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Results for “Absolute quantification”

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At least 19 recordsLinked to original sources

PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗

PubMed2025

Absolute quantification of the living skin microbiome overcomes relic-DNA bias and reveals specific patterns across volunteers.

BACKGROUND: As the first line of defense against external pathogens, the skin and its resident microbiota are responsible for protection and eubiosis. Innovations in DNA sequencing have significantly increased our knowledge of the skin microbiome. However, current characterizations do not discriminate between DNA from live cells and remnant DNA from dead organisms (relic DNA), resulting in a combined readout of all microorganisms that were and are currently present on the skin rather than the actual living population of the microbiome. Additionally, most methods lack the capability for absolute quantification of the microbial load on the skin, complicating the extrapolation of clinically relevant information. RESULTS: Here, we integrated relic-DNA depletion with shotgun metagenomics and bacterial load determination to quantify live bacterial cell abundances across different skin sites. Though we discovered up to 90% of microbial DNA from the skin to be relic DNA, we saw no significant effect of this on the relative abundances of taxa determined by shotgun sequencing. Relic-DNA depletion prior to sequencing strengthened underlying patterns between microbiomes across volunteers and reduced intraindividual similarity. We determined the absolute abundance and the fraction of population alive for several common skin taxa across body sites and found taxa-specific differential abundance of live bacteria across regions to be different from estimates generated by total DNA (live + dead) sequencing. CONCLUSIONS: Our results reveal the significant bias relic DNA has on the quantification of low biomass samples like the skin. The reduced intraindividual similarity across samples following relic-DNA depletion highlights the bias introduced by traditional (total DNA) sequencing in diversity comparisons across samples. The divergent levels of cell viability measured across different skin sites, along with the inconsistencies in taxa differential abundance determined by total vs live cell DNA sequencing, suggest an important hypothesis for certain sites being susceptible to pathogen infection. Overall, our study demonstrates a characterization of the skin microbiome that overcomes relic-DNA bias to provide a baseline for live microbiota that will further improve mechanistic studies of infection, disease progression, and the design of therapies for the skin. Video Abstract.

Humans↗

PubMedDate not supplied

Direct absolute quantification of metabolites in the human brain with in vivo localized proton spectroscopy.

The absolute concentrations of the three major brain metabolites observable by in vivo proton spectroscopy--N-acetylaspartate(NAA), creatine and phosphocreatine (Cr and PCr) and choline (Cho)--have been measured at four standardized localizations in 34 healthy volunteers by in vivo localized proton spectroscopy using an external reference sample. The results show that the concentration of Cr and PCr as observed by in vivo MRS (5-6 mmol/L) is lower than that measured by other methods. The results are concordant with the hypothesis, that the Cr and PCr resonance as observed by proton spectroscopy is due mainly to PCr, whereas Cr remains invisible by being attached to a larger molecule. It is also demonstrated, that Cr and PCr is higher in the cerebellum than in the cerebrum, whereas NAA remains constant within the margin of error (8-9 mmol/L).

Aspartic Acid↗

PubMedDate not supplied

Absolute quantification of 31P liver metabolites in rat using an external reference and a surface spoiling magnetic field gradient.

A simple method for measuring absolute concentrations of 31P metabolites in rat liver using an external reference is described. It neutralizes systematic errors due to conductive losses by calibration of the matching capacitor and selects liver signals via an electrically driven, surface spoiling magnetic field gradient. The technique avoids the chemical shift problems associated with linear field gradient localization methods at 4.7 T and allows combination with the double standard method for absolute metabolite concentration determination. Application of the method to the in vivo measurement of the absolute concentrations of ATP and P(i) in eight rat livers yields results that are in good agreement with literature values. Absolute phosphomonoester concentrations were also obtained, but no literature data were available.

Adenosine Triphosphate↗

PubMed1991

Absolute quantification of 31P muscle metabolites using NMRS with an internal standard and a high-Q, double-tuned coil.

31P-nuclear magnetic resonance spectroscopy (NMRS) is used to determine absolute concentrations of 31P metabolites in rat muscle. A technique exploiting tissue water 1H as internal concentration reference and a highly sensitive, double-tuned coil is described and evaluated experimentally. On KH2PO4 solutions of known concentration and varying coil loading it is shown to allow neutralization of systematic errors due to conductive losses, which normally range up to 20% or more. In vivo application in the determination of the absolute concentrations of ATP, PCr and Pi in eight rat thighs yields results that are in good agreement with literature values.

Adenosine Triphosphate↗

PubMed1992

Concurrent quantification of tissue metabolism and blood flow via 2H/31P NMR in vivo. I. Assessment of absolute metabolite quantification.

In a series of three papers, we demonstrate and validate an approach for concurrent absolute quantification in situ of blood flow and energy metabolism with a modification of the NMR method for absolute concentration determination put forth by Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] and later expanded upon by Tofts and Wray. In this first paper of the series, we briefly review the theoretical basis for the concentration measurement and present, for the first time, a successful paired validation of metabolite quantification via 31P surface-coil NMR through corroborative in vitro enzymatic assays. The paired radiolabeled microsphere validation of blood flow measurement via 2H surface-coil NMR employing D2O as a freely diffusible tracer and the concurrent determination of blood flow and energy metabolism in a septic rat model are presented in the accompanying second and third paper to complete the series. In this article a classical RF tank circuit is employed to describe the effect of conductive sample loading on the NMR receiver by considering its apparent series resistance. It is shown in an easily visualized generalizable manner that the effect of sample loading on the observed NMR signal intensity can be accounted for quantitatively by monitoring changes in 90 degrees pulse width at constant power at a fixed reference point, i.e., Ssample = Sphantom (PW90phantom/PW90sample). In a series of paired experiments the absolute concentrations of high energy phosphates obtained from resting rat leg muscle (n = 4) in situ (NMR) and in vitro (enzymatic) were determined as follows: [PCr]NMR = 17.2 +/- 0.8 SD, [PCr]enzymatic = 17.3 +/- 2 SD, [ATP]NMR = 5.1 +/- 0.8 SD, [ATP]enzymatic = 5.0 +/- 0.2 SD mmol/kg tissue wet wt. Results of these two independent methods of concentration determination were not statistically different (P = 0.94 and P = 0.74 respectively) and serve to rigorously validate the Thulborn approach for absolute quantification of phosphorous metabolites in situ via NMR. Furthermore, these results strongly suggest that ATP and PCr in resting rat leg muscle under normal physiologic conditions are 100% NMR visible. The free cytosolic [ADP]NMR was estimated from the creatine kinase reaction equilibrium expression to be 0.022 +/- 0.003 SD mmol/kg tissue wet wt.

Adenosine Triphosphate↗

PubMed1989

Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY).

The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.

Base Sequence↗

PubMed1992

A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates.

A method is described for the absolute quantification by polymerase chain reaction (PCR) of nucleic acids present in low abundance. The method entails the addition to the sample of competitor DNA molecules that share the same sequence as the amplified target (including primer recognition sites), except for a 20-bp insertion in the middle, which allows easy resolution by gel electrophoresis (competitive PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial rate of targets, rendering the reaction independent of the number of amplification cycles. An easy and reliable method for the construction and quantification of competitive templates obtained as recombinant PCR products was developed. The technique was used for the absolute quantification of human genomic DNA with primers from a single copy, subtelomeric region of chromosome 19.

Base Sequence↗

PubMed1991

Quantification of absolute myocardial perfusion at rest and during exercise with positron emission tomography after human cardiac transplantation.

The maximal exercise capacity of cardiac transplant recipients is reduced compared with that of normal subjects. To determine if this reduced exercise capacity is related to inadequate myocardial perfusion during exercise, myocardial perfusion was measured noninvasively with use of positron emission tomography and nitrogen (N)-13 ammonia. Twelve transplant recipients with no angiographic evidence of accelerated coronary atherosclerosis were studied. Serial N-13 ammonia imaging was performed at rest and during supine bicycle exercise. The results were compared with those from 10 normal volunteers with a low probability of having cardiac disease. A two-compartment kinetic model for estimating myocardial perfusion was applied to the data. Transplant recipients achieved a significant lower exercise work load than did the volunteers (42 +/- 16 vs. 128 +/- 22 W), but a higher venous lactate concentration (31.3 +/- 14.9 vs. 13.7 +/- 4.1 mg/100 ml). Despite the difference in exercise work load, there was no significant difference in the cardiac work achieved by transplant recipients and normal subjects as evidenced by similar rate-pressure products of 24,000 +/- 3,400 versus 21,300 +/- 2,800 betas/min per mm Hg, respectively. In addition, myocardial blood flow during exercise was not significantly different between the two groups (1.70 +/- 0.60 vs. 1.56 +/- 0.71 ml/min per g, respectively). This study demonstrates that the myocardial flow response to the physiologic stress of exercise is appropriate in transplant recipients and does not appear to explain the decreased exercise capacity in these patients.

Ammonia↗

PubMedDate not supplied

Assessing adequacy of collateral circulation during balloon test occlusion of the internal carotid artery with 99mTc-HMPAO SPECT.

A balloon test occlusion of the internal carotid artery was performed in 11 patients with internal carotid artery aneurysms. Tolerance by patients was assessed by a combination of clinical examination; angiography; electroencephalography; 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) single-photon emission computed tomography (SPECT) with relative quantification; and, in four patients, 99mTc-HMPAO SPECT with absolute quantification of cerebral blood flow. During test occlusion, angiography showed a patent circle of Willis in all patients. No patient developed new clinical findings or electroencephalographic changes. The SPECT studies of five patients in whom 99mTc-HMPAO was injected during test occlusion demonstrated changes from their baseline SPECT studies. The internal carotid artery was permanently occluded in two of these patients, neither of whom became symptomatic because of the occlusion. Three patients who demonstrated no changes between baseline and test occlusion SPECT studies underwent permanent occlusion of the internal carotid artery without incident, and postoperative SPECT images were unchanged from baseline. Our preliminary results suggest that patients who have no changes between baseline and test occlusion 99mTc-HMPAO SPECT studies should have adequate collateral circulation to sustain cerebral blood flow after occlusion of the internal carotid artery if no thromboembolic episodes occur. In contrast, a patient's tolerance of permanent occlusion cannot be consistently and reliably predicted if there are changes between baseline and test occlusion SPECT studies. In these patients, absolute quantitation of cerebral blood flow is important. Greater numbers of patients are required to confirm these initial results.

Carotid Artery, Internal↗

PubMed2024

Combining Data Independent Acquisition With Spike-In SILAC (DIA-SiS) Improves Proteome Coverage and Quantification.

Data-independent acquisition (DIA) is increasingly preferred over data-dependent acquisition due to its higher throughput and fewer missing values. Whereas data-dependent acquisition often uses stable isotope labeling to improve quantification, DIA mostly relies on label-free approaches. Efforts to integrate DIA with isotope labeling include chemical methods like mass differential tags for relative and absolute quantification and dimethyl labeling, which, while effective, complicate sample preparation. Stable isotope labeling by amino acids in cell culture (SILAC) achieves high labeling efficiency through the metabolic incorporation of heavy labels into proteins in vivo. However, the need for metabolic incorporation limits the direct use in clinical scenarios and certain high-throughput experiments. Spike-in SILAC (SiS) methods use an externally generated heavy sample as an internal reference, enabling SILAC-based quantification even for samples that cannot be directly labeled. Here, we combine DIA-SiS, leveraging the robust quantification of SILAC without the complexities associated with chemical labeling. We developed DIA-SiS and rigorously assessed its performance with mixed-species benchmark samples on bulk and single cell-like amount level. We demonstrate that DIA-SiS substantially improves proteome coverage and quantification compared to label-free approaches and reduces incorrectly quantified proteins. Additionally, DIA-SiS proves effective in analyzing proteins in low-input formalin-fixed paraffin-embedded tissue sections. DIA-SiS combines the precision of stable isotope-based quantification with the simplicity of label-free sample preparation, facilitating simple, accurate, and comprehensive proteome profiling.

Isotope Labeling↗

PubMed1986

Potential problems in serum protein electrophoresis.

Potential problems are described that one could encounter in carrying out an electrophoretic procedure including its ancillary phases of visualization (staining) and quantification (densitometry). Endpoint-like measurements for separated isoenzymes may provide artifactual kinetic values as well, because stain measurement is fixed at a single time whereas reagent blanking in the electrophoretic medium is substituted for the conventional serum initial absorbance readings of test-tube determinations. Truncation of separated electrophoretic zones or opacity of an electrophoretic anti-convection medium such as uncleared cellulose acetate may also interfere with absolute quantification procedures.

Albumins↗

PubMed1992

SPECT quantification: a simplified method of attenuation and scatter correction for cardiac imaging.

The quantitative and visual interpretation of SPECT myocardial perfusion images is limited by physical factors such as photon attenuation, Compton scatter, and finite resolution effects. A method of attenuation correction is described for use in nonhomogeneous media and applied to cardiac SPECT imaging. This method, termed multiplicative variable attenuation compensation (MVAC), uses tissue contours determined from segmentation of a transmission scan to assign a priori determined attenuation coefficients to different tissue regions of the transaxial images. An attenuation correction map is then constructed using a technique inspired by Chang's method that includes regionally dependent attenuation within the chest cavity and is applied after reconstruction by filtered backprojection. Scatter correction using the subtraction of a simultaneously acquired scatter window image enables the use of narrow beam attenuation coefficients. Experimental measurements to evaluate these methods were conducted for 201Tl and 99mTc SPECT using a homomorphic cardiac phantom. Finite resolution effects were included in the evaluation of results by computer simulation of the three-dimensional activity distribution. The correction methodology was shown to substantially improve both relative and absolute quantification of uniform and nonuniform regions of activity in the phantom's myocardial wall.

Heart↗

PubMed1992

Absolute quantitation of gallium-67 citrate accumulation in the lungs and its importance for the evaluation of disease activity in pulmonary sarcoidosis.

Our modification of a method for the absolute quantification of gallium-67 uptake in lungs with a scintillation camera and computer is described. The uptake of 67Ga in lungs, expressed in percentage of administered radioactivity, was determined by the transmission-emission method. We proved theoretically and experimentally that a 67Ga planar source could be replaced with a 57Co planar source. The performance of lung perfusion scans allows a more accurate delineation of the regions of interest on gallium scans. The method was applied to control subjects (n = 27) and to patients (n = 114) suffering from biopsy-proven pulmonary sarcoidosis (28 with inactive and 86 with active disease). The obtained results were compared with chest X-ray findings, the percentage of lymphocytes in the bronchoalveolar fluid (BAF-ly%), and serum angiotensin-converting enzyme (SACE) values. The method seems suitable for the assessment of disease activity in sarcoidosis. It is more accurate in detecting parenchymal involvement in lung sarcoidosis than the commonly used X-ray criteria. No correlation was found between 67Ga uptake and the BAF-ly% and SACE values.

Bronchoalveolar Lavage Fluid↗

PubMed2026

Comprehensive quality profiling and comparative metabolic characterization of seven dominant fresh-eating Chinese olive (Canarium album Lour.) cultivars in Southern China.

Fresh-eating Chinese olive (Canarium album Lour.) is a subtropical fruit endemic to southern China with considerable commercial value, yet systematic quality characterization of dominant cultivars remains scarce. This study established a multi-dimensional quality dataset for seven dominant cultivars from Fujian and Guangdong provinces, integrating nutritional components, soluble sugars, organic acids, mineral elements, volatile profiles, and non-targeted metabolomics. Significant cultivar-specific differences were observed across all evaluated dimensions: "Lingfeng" exhibited a sugar-dominant low-acid profile, whereas "Sanleng" showed elevated phenolic constituents accumulation. Volatile profiling identified terpenoid-based candidate discriminatory biomarkers, and metabolomic analysis revealed phenylpropanoid biosynthesis, tryptophan metabolism, and starch and sucrose metabolism as the most variable pathways. Correlations between untargeted profiling and targeted absolute quantification validated untargeted result reliability and revealed their complementarity in nutritional evaluation. These findings provide baseline data for FECO germplasm evaluation and targeted industrial utilization.

China↗

PubMed2026

Quantitative proteomics of molybdenum cofactor biosynthesis and utilization in Caenorhabditis elegans.

The molybdenum cofactor (Moco) is a chemically labile prosthetic group required by a small but essential set of metazoan enzymes, including sulfite oxidase, xanthine dehydrogenase, aldehyde oxidases, and the mitochondrial amidoxime reducing components (MARC). Disruption of Moco biosynthesis in humans causes Molybdenum Cofactor Deficiency (MoCD), a severe neonatal encephalopathy. Caenorhabditis elegans is unique among animals studied so far in that it can meet its Moco requirement through both endogenous biosynthesis and direct uptake of mature Moco from its bacterial diet. However, the organism-wide abundance of the Moco biosynthetic machinery and Moco-dependent enzymes, and their response to altered Moco supply, have remained unknown. Here, using data independent acquisition proteomics with histone anchored absolute quantification, we generated an organism wide quantitative atlas of Moco biosynthesis and utilization in C. elegans under standard and Moco limiting conditions. Components of the biosynthetic pathway showed a strikingly asymmetric abundance. The mitochondrial enzyme MOC-5, which catalyzes the first committed step in Moco biosynthesis, was present at only about 120 copies per genome equivalent, roughly fifty-fold below the downstream cytoplasmic biosynthetic machinery, which ranged from about 5,000 to 8,500 copies per genome equivalent, identifying MOC-5 as a stoichiometric bottleneck. On the utilization side, the MARC paralogs were the dominant Moco consumers, with MARC-1 exceeding 20,000 copies per genome equivalent. Loss of dietary or endogenous Moco selectively depleted the nonsulfurated clients SUOX-1 and MARC-1, whereas biosynthetic proteins remained unchanged, indicating that protein stability, rather than compensatory expression, is the main response to Moco limitation.

Caenorhabditis elegans↗

PubMed1991

[Modern nuclear medical diagnosis as applied pathophysiology. Basis and outlook].

In their capacity as "image-assisted functional diagnostics", methods of nuclear medicine link morphological patterns of radiology with clinical presentation. Based on pathophysiology they supply an insight into both global and regional parameters, present as basal values or as reserves. Both, single photon emission computed tomography (SPECT) or highly defined positron ECT (PET), enabled computer-assisted topographical overlay and thus an exact comparative evaluation of regional function versus morphology. In addition, PET gives access to a true physiological, absolute quantification employing process specific, carrier-free substrates. Novel tracers (markers of metabolism or perfusion, receptors or transmitter substances, monoclonal antibodies), an intraluminal transport model and the concept of buffer or working reserves aim at conducting early diagnosis, determination of disease severity and parameters that are relevant for therapy, on a level that is pathophysiologically oriented.

Brain Diseases↗

PubMed2025

StrainR2 accurately deconvolutes strain-level abundances in synthetic microbial communities.

MOTIVATION: Synthetic microbial communities offer an opportunity to conduct reductionist research in tractable model systems. However, deriving abundances of highly related strains within these communities is currently unreliable. 16S rRNA gene sequencing does not resolve abundance at the strain level and other methods such as quantitative polymerase chain reaction (qPCR) scale poorly and are resource prohibitive for complex communities. We present StrainR2, which utilizes shotgun metagenomic sequencing to provide high accuracy strain-level abundances for all members of a synthetic community, provided their genomes. RESULTS: Both in silico, and using sequencing data derived from gnotobiotic mice colonized with a synthetic fecal microbiota, StrainR2 resolves strain abundances with greater accuracy and efficiency than other tools utilizing shotgun metagenomic sequencing reads. We demonstrate that StrainR2's accuracy is comparable to that of qPCR on a subset of strains resolved using absolute quantification. AVAILABILITY AND IMPLEMENTATION: Software is available at GitHub and implemented in C, R, and Bash. Software is supported on Linux and MacOS, with packages available on Bioconda or as a Docker container. The source code at the time of publication is also available on figshare at the doi: 10.6084/m9.figshare.29420780.

Mice↗