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PubMed2000

Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport.

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.

3T3 Cells↗

PubMed2005

Quantification of neurocognitive changes before, during, and after hyperbaric oxygen therapy in a case of fetal alcohol syndrome.

Fetal alcohol syndrome (FAS) is the most common nonhereditary cause of mental retardation, with deficits in general intellectual functioning, learning, memory, attention, and problem-solving. Presented here is the first case in which measured neurocognitive abilities were determined before, during, and after hyperbaric oxygen therapy in a case of FAS involving a teenage male patient. Memory, reaction time, and visual motor speed assessments were compared. After 40 hyperbaric treatments with 100% oxygen at 1.5 atmospheres absolute, the patient's performance in 6 of 6 categories of the computer-administered test battery improved. Word composite (verbal) scores improved from 55% to 73%, memory composite (visual) scores improved from 38% to 55%, reaction time composites improved from 1.03 to 0.53 seconds, impulse control composite scores improved from 8 to 5, and visual motor speed scores improved from 18.6 to 19.03. The patient's subjective symptoms diminished 94%. Six months after these treatments, the patient's verbal memory was maintained at 73% without any other interventions; impulsivity continued to improve, whereas other indices did not. Thirty-three additional treatments continued to improve test performance, with verbal memory at 95%, visual memory at 57%, and a 100% reduction of subjective symptoms. This patient, with 15-year-matured FAS, benefited from a short course of low-pressure hyperbaric oxygen therapy, sustained durable cognitive improvements, and continued to exhibit improvement with another short course of treatments.

Adolescent↗

PubMed1993

Ontogeny of T-maze behavioral lateralization in rats.

Behavioral lateralization has been reported in both humans and animals. In humans, lateralization can be detected in neonates and increases to adult levels during postnatal development. Recently we reported lateralization of head and tail movements in neonatal rats. However, the postnatal ontogeny of lateralization in animals has not been previously studied. This work presents a study of rat behavioral lateralization in the T-maze test during postnatal development (from day 30 to day 60). A decrease was found in absolute (percent preferred-side choices) and population (right-left arm choices) laterality between day 30 and day 45 of postnatal life. The lateralization degree remained unchanged between days 45 and 60. Because behavioral alternation increases from day 30 to day 45, the present data suggest that animal lateralization of behavior is a phenomenon that remains throughout the subject's life span, but whose behavioral quantification could be concealed by the ontogenic increases of other phenomena such us behavioral alternation. This hypothesis could explain the high level of lateralization in neonatal rats, the low level of rat lateralization during adulthood, and the increases in lateralization induced by stress.

Aging↗

PubMed1985

An improved technique for extracting catecholamines from body fluids.

This paper describes a technique for selectively extracting catecholamines from body fluids prior to quantification by high-performance liquid chromatography with electrochemical detection. The technique is a two-stage process, the first stage involves the extraction of cations from the sample whilst the second stage is a liquid-liquid extraction involving the complexation of the cationic catecholamines with diphenylborate. This technique provides a very specific extraction procedure which results in chromatograms with no interfering compounds, and gives absolute recoveries of 70-80% for noradrenaline (NA), adrenaline (A) and dihydroxybenzylamine (internal standard), with similar relative recoveries of the 3 compounds. The intra-assay coefficients of variation for the measurement of catecholamines in venous plasma taken from resting subjects, are 8-9% for both NA and A, whilst the inter-assay values are 8% for NA and 20% for A.

Aluminum Oxide↗

PubMed2006

Simultaneous determination of mandelic acid enantiomers and phenylglyoxylic acid in urine by high-performance liquid chromatography with precolumn derivatization.

A reversed-phase HPLC method for the simultaneous quantitative determination of mandelic acid enantiomers (MA) and phenylglyoxylic acid (PGA) in urine is described. MA and PGA were extracted with ethyl acetate from urine at acidic pH and derivatized with S-(-)-1-(1-naphthyl) ethylamine. A ZORBAX SB-C(18) column (250 mm x 4.6mm i.d., 5 microm, Agilent, USA) was used with a mobile phase composed of methanol-10 mmol/L phosphate buffer [pH 2.5 (65:35, v/v)] at a flow-rate of 0.8 ml/min. Detection was set at UV wavelength of 254 nm. The mean absolute recoveries were 94.2%, 91.9%, 92.5% and 86.3% for S-MA, R-MA, PGA and salicylic acid (I.S.), respectively. The intra- and inter-day precisions determined at three different concentrations ranged from 2.8% to 4.8%, 0.7% to 7.7% and 1.3% to 6.8%, respectively. The lower limits of detection for MA enantiomers and PGA in urine were 1 microg/ml and the lower limits of quantification were 5 microg/ml (R.S.D.<10%, n=5). The method has been applied to determine the urinary excretion of MA enantiomers and PGA from Sprague-Dawley rats after orally administered with styrene.

Animals↗

PubMed1998

Volume-referent bone turnover estimated from the interlabel area fraction after sequential labeling.

In the compact bony otic capsule remodeling is low, and bone remodeling units are distributed centrifugally in relation to inner ear tissues and spaces. Fluorochrome-labeled bone remodeling units are scarce, abortive, and tortuous with no uniform direction of movement. This study presents a method for the estimation of volume-referent bone turnover based on measurements of the fractional area between labels after sequential labeling with osteofluorochromes. The applicability of this method is tested against a classical quantification method in undecalcified cortical specimens from the canine humerus, where both methods can be used. The estimate of bone turnover derived from the new sequential labeling in eight dogs is 7.4% (SEM 2.1%) per year and the classic estimate derived from calculations of the formative osteonal area and the formative period yields 6.9% (SEM 2.1%) per year. Agreement is sufficient to justify future measurements of absolute bone turnover in sequentially labeled perilabyrinthine bone.

Animals↗

PubMed1997

Right ventricular volumes overestimate left ventricular preload in critically ill patients.

BACKGROUND: Studies have shown right ventricular end-diastolic volume (RVEDV) to be a more accurate estimate of left ventricular preload than pulmonary artery wedge pressure. We prospectively evaluated the ability of RVEDV to predict left ventricular end-diastolic volume (LVEDV) in critically ill patients. METHODS: Thirty critically ill patients in the surgical intensive care unit underwent concurrent measurement of RVEDV and LVEDV. RVEDV was measured using a residual fraction Swan-Ganz catheter (RF Swan). LVEDV was measured using transesophageal echocardiography with acoustic quantification. Intracardiac, intra-abdominal, and ventilatory pressures were also measured. RESULTS: RVEDV as measured by the RF Swan was significantly larger (by a factor of 2) than LVEDV (p < 0.0001 analysis of variance). However, the RVEDV and LVEDV were strongly correlated (r = 0.71, p < 0.0001, Pearson's correlation). CONCLUSIONS: RVEDV from the RF Swan markedly overestimated left ventricular preload. If RVEDV is used as an absolute value for determining preload, patients may be underresuscitated. Transesophageal echocardiography in conjunction with RF Swan can be used to more accurately determine preload and cardiac performance than RF Swan alone in critically ill patients.

Analysis of Variance↗

PubMed2001

Repeatability and validity of a fluorimetric HPLC method in the quantification of yessotoxin in blue mussels (Mytilus edulis) related to the mouse bioassay.

Repeatability and validity of a fluorimetric HPLC method in quantification of yessotoxin (YTX) in mussels related to the mouse bioassay was studied. Blue mussels (Mytilus edulis) from the Sognefjord, Norway were sampled from March to November, 1997, and October to December, 1998. A total of 75 samples were analysed for YTX by HPLC using 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3, 4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3, 5-dione (DMEQ-TAD) as a fluorimetric derivatization agent. Among these, 28 of the samples were analysed by HPLC in duplicate. All samples were analysed by the mouse bioassay using both chloroform and ether in the final step of extraction. The duplicate measurements using HPLC was found equal and the method repeatable (p<0.05). The absolute difference between the two measurements was found to increase with increasing level of measurements. This significant positive correlation (p<0.05) was mainly due to concentrations of YTX higher than 200 microg/100g mussel meat. However the precision of the results obtained was not found to be less in the upper level than in the lower level. Based on the internal correlation analysis including the mouse bioassay and the HPLC method a cut-off value of < or =10 microg YTX/5 g digestive gland was found preferable. The mouse bioassay of ether extracts often failed to detect high levels of YTX, and as demonstrated by the low kappa-values, the agreement between the mouse bioassay of ether extracts and the HPLC method was very weak. The HPLC method was found to give repeatable results and thereby found to be reliable. Consequently, the HPLC method seems to the method of choice for detection and quantification of YTX in mussels when compared with the mouse bioassay.

Animals↗

PubMed1983

Measurement of leucine enkephalin in caudate nucleus tissue with fast atom bombardment-collision activated dissociation-linked field scanning mass spectrometry.

The endogenous amount of the opioid pentapeptide leucine enkephalin was measured in a canine caudate nucleus tissue extract using mass spectral analytical methods which retain absolute molecular specificity. Fast atom bombardment mass spectrometry generation of the protonated molecular ion of leucine enkephalin followed by collision activated dissociation produced amino acid sequence-determining ions. These amino acid sequence-determining ions were analyzed by a linked field (B/E) scan. One amino acid sequence-determining ion was selected to measure endogenous leucine enkephalin. This novel measurement mode offers optimal molecular specificity for quantification of an endogenous amount (451 pmol g-1 tissue) of leucine enkephalin in a biologic tissue extract of canine caudate nucleus.

Animals↗

PubMed2001

A reliable method of determining wound healing rate.

Several wound healing rate measures have been introduced with the main goal of enabling quantification of the effects of various therapeutic modalities on the healing of open wounds. Different definitions of wound healing rate render comparison of clinical results difficult. The goal of the present study was to propose a measure of wound healing rate that is independent of initial wound extent and to present a method of wound healing rate prediction. Comparisons were made of wound healing rate defined as absolute area healed per day, percentage of initial area healed per day and advance of the wound margin towards the wound centre per day. Analysis was performed on 300 wound cases. A disadvantage of wound healing measures that either use absolute area healed per day or percentage of initial area healed per day is their very limited use for comparing healing rates of wounds with different initial sizes. This disadvantage was overcome by incorporating a wound perimeter; thus obtaining a measure of the advance of the wound margin towards the wound centre. A definition of healing rate expressed as the greatest average wound margin distance from the wound centre divided by the time to complete wound closure is proposed. Because not all wounds are closed in the observation period, the time to complete wound closure has to be predicted. A method of wound healing rate prediction is presented based on a delayed exponential model the parameters of which are obtained from at least five weekly wound area measurements. Paired t-tests between actual time needed to complete wound closure and the predicted time resulted in p = 0.062 after four, 0.484 after five and 0.900 after six weeks of observation.

Chronic Disease↗

PubMedDate not supplied

Comparison of post-stress ejection fraction and relative left ventricular volumes by automatic analysis of gated myocardial perfusion single-photon emission computed tomography acquired in the supine and prone positions.

BACKGROUND: We have previously described an automatic method for measuring left ventricular ejection fraction (LVEF) for myocardial perfusion single-photon emission computed tomography (SPECT). The repeatability of this method has not been previously described. METHODS AND RESULTS: This study compares LVEF and relative end-systolic and end-diastolic volumes assessed from myocardial perfusion SPECT by our automatic method in 180 consecutive patients undergoing gated myocardial perfusion SPECT with injection of 99mTc-labeled sestamibi in whom the acquisitions were performed sequentially in supine and prone positions. The algorithm operated completely automatically in the prone and supine positions in 178 of the 180 patients. Very high correlations were observed for LVEF (r = 0.93), relative left ventricular end-systolic volume (r = 0.98), and relative left ventricular end-diastolic volume (r = 0.97). The mean paired absolute difference between LVEFs in the prone and supine position was 3.8+/-3.2, for left ventricular end-systolic volume was 4.9+/-4.8 ml, and for left ventricular end-diastolic volume was 7.4+/-6.7 ml. When patients were classified by the extent and severity of stress perfusion defect, there was no significant difference in repeatability for the measurements in any category. CONCLUSIONS: Our algorithm for automatic quantification of LVEF and relative end-systolic and end-diastolic volumes from gated 99mTc sestamibi myocardial perfusion SPECT is repeatable. When performed in the prone position, values of ejection fractions and ventricular volumes are essentially identical to those obtained in the supine position.

Adult↗

PubMed2001

Molecular quantification and mapping of lymph-node micrometastases in cervical cancer.

BACKGROUND: A proportion of patients with cancer and lymph nodes negative on histology will develop recurrence. Reverse-transcriptase PCR (RT-PCR) is a highly sensitive method for detection of lymph-node micrometastases, but accurate quantitative assessment has been difficult. METHODS: We studied primary tumours and 156 lymph nodes from 32 patients with cervical cancer (stage IA2, IB1, and IB2) and 32 lymph nodes from nine patients with benign disease. A fully quantitative, real-time RT-PCR assay was used to document absolute copy numbers of the epithelial marker cytokeratin 19. Primers and probe were designed not to amplify either of the two cytokeratin 19 pseudogenes. FINDINGS: All primary tumours and histologically involved lymph nodes (six) had more than 106 copies of cytokeratin 19 mRNA per microg total RNA. Expression of cytokeratin 19 (up to 1.1 x 10(5) copies per microg RNA) was detected in 66 (44%) of 150 histologically uninvolved lymph nodes, and in nodes from 16 of 32 patients with cervical cancer. 15 of these 16 patients with evidence of micrometastases had the highest cytokeratin 19 transcription level in a first lymph-node drainage station (three obturator, six internal, and six external iliac node). Transcription of cytokeratin 19 was found at a low level in just one of 32 lymph nodes obtained from nine patients with benign disease. Median copy number of cytokeratin 19 transcription was significantly higher (>10(3) copies) in association with adverse prognostic features. INTERPRETATION: The results suggest that about 50% of early-stage cervical cancers shed tumour cells to the pelvic lymph nodes. The amount of cytokeratin 19 expression was related to clinicopathological features. Further studies are required to document the clinical implications of molecular micrometastases.

Adult↗

PubMed2003

In vivo quantification of optical contrast agent dynamics in rat tumors by use of diffuse optical spectroscopy with magnetic resonance imaging coregistration.

We present a study of the dynamics of optical contrast agents indocyanine green (ICG) and methylene blue (MB) in an adenocarcinoma rat tumor model. Measurements are conducted with a combined frequency-domain and steady-state optical technique that facilitates rapid measurement of tissue absorption in the 650-1000-nm spectral region. Tumors were also imaged by use of contrast-enhanced magnetic resonance imaging (MRI) and coregistered with the location of the optical probe. The absolute concentrations of contrast agent, oxyhemoglobin, deoxyhemoglobin, and water are measured simultaneously each second for approximately 10 min. The differing tissue uptake kinetics of ICG and MB in these late-stage tumors arise from differences in their effective molecular weights. ICG, because of its binding to plasma proteins, behaves as a macromolecular contrast agent with a low vascular permeability. A compartmental model describing ICG dynamics is used to quantify physiologic parameters related to capillary permeability. In contrast, MB behaves as a small-molecular-weight contrast agent that leaks rapidly from the vasculature into the extravascular, extracellular space, and is sensitive to blood flow and the arterial input function.

Adenocarcinoma↗

PubMed1999

Quantification of ovine and bovine calpain I, calpain II, and calpastatin mRNA by ribonuclease protection assay.

We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.

Animals↗

PubMed1983

Radionuclide analysis of ejection time, peak ejection rate, and time to peak ejection rate: response to supine bicycle exercise in normal subjects and in patients with coronary heart disease.

Using equilibrium radionuclide angiography, we evaluated the ejection time (ET), peak ejection rate (PER), and time to peak ejection rate (TTp) at test and during supine bicycle exercise in 39 subjects, divided into three groups: group 1 = 13 normal subjects; group 2 = 10 patients with a previous infarction (MI); and group 3 = 16 patients with coronary disease without a previous MI. Normal subjects had greater ejection fractions and PERs than the other two groups at rest or peak exercise (p less than 0.05). PER was no more useful than ejection fraction in identifying cardiac dysfunction at either rest or exercise. The time of its occurrence varied with the group studied, and was slightly but significantly later in systole in groups 2 and 3 when compared to normals (p less than 0.05), though substantial overlap between groups occurred. During exercise, absolute ET shortened in all groups, but actually increased as a function of the R-R interval. The time to peak ejection rate (normalized for the R-R interval) was greater in the noninfarct group (group 3) patients (p less than 0.05) when compared to the group 1 or group 2 individuals at peak exercise. In conclusion, equilibrium radionuclide angiography is a useful technique for the quantification and characterization of events during systole, and is capable of providing information on the timing of events during ejection. Tardokinesis, or the delay of ventricular ejection, is not seen in the response of global indices of left ventricular function to exercise stress. While global early systolic indexes may not detect regional dyssynchrony, their timing during stress may occasionally aid in discerning the presence of cardiac dysfunction.

Adult↗

PubMed1994

Measurement of lactate in acutely ischemic rat kidneys using magnetic resonance spectroscopy.

RATIONALE AND OBJECTIVES: Quantification of lactate in the kidney by 1H magnetic resonance spectroscopy (MRS) is a difficult task because of the presence of large amounts of peri-renal fat. When an editing scheme is used to detect lactate that filters out all resonances except lactate, there is no suitable metabolite to serve as an internal standard. In this study, the authors evaluate the potential of MRS to measure the absolute lactate concentration in rat kidneys during acute ischemia using MRS. MATERIALS AND METHODS: The authors propose a method based on a double resonance lactate editing scheme used in combination with the fully relaxed water peak as an internal standard. Experiments were performed on the left kidney rendered ischemic in eight rats. RESULTS: Renal lactate concentrations measured by MRS were compared with values derived from chemical analysis. The mean (+/- standard deviation) renal lactate concentrations measured by MRS and determined chemically were 12 +/- 1.2 umol/g, and 12.94 +/- 1.07 umol/g wet weight, respectively. The coefficient of variation for paired observations was 2.96%, indicating excellent agreement between the two methods used for measuring lactate. DISCUSSION: The study results demonstrate that it is possible to assess the lactate concentration in a rat model of ischemic kidney with MRS and suggest that the total lactate pool is detectable by this method under these experimental conditions.

Animals↗

PubMed1979

Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.

Isolated rat livers were perfused for four hours in a recirculating system containing washed rat erythrocytes. Biologically screened radioiodinated rat high density lipoproteins (1.090 < d < 1.21 g/ml) were added to the perfusate with different amounts of whole serum to supply unlabeled rat high density lipoproteins. The protein moiety of the lipoprotein contained more than 95% of the radioiodine. The fraction of apolipoprotein mass degraded during the perfusion was quantified by the linear increment of non-protein-bound radioiodine in the perfusate, corrected for the increment observed during recirculation of the perfusate in the absence of a liver. The small amount of (131)I secreted into bile was added to calculate the fractional catabolic rate. The fractional catabolic rate ranged from 0.22 to 0.63% per hour in 12 experiments and was inversely related to the size of the perfusate pool of high density apolipoprotein. The absolute catabolic rate of high density apolipoprotein (fractional catabolic rate x pool size) in three livers in which the concentration of rat HDL in the perfusate approximated that in intact rats was 69.5 +/- 10.4 micro g hr(-1) (mean +/- SD). The rate of disappearance of cholesteryl esters of rat high density lipoproteins (labeled biologically by injecting donor rats with [5-(3)H]mevalonic acid) from the liver perfusate did not exceed that of the apoprotein component. These rates were compared with catabolic rates for rat high density lipoproteins in intact rats. Fractional catabolic rate in vivo, obtained by multicompartmental analysis of the disappearance curve of (131)I-high density apolipoprotein from blood plasma, was 11.9 +/- 1.3% hr(-1) (mean +/- SD). Total catabolic rate in vivo (fractional catabolic rate x intravascular pool of high density apolipoprotein) was 986 +/- 145 micro g hr(-1) (mean +/- SD). The results suggest that only a small fraction of high density lipoproteins in blood plasma of rats is degraded directly by the liver.-Sigurdsson, G., S-P. Noel, and R. J. Havel. Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.

Animals↗

PubMed1981

Quantitative mass spectrometry.

The basic principle in quantitative analysis by mass spectrometry is the measurement of a signal representative of the mass of the analyte relative to a known amount of an internal mass standard. To ensure that the signal is representative of the particular substance to be measured and not a result of some other substance in the biological matrix, a range of analytical methods have been employed. Combination of mass spectrometry with chromatographic (gas or liquid) separations and selective chemical derivatization frequently allows quantification of substances at the ppm and ppb level. Below the ppb level, new problems arise due to mass spectrometric sensitivity and chromatographic separation resolution with resulting debate regarding the specificity and significance of quantitative data. Successful high sensitivity (ppt) plasma analysis of the pineal hormone melatonin is described and critically compared with conflicting analytical data. A second area of application of quantitative mass spectrometry is the establishment of absolute or definitive reference measurements which require rigorous error minimization. Conflicting results published by two laboratories regarding methods for the definitive measurement of serum cholesterol are compared.

Cholesterol↗