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PubMed2004

Evaluation of agar diffusion bioassay for nisin quantification.

The agar diffusion bioassay is the most widely used method for the quantification of nisin, due to its high sensitivity, simplicity, and cost-effectiveness. This method is based on the measurement of the inhibition zone produced in nisin-sensitive microorganisms. The size of the zone is affected by many factors, such as nisin-sensitive strain, amount of added agar and surfactant, and pre-diffusion step. This research aims to evaluate the effects of nisin-sensitive strains and pre-diffusion on the accuracy and precision of nisin quantification. Three strains of nisin-sensitive microorganisms (Micrococcus luteus, Lactobacillus sakei, Brochothrix thermosphacta) were tested along with three different incubation processes. The best combination was the method using L. sakei as an indicator strain with pre-diffusion at 4 degrees C for 24 h. Compared with M. luteus and B. thermosphacta, L. sakei gave more accurate and reproducible results. Moreover, the pre-diffusion step resulted in larger inhibition zones and more precise results. Finally, the best combination was validated and compared with the method that is usually used and the result showed that the method using L. sakei with pre-diffusion gave more accurate and precise results.

Agar↗
PubMed2004

Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion.

Methods are described which facilitate quantification of supplemental cellulase, protease and alpha-amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t(-1) cellulase, protease and alpha-amylase yielded net supplemental activity recoveries of 104+/-11.7%, 91.3+/-6.74% and 126+/-29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and alpha-amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4+/-0.47%, 6.6+/-0.82% and 29.0+/-14.1%, respectively, of a 1 kg t(-1) supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.

Animal Feed↗
PubMed2000

A simplification of the protein assay method of Ramsay et al. for the quantification of Thiobacillus ferrooxidans in the presence of ferric precipitates.

A variation on Ramsay's method for microbial protein determination has been developed in order to quantify Thiobacillus ferrooxidans attached to ferric precipitates or in aqueous suspensions containing such precipitates. Some modifications have been introduced to provide a method that is more sensitive, simple and rapid. A linear standard curve is presented to permit a direct correlation between the protein concentration (mg/l) and the cell concentration (10(6) cells/ml). An application of this method has been demonstrated in the quantification of biomass immobilized on the surface of polyurethane foam particles in a packed bed reactor, several experiments having been conducted to establish the best conditions for the quantification studies.

Bacterial Proteins↗
PubMed2001

Quantification of 5-HT(1A) receptors in human brain using p-MPPF kinetic modelling and PET.

Serotonin-1A (5-HT(1A)) receptors are implicated in neurochemical mechanisms underlying anxiety and depression and their treatment. Animal studies have suggested that 4-(2'-methoxyphenyl)-1-[2'-[ N-(2"-pyridinyl)- p-[(18)F]fluorobenzamido] ethyl] piperazine ( p-MPPF) may be a suitable positron emission tomography (PET) tracer of 5-HT(1A) receptors. To test p-MPPF in humans, we performed 60-min dynamic PET scans in 13 healthy volunteers after single bolus injection. Metabolite quantification revealed a fast decrease in tracer plasma concentration, such that at 5 min post injection about 25% of the total radioactivity in plasma corresponded to p-MPPF. Radioactivity concentration was highest in hippocampus, intermediate in neocortex and lowest in basal ganglia and cerebellum. The interactions between p-MPPF and 5-HT(1A) receptors were described using linear compartmental models with plasma input and reference tissue approaches. The two quantification methods provided similar results which are in agreement with previous reports on 5-HT(1A) receptor brain distribution. In conclusion, our results show that p-MPPF is a suitable PET radioligand for 5-HT(1A) receptor human studies.

Adult↗
PubMed2006

Quantification of [123I]FP-CIT SPECT brain images: an accurate technique for measurement of the specific binding ratio.

PURPOSE: A technique is described for accurate quantification of the specific binding ratio (SBR) in [(123)I]FP-CIT SPECT brain images. METHODS: Using a region of interest (ROI) approach, the SBR is derived from a measure of total striatal counts that takes into account the partial volume effect. Operator intervention is limited to the placement of the striatal ROIs, a task facilitated by the use of geometrical template regions. The definition of the image for the analysis is automated and includes transaxial slices within a "slab" approximately 44 mm thick centred on the highest striatal signal. The reference region is automatically defined from the non-specific uptake in the whole brain enclosed in the slab, with exclusion of the striatal region. A retrospective study consisting of 25 normal and 30 abnormal scans-classified by the clinical diagnosis reached with the scan support-was carried out to assess intra- and inter-operator variability of the technique and its clinical usefulness. Three operators repeated the quantification twice and the variability was measured by the coefficient of variation (COV). RESULTS: The COVs for intra- and inter-operator variability were 3% and 4% respectively. A cutoff approximately 4.5 was identified that separated normal and abnormal groups with a sensitivity, specificity and diagnostic concordance of 97%, 92% and 95% respectively. CONCLUSION: The proposed technique provides a reproducible and sensitive index. It is hoped that its independence from the partial volume effect will improve consistency in quantitative measurements between centres with different imaging devices and analysis software.

Aged↗
PubMed1997

Quantification of 4-hydroxyifosfamide in plasma of ifosfamide-treated mice.

PURPOSE: Ifosfamide is becoming an important clinical anticancer drug. Meaningful pharmacology studies require quantification of its activated and active metabolites, 4-hydroxyifosfamide (HOIfos) and isophosphoramide mustard (IPM), respectively. METHODS: Current methodology for quantifying the unstable HOIfos in biological fluids consists of trapping acrolein as it is produced during the decomposition of this metabolite. However, unlike cyclophosphamide, ifosfamide is extensively metabolized to two dechloroethylated metabolites, which are susceptible to 4-hydroxylation and similarly are capable of yielding acrolein upon decomposition. Because the current method has the potential to yield higher than actual values for HOIfos, it was compared with an HOIfos-specific method that traps the first stage degradation product of HOIfos, aldoifosfamide, as its semicarbazone, and depends on the use of radiolabeled drug for quantification. Six experiments in mice were conducted with blood collection 15 or 30 min after drug treatment followed by determination of HOIfos in plasma by the two methods. RESULTS: Comparison of plasma levels of HOIfos determined by the two methods indicated only minor differences between the two. CONCLUSION: These results suggest the possibility that the nonspecific method may be acceptable as a first estimation of levels of this metabolite in biological fluids until the development of a specific method that does not require radiolabeled drug, such as high-performance liquid chromatography or gas chromatography/mass spectrometry, has been developed.

Animals↗
PubMed2002

Reproducibility of coronary calcium quantification in repeat examinations with retrospectively ECG-gated multisection spiral CT.

High reproducibility is a key requirement for coronary calcium scoring in follow-up examinations. We investigated the inter-examination reproducibility of calcium scoring with retrospectively ECG-gated multisection spiral CT (MSCT). Fifty patients were examined twice with MSCT. Slices were reconstructed with retrospective ECG gating in the diastolic phase with 3-mm slice width and up to 125-ms temporal resolution. We calculated the Agatston score, calcium volume with and without isotropic interpolation, and calcium mass, and derived the mean and median variability. We investigated the change of variability with use of 3-mm non-overlapping and overlapping increments (2, 1.5, 1 mm). Use of overlapping increment results in considerably reduced interscan variability. We observed a minimum mean variability of 12% and a minimum median variability of 9% for the Agatston score. For volume and mass quantification we obtained a minimum mean variability of 7.5% and a minimum median variability of 5%. Multisection spiral CT enables coronary calcium quantification with high reproducibility in follow-up examinations mainly founded on image data with reduced partial-volume errors due to overlapping increment.

Adult↗
PubMed2000

[Echocardiographic online quantification of left ventricular systolic function in children: comparison with conventional off-line determination].

Accurate and efficient echocardiographic on-line determination of left ventricular volume would be advantageous in the care of children with congenital heart disease and children with hemodynamic instability. The prospective study was performed to evaluate the clinical usefulness of the on-line automatic border detection system (acoustic quantification: AQ) for determination of left ventricular volumes and ejection fraction in comparison to the conventional off-line method (manual tracing). 107 patients were enrolled in the study. The ages ranged from 0.1 to 18.8 years (mean 8.3 +/- 5.6). All patients were studied in the apical four-chamber plane for acoustic quantification (AQ) and manual tracing as well. Left ventricular volumes were determined using the mono-plane Simpson's rule. Left ventricular end-diastolic volumes obtained by AQ correlated well but were slightly underestimated compared to those determined by manual tracing (r = 0.99). Left ventricular endsystolic volumes by AQ correlated well but were also slightly underestimated compared to those obtained by manual tracing (r = 0.98). Mean ejection fraction was 61.1 +/- 6.8% by AQ compared with 61.5 +/- 5.9% by manual tracing. Linear regression analysis demonstrated good correlation: y = 0.77x + 14.1, r = 0.89; p < 0.001. Measurement of left ventricular volumes and ejection fraction by AQ using automatic border detection compares well with measurements done by manual tracing. However, AQ tends to underestimate to some degree. The time necessary for acquisition of data was similar in both methods. AQ seems to be a promising method for real-time estimation of left ventricular volume, even in children.

Adolescent↗
PubMed1998

A sensitive assay for the quantification of glucose and lactate in the human cornea using a modified bioluminescence technique.

BACKGROUND: Quantification of glucose and lactate concentrations in human corneal extracts has been performed using spectrophotometry. We employed a bioluminescence technique to obtain a more sensitive assay for glucose and lactate and to reduce the volume of the test sample. MATERIALS AND METHODS: The NAD(P)H bioluminescence assay (Boehringer Mannheim, Germany) was modified for glucose and lactate. Standard curves were established using a standard solution with 0.004 mM and 0.01 mM concentrations of glucose and lactate, respectively. RESULTS: Linear standard curves ranging from 0 to 200 pmol for glucose and from 0 to 250 pmol for lactate were established. The sample volume was reduced from 100 microliters to 25 microliters compared with spectrophotometry. DISCUSSION: The modified bioluminescence technique provides a highly sensitive quantification of glucose and lactate in the human cornea and thus reveals more details of the overall metabolic status of the tissue.

Aged↗
PubMed2001

Quantification of alpha 1-fetoprotein mRNA in peripheral blood and bone marrow: a tool for perioperative evaluation of patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Quantification of alpha 1-fetoprotein (AFP) mRNA in the blood using reverse transcriptase polymerase chain reaction (RT-PCR) could be a useful tool in monitoring the dynamics of minimal residual disease in patients with hepatocellular carcinoma (HCC). Since all available assays do not take into account the efficiency of cell separation, RNA extraction and reverse transcription, a competitive RT-PCR assay for quantification of AFP mRNA in relation to the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH) was established. PATIENTS AND METHODS: Peripheral blood of 22 patients and bone marrow aspirates of 11 patients with hepatocellular carcinoma was monitored perioperatively. Eighteen patients with other hepatic tumours or non-malignant hepatic diseases and 26 healthy blood donors served as controls. Messenger RNA contents were calculated relative to the content of GAPDH mRNA as an indicator of total cell count. RESULTS: Among HCC patients, 6 of 22 (26%) were positive for AFP mRNA before operation with values ranging from 2 ag/100 fg to 36 ag/100 fg GAPDH mRNA (mean 14). Among bone marrow samples, AFP mRNA was detectable in 5 of 11 (45%) cases, with 4 ag/100 fg to 23 ag/100 fg GAPDH (mean 9). However, AFP mRNA was also detectable in 3 of 18 (17%) control patients and in 2 of 26 (8%) healthy blood donors. Perioperative findings were highly variable. CONCLUSION: AFP mRNA is not a specific marker for circulating malignant hepatocytes. Whether definition of a cut-off level or the use of a multimarker-PCR will provide more useful data remains to be established.

Adult↗
PubMed2002

Quantification of cumulated physical fatigue at the workplace.

Quantification of physical fatigue remains a challenge. We hypothesized that its effects on central autonomic nervous system activity could be explored for such a quantification. To test this relationship, we prospectively measured central autonomic nervous activity through nocturnal heart rate variability (HRV) in six French garbage collectors, aged 32.1+/-4.3 years, twice a week during 3 consecutive weeks of work, and during the following week of rest. Eight healthy sedentary males formed a control group. HRV indices were calculated by applying standard temporal domain and wavelet transform analyses to standard ECG recordings. During the 3 consecutive weeks of work, there was a significantly progressive decrease in HRV indices, particularly pNN50 (-34.2%, P<0.05), as well as the high (-33.3%, P<0.05) and low (-22.2%, P<0.01) frequency components of wavelet transform, while there was an increase, although non-significant, of the ratio of low to high frequencies (9.1%). During the resting period, there was a significant recovery of HRV indices, notably of its high (50.0%, P<0.05) and low (28.6%, P<0.05) frequency components. No such changes occurred in the control group. A central signature of cumulated physical fatigue can thus be detected and quantified through nocturnal autonomic nervous system activity. Its characteristics are those of a progressive parasympathetic withdrawal.

Adult↗
PubMed1998

Evaluation of flow-cytometric three-parameter analysis for EGFR quantification and DNA assessment in human bladder carcinomas.

Flow-cytometric multi-parameter staining is an excellent method for defining tumour subpopulations. This provides further understanding of tumour heterogeneity and defines the biological relevance of tumour subpopulations. A method of quantifying the epidermal growth factor receptor (EGFR) in parallel with DNA staining, which was previously established in bladder carcinoma cell lines, was applied to twenty-five biopsies of urothelium and urothelial neoplasms. Uro5, a surface glycoprotein, was used to identify urothelial cells. Objective quantification of receptor content via flow cytometry was achieved with beads of defined numbers of antigen-binding sites, and receptor numbers obtained from urothelial and nonurothelial cells were compared with staining intensity in a three-step immunoperoxidase detection of the EGFR. The data obtained matched the immunohistochemical findings and were more sensitive in the low range (ca. 5x103) of receptors. Parallel definition of the proliferative fraction and DNA-ploidy of tumour cells means that this method satisfies the requirements of objective quantification for oncological diagnosis.

DNA, Neoplasm↗
PubMed1997

A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces.

The determination of total protein is often a key step for the quantitative analysis of various parameters in tissue and general biochemical research. The classical protocols are restricted to a few compatible buffers, and protocols for the determination of protein in solutions containing protein agglomerates or of protein immobilized on solid surfaces are not available. In such cases, quantification may be complicated. Here, we describe a simple sensitive method for protein quantification circumventing all these restrictions. Proteins in solution or suspension in any buffer are spotted onto cellulose acetate, dried, and stained with Amido Black. After washing off the excess dye, bound Amido Black is solubilized in an acidic solution and determined photometrically. Tissue slices (fixed or native), adherent cell cultures, or Western blots can also be stained and their protein content determined irrespective of the supporting material. A micro-version of the protocol for proteins in solution allows large numbers of samples to be evaluated at a time in microtitration plates and requires only 1-2 microl per sample. A linear concentration dependency (r2=0.950-0.999) was obtained for all samples in all cases investigated. The method presented here permits the exact determination of soluble protein in a large variety of buffers, of insoluble or immobilized protein present on a wide variety of supports, and even of whole cells or tissue slices.

Amido Black↗
PubMed1997

Quantification and genotyping of serum HCV-RNA in patients with chronic hepatitis C undergoing interferon treatment.

Quantification of serum HCV-RNA and HCV genotyping was studied in 27 patients with chronic hepatitis C undergoing interferon treatment. Pretreatment serum HCV-RNA levels were quantified using competitive RT-PCR and compared to a quantitative RT-PCR assay based on co-amplification of HCV-RNA with a synthetic RNA standard. HCV genotyping was performed using a line probe reversed hybridisation assay or direct solid-phase sequencing. This study shows the feasibility of performing HCV-RNA quantification. RT-PCR based on co-amplification HCV-RNA titer less than 6 x 10(4) genome equivalents/ml serum did correlate with a complete sustained response to alpha interferon in chronic hepatitis C. HCV genotype 1b was alpha predominantly associated with a high non-responder rate. Future prospective trials will be required to evaluate quantitative HCV-RNA levels and HCV genotyping as response predicting parameters for interferon-treatment.

Adult↗
PubMed2007

Detection and quantification of hepatitis B virus DNA by SYBR green real-time polymerase chain reaction.

A single-round real-time polymerase chain reaction (PCR) assay based on SYBR green dye technology for the detection and quantification of hepatitis B virus (HBV) DNA in serum was evaluated and compared with a qualitative nested PCR and the Cobas Amplicor HBV Monitor assay (Roche Molecular Diagnostics, Milan, Italy). The performance of the real-time PCR assay was evaluated in a routine clinical laboratory setting with a total of 212 clinical specimens. The sensitivity of the real-time PCR corresponded to 31 IU/ml (70 copies/ml), and comparison with the qualitative nested PCR showed significant concordance for 94% of samples. The linear curve over 7 log units, spanning 10(3)-10(9) IU/ml (2.28 x 10(3) to 2.28 x 10(9) copies/ml), was observed in the quantitative determination. The interexperimental variability coefficient of the assay ranged from 0.22 to 0.39 and the intraexperimental variability coefficient from 0.24 to 0.41. By excluding values outside of the dynamic ranges of both tests, the HBV Monitor and the real-time PCR gave an agreement within +/-1 log unit for 90% of samples, while those for the remaining 10% were found to be above 1 log unit but less than 1.5 log units. When the results inside and outside the dynamic range of the HBV Monitor were examined, 90% of the results were in agreement. In conclusion, the real-time PCR based on SYBR green technology proved suitable for routine diagnostic purposes, showing good sensitivity, high specificity, high reproducibility, and good linearity over a broad dynamic range of quantification.

Benzothiazoles↗
PubMed2005

Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by Real-time PCR assay.

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella-specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense-specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.

Animals↗
PubMedDate not supplied

Intraoperative quantification using finger force for involutional blepharoptosis without postoperative lagophthalmos.

PURPOSE: To report intraoperative quantification using finger force for involutional blepharoptosis, which helps in the prevention of postoperative lagophthalmos. METHODS: We carried out levator resection on 20 involutional blepharoptic eyelids. Fissure height was examined intraoperatively to evaluate the extent of resection. If a patient presented more than 3 mm of lagophthalmos in voluntary eyelid closure but could fully open the eye, we forcibly closed the eyelid, using a finger, after voluntary eyelid closure. If more than 3 mm of lagophthalmos was still observed after forced eyelid closure, we corrected eyelid tension until lagophthalmos became less than 2 mm. RESULTS: Six of the ten patients (20 eyelids) presented with full eyelid opening but more than 3 mm of lagophthalmos in voluntary eyelid closure. After the upper eyelids were forcibly lowered, all six eyelids showed less than 2 mm of lagophthalmos. There were no cases of lagophthalmos 1 month postoperatively. CONCLUSIONS: Additional finger force makes precise quantification of blepharoptosis surgery possible and prevents postoperative lagophthalmos.

Aged↗
PubMed2006

Quantification of S-adenosyl methionine in microbial cell extracts.

A sensitive method for quantification of S-adenosyl methionine (SAM) in microbial cell extracts was developed and applied to Corynebacterium glutamicum. The method is based on SAM being completely hydrolyzed into (18)O-homoserine when extracted in boiling H(2) (18)O and thus can be clearly distinguished by GC-MS analysis from naturally labeled homoserine present in the cell extract. Additional quantification of the total homoserine pool, representing both SAM and homoserine, via HPLC allows separate determination of both metabolites.

Corynebacterium glutamicum↗