADH at 340 nm with six aqueous samples. Recoveries were 97.1–100.3%. The reaction was linear up to 20.28 mmol/L. The mean within-day (n = 20) and between-day (n = 10) imprecision (CV) was 0.29–0.43% and 0.22–0.61%, respectively. No interference by bilirubin, hemoglobin, ascorbic acid, and other reducing agents was observed. The equation obtained in comparison with the modified Abell-Levy-Brodie-Kendall method was: y = 0.992x − 0.0058 mmol/L; r = 0.997; Sy|x = 0.117 mmol/L; n = 50.Conclusion: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.

The serum sialic acid (SA) concentration has been reported to be a potentially useful but nonspecific disease marker. We wanted to study which factors influence SA concentration in a well-characterized healthy population.SA was determined in 97 women and 96 men with a colorimetric Warren method.The mean +/- SD concentrations of SA were 634 +/- 109 (95% confidence interval, 612-656) and 630 +/- 106 (95% confidence interval, 608-651) mg/L for women and men, respectively. The serum SA showed a significant positive association with body mass index and with systolic and diastolic blood pressure among both women and men. SA also correlated significantly with the use of contraceptive pills and age among women and with smoking among men.Our study suggests that SA does not increase with age in men but appears to increase with female menopause. The strong positive association with blood pressure may explain why SA predicts cardiovascular mortality.

The hepatic iron concentration (HIC) is widely used in clinical practice and in research; however, data on the variability of HIC among biopsy sites are limited. One aim of the present study was to determine the variability of HIC within both healthy and cirrhotic livers.Using colorimetric methods, we determined HIC in multiple large (microtome) and small (biopsy-sized) paraffin-embedded samples in 11 resected livers with end-stage cirrhosis. HIC was also measured in multiple fresh samples taken within 5 mm of each other ("local" samples) and taken at sites 3-5 cm apart ("remote" samples) from six livers with end-stage cirrhosis and two healthy autopsy livers.The within-organ SD of HIC was 13-1553 microg/g (CV, 3.6-55%) for microtome samples and 60-2851 microg/g (CV, 15-73%) for biopsy-sized samples. High variability of HIC was associated with mild to moderate iron overload, because the HIC SD increased with increasing mean HIC (P0.002). Livers with mean HIC1000 microg/g exhibited significant biological variability in HIC between sites separated by 3-5 cm (remote sites; P0.05). The SD was larger for biopsy-sized samples than for microtome samples (P = 0.02).Ideally, multiple hepatic sites would be sampled to obtain a representative mean HIC.

Three assays were compared for the determination of total antioxidant capacity in human serum: the oxygen radical absorbance capacity (ORAC) assay, the Randox Trolox-equivalent antioxidant capacity (Randox-TEAC) assay, and the ferric reducing ability (FRAP) assay. There was a weak but significant linear correlation between serum ORAC and serum FRAP. There was no correlation either between serum ORAC and serum TEAC or between serum FRAP and serum TEAC. The effect of dilution on the serum TEAC value and the use of inhibition percentage at a fixed time, without considering the length of inhibition time in the quantitation of results, adversely affected the Randox-TEAC assay. The FRAP assay is simple and inexpensive but does not measure the SH-group-containing antioxidants. The ORAC assay has high specificity and responds to numerous antioxidants. By utilizing different extraction techniques in the ORAC assay, one can remove serum proteins and also make some gross differentiation between aqueous and lipid-soluble antioxidants. However, the ORAC assay requires approximately 60 min more than the FRAP or Randox-TEAC assay to quantitate results.

A new antibody to lysergic acid diethylamide (LSD) was used to develop a novel indirect ELISA for the quantification of drug in urine. Evaluation of the new assay with the commercially available LSD ELISA (STC Diagnostics) shows improved performance. The test requires 50 microL of urine, which is used to measure concentrations of drug in the microg/L to ng/L range. The limit of detection was 8 ng/L compared with 85 ng/L in the commercial assay, and analytical recoveries were 98-106%. Our test detected 0.1 microg/L of LSD in urine with an intraassay CV of 2.4% (n = 8) compared with 6.0% for a 0.5 microg/L sample in the commercial assay (n = 20). The upper and lower limits of quantification were estimated to be 7 microg/L and 50 ng/L, respectively. Specificity was evaluated by measuring the extent of cross-reactivity with 24 related substances. Drug determination using the new assay offers both improved sensitivity and precision compared with existing methods, thus facilitating the preliminary quantitative estimation of LSD in urine at lower concentrations with a greater degree of certainty.

The use of pressure to disrupt immune complexes was demonstrated in two model systems: prostate-specific antigen (PSA) and anti-PSA antibody; and epiglycanin, a mucin glycoprotein, and an antibody specific to that protein. Dissociation of the anti-PSA antibody from the immobilized PSA antigen was observed when pressures of 415 MPa and 550 MPa (1 MPa approximately 144 psi) were applied at room temperature (approximately 21 degrees C). Application of pressures ranging from 140 MPa to 550 MPa resulted in dissociation of antibody from epiglycanin. In both cases, the rebinding of dissociated antibody to immobilized antigen indicated that the effect of high pressure on the binding of the immune complexes was reversible. These findings suggest that application of high hydrostatic pressure has the potential to be used to significantly improve the sensitivity and specificity of clinical assays.