Blood osmolality in vitro: dependence on base addition, buffer value, and temperature

Blood osmolality (Osm) increases with PCO2 because of CO2 absorption. The influences of NaOH addition, equilibration temperature, and hemoglobin concentration on these respiratory changes of Osm were measured by freezing-point determination in true plasma. Addition of NaOH increases Osm by 2 mosmol X kg H2O-1 X mmol base-1 X l at constant PCO2 due to the osmotic effects of Na+ and produced bicarbonate. Respiratory compensation of the pH change further increases Osm. This contrasts to the respiratory compensation of the osmolar disturbance caused by fixed acid. Raising the equilibration temperature reduces Osm by 0.5 mosmol X kg H2O-1 X degrees C-1 at constant pH mainly caused by a lower absorption coefficient for CO2 and changed pK value for H2CO3. The slope of the linear regression lines between Osm and pH during CO2 equilibration increases with hemoglobin; the value of the quotient delta Osm/delta pH depends directly on the nonbicarbonate buffer value. The use of this quotient for the estimation of the mean nonbicarbonate buffer value of the whole body is suggested. The osmotic effects of therapeutic base infusion should be regarded with caution.

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Blood osmolality in vitro: dependence on PCO2, lactic acid concentration, and O2 saturation

Journal of Applied Physiology ◽

10.1152/jappl.1983.54.1.118 ◽

1983 ◽

Vol 54 (1) ◽

pp. 118-122 ◽

Cited By ~ 5

Author(s):

D. Boning ◽

N. Maassen

Keyword(s):

Lactic Acid ◽

Acid Concentration ◽

Freezing Point ◽

Co2 Absorption ◽

Lactic Acid Concentration ◽

Co2 Partial Pressure ◽

Constant Ph ◽

Acid Titration ◽

Point Determination

Changes of osmolality (Osm) were measured by freezing-point determination in true plasma of 10 healthy subjects. This was done after equilibration with CO2 (0.5–10.0%), after the addition of lactic acid (10 and 20 mmol/l), and after deoxygenation. The graph for the dependence of Osm on CO2 partial pressure (PCO2) in oxygenated blood resembles the classical CO2 absorption curve. The increase of Osm with PCO2 (approximately 0.2 mosmol . kg H2O-1 . Torr-1) is almost as great as the increase in dissolved CO2 plus bicarbonate (HCO-3). Addition of lactic acid shifts the curve upward by only 0.6 mosmol/mmol because of displacement of HCO-3. Deoxygenation has no significant effect at constant PCO2 despite an increase in [HCO-3]. This is probably due to the binding of 2,3-diphosphoglycerate to hemoglobin. It can be seen in the Osm-pH diagram that differences between CO2 and lactic acid titration largely disappear. For each lactic acid concentration there is a linear dependence corresponding to the linear [HCO-3]-pH relation in plasma. At constant pH, Osm increases after deoxygenation. The observed in vitro relation might explain part of the osmolality increase during physical exercise.

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Minimal contribution of cell-bound antibodies to the immunoscintigraphy of inflamed joints with 99mTc-anti-CD4 monoclonal antibodies

Nuklearmedizin ◽

10.1055/s-0038-1623888 ◽

2002 ◽

Vol 41 (03) ◽

pp. 129-134 ◽

Cited By ~ 4

Author(s):

A. Wolski ◽

E. Palombo-Kinne ◽

F. Wolf ◽

F. Emmrich ◽

W. Becker ◽

...

Keyword(s):

T Cells ◽

Monoclonal Antibodies ◽

Synovial Membrane ◽

In Vitro Release ◽

Peritoneal Macrophages ◽

Lymphoid Organs ◽

Whole Body ◽

Free Form ◽

Ankle Joints

Summary Aim: The cellular joint infiltrate in rheumatoid arthritis patients is rich in CD4-positive T-helper lymphocytes and macrophages, rendering anti-CD4 monoclonal antibodies (mAbs) suitable for specific immunoscintigraphy of human/ experimental arthritis. Following intravenous injection, however, mAbs are present both in the free form and bound to CD4-positive, circulating monocytes and T-cells. Thus, the present study aimed at analyzing the relative contribution of the free and the cell-bound component to the imaging of inflamed joints in experimental adjuvant arthritis (AA). Methods: AA rat peritoneal macrophages or lymph node T-cells were incubated in vitro with saturating amounts of 99mTc-anti-CD4 mAb (W3/25) and injected i.v. into rats with AA. Results: In vitro release of 99mTc-anti-CD4 mAb from the cells was limited (on average 1.57%/h for macrophages and 0.84%/h for T-cells). Following i.v. injection, whole body/joint scans and tissue measurements showed only negligible accumulation of radioactivity in inflamed ankle joints (tissue: 0.22 and 0.34% of the injected activity, respectively), whereas the radioactivity was concentrated in liver (tissue: 79% and 71%, respectively), kidney, and urinary bladder. Unlike macrophages, however, anti-CD4 mAb-coated T-cells significantly accumulated in lymphoid organs, the inflamed synovial membrane of the ankle joints, as well as in elbow and knee joints. Conclusion: While the overall contribution of cell-bound mAbs to the imaging of arthritic joints with anti-CD4 mAbs is minimal, differential accumulation of macrophages and T-cells in lymphoid organs and the inflamed synovial membrane indicates preferential migration patterns of these 2 cell populations in arthritic rats. Although only validated for 99mTc-anti-CD4 mAbs, extrapolation of the results to other anticellular mAbs with similar affinity for their antigen may be possible.

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A Simple and Inexpensive Clinical Whole Body Counter

Nuklearmedizin ◽

10.1055/s-0038-1624972 ◽

1976 ◽

Vol 15 (05) ◽

pp. 248-253

Author(s):

A. K. Basu ◽

S. K. Guha ◽

B. N. Tandon ◽

M. M. Gupta ◽

M. ML. Rehani

Keyword(s):

The Body ◽

Whole Body ◽

Vitro Assay ◽

Body Counter ◽

Optimum Parameters ◽

Whole Body Counter ◽

Single Detector ◽

Background Index ◽

Iodide Crystal

SummaryThe conventional radioisotope scanner has been used as a whole body counter. The background index of the system is 10.9 counts per minute per ml of sodium iodide crystal. The sensitivity and derived sensitivity parameters have been evaluated and found to be suitable for clinical studies. The optimum parameters for a single detector at two positions above the lying subject have been obtained. It has been found that for the case of 131I measurement it is possible to assay a source located at any point in the body with coefficient of variation less than 5%. To add to the versatility, a fixed geometry for in-vitro counting of large samples has been obtained. The retention values obtained by the whole body counter have been found to correlate with those obtained by in-vitro assay of urine and stool after intravenous administration of 51Cr-albumin.

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Evaluation of a New Preparation of Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649107 ◽

1973 ◽

Vol 30 (01) ◽

pp. 114-122

Author(s):

C.R.M Prentice ◽

K.M Rogers ◽

G.P McNicol

Keyword(s):

Body Weight ◽

Maintenance Therapy ◽

Initial Dose ◽

Fibrinolytic Activity ◽

Pharmacological Effect ◽

Whole Body ◽

Fibrinolytic Agent ◽

Thromboplastic Activity

SummaryThe pharmacological effect of a new preparation of urokinase (Leo) has been studied, both in vitro and in six patients suffering from thrombo-embolic disorders. It was a non-toxic, effective fibrinolytic agent if given in sufficient dosage. A regimen consisting of an initial dose of 7,200 ploug units per kg body weight, followed by hourly maintenance therapy with 3,600 ploug units per kg intravenously, gave satisfactory evidence of whole body fibrinolytic activity. The preparation had minor but insignificant thromboplastic activity both when assayed in the laboratory and when given to patients.

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Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

10.26434/chemrxiv.12235361 ◽

2020 ◽

Author(s):

Lucas S. Ryan ◽

Jeni Gerberich ◽

Uroob Haris ◽

ralph mason ◽

Alexander Lippert

Keyword(s):

Energy Transfer ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Whole Body ◽

Photon Flux ◽

Ph Homeostasis ◽

Ph Imaging ◽

Confounding Variables ◽

Significant Difference

Regulation of physiological pH is integral for proper whole-body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.

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Use of Toxicokinetics in Risk Assessment Based on In Vitro Data

Alternatives to Laboratory Animals ◽

10.1177/026119299302100209 ◽

1993 ◽

Vol 21 (2) ◽

pp. 173-180

Author(s):

Gunnar Johanson

Keyword(s):

Risk Assessment ◽

Whole Body ◽

External Exposure ◽

Target Dose ◽

Toxic Response ◽

Route Of Exposure ◽

Vitro Data ◽

In Vitro Data

This presentation addresses some aspects of the methodology, advantages and problems associated with toxicokinetic modelling based on in vitro data. By using toxicokinetic models, particularly physiologically-based ones, it is possible, in principle, to describe whole body toxicokinetics, target doses and toxic effects from in vitro data. Modelling can be divided into three major steps: 1) to relate external exposure (applied dose) of xenobiotic to target dose; 2) to establish the relationship between target dose and effect (in vitro data, e.g. metabolism in microsomes, partitioning in tissue homogenates, and toxicity in cell cultures, are useful in both steps); and 3) to relate external exposure to toxic effect by combining the first two steps. Extrapolations from in vitro to in vivo, between animal and man, and between high and low doses, can easily be carried out by toxicokinetic simulations. In addition, several factors that may affect the toxic response by changing the target dose, such as route of exposure and physical activity, can be studied. New insights concerning the processes involved in toxicity often emerge during the design, refinement and validation of the model. The modelling approach is illustrated by two examples: 1) the carcinogenicity of 1,3-butadiene; and 2) the haematotoxicity of 2-butoxyethanol. Toxicokinetic modelling is an important tool in toxicological risk assessment based on in vitro data. Many factors, some of which can, and should be, studied in vitro, are involved in the expression of toxicity. Successful modelling depends on the identification and quantification of these factors.

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Adipocyte PHLPP2 inhibition prevents obesity-induced fatty liver

Nature Communications ◽

10.1038/s41467-021-22106-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

KyeongJin Kim ◽

Jin Ku Kang ◽

Young Hoon Jung ◽

Sang Bae Lee ◽

Raffaela Rametta ◽

...

Keyword(s):

Fatty Liver ◽

Whole Body ◽

Acid Oxidation ◽

Chow Diet ◽

Peroxisome Proliferator ◽

Obese Mice ◽

Ph Domain ◽

Peroxisome Proliferator Activated Receptor

AbstractIncreased adiposity confers risk for systemic insulin resistance and type 2 diabetes (T2D), but mechanisms underlying this pathogenic inter-organ crosstalk are incompletely understood. We find PHLPP2 (PH domain and leucine rich repeat protein phosphatase 2), recently identified as the Akt Ser473 phosphatase, to be increased in adipocytes from obese mice. To identify the functional consequence of increased adipocyte PHLPP2 in obese mice, we generated adipocyte-specific PHLPP2 knockout (A-PHLPP2) mice. A-PHLPP2 mice show normal adiposity and glucose metabolism when fed a normal chow diet, but reduced adiposity and improved whole-body glucose tolerance as compared to Cre- controls with high-fat diet (HFD) feeding. Notably, HFD-fed A-PHLPP2 mice show increased HSL phosphorylation, leading to increased lipolysis in vitro and in vivo. Mobilized adipocyte fatty acids are oxidized, leading to increased peroxisome proliferator-activated receptor alpha (PPARα)-dependent adiponectin secretion, which in turn increases hepatic fatty acid oxidation to ameliorate obesity-induced fatty liver. Consistently, adipose PHLPP2 expression is negatively correlated with serum adiponectin levels in obese humans. Overall, these data implicate an adipocyte PHLPP2-HSL-PPARα signaling axis to regulate systemic glucose and lipid homeostasis, and suggest that excess adipocyte PHLPP2 explains decreased adiponectin secretion and downstream metabolic consequence in obesity.

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IMMU-31. QUANTITATIVE EVALUATION OF ROUTES OF ADMINISTRATION OF IMMUNOTHERAPEUTIC T CELLS TO ORTHOTOPIC GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.461 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii111-ii111

Author(s):

Lan Hoang-Minh ◽

Angelie Rivera-Rodriguez ◽

Fernanda Pohl-Guimarães ◽

Seth Currlin ◽

Christina Von Roemeling ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Ex Vivo ◽

Adoptive T Cell Therapy ◽

Whole Body ◽

T Cell Therapy ◽

Clinical Responses

Abstract SIGNIFICANCE Adoptive T cell therapy (ACT) has emerged as the most effective treatment against advanced malignant melanoma, eliciting remarkable objective clinical responses in up to 75% of patients with refractory metastatic disease, including within the central nervous system. Immunologic surrogate endpoints correlating with treatment outcome have been identified in these patients, with clinical responses being dependent on the migration of transferred T cells to sites of tumor growth. OBJECTIVE We investigated the biodistribution of intravenously or intraventricularly administered T cells in a murine model of glioblastoma at whole body, organ, and cellular levels. METHODS gp100-specific T cells were isolated from the spleens of pmel DsRed transgenic C57BL/6 mice and injected intravenously or intraventricularly, after in vitro expansion and activation, in murine KR158B-Luc-gp100 glioma-bearing mice. To determine transferred T cell spatial distribution, the brain, lymph nodes, heart, lungs, spleen, liver, and kidneys of mice were processed for 3D imaging using light-sheet and multiphoton imaging. ACT T cell quantification in various organs was performed ex vivo using flow cytometry, 2D optical imaging (IVIS), and magnetic particle imaging (MPI) after ferucarbotran nanoparticle transfection of T cells. T cell biodistribution was also assessed in vivo using MPI. RESULTS Following T cell intravenous injection, the spleen, liver, and lungs accounted for more than 90% of transferred T cells; the proportion of DsRed T cells in the brains was found to be very low, hovering below 1%. In contrast, most ACT T cells persisted in the tumor-bearing brains following intraventricular injections. ACT T cells mostly concentrated at the periphery of tumor masses and in proximity to blood vessels. CONCLUSIONS The success of ACT immunotherapy for brain tumors requires optimization of delivery route, dosing regimen, and enhancement of tumor-specific lymphocyte trafficking and effector functions to achieve maximal penetration and persistence at sites of invasive tumor growth.

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Maternal obesity during pregnancy leads to adipose tissue ER stress in mice via miR-126-mediated reduction in Lunapark

Diabetologia ◽

10.1007/s00125-020-05357-4 ◽

2021 ◽

Author(s):

Juliana de Almeida-Faria ◽

Daniella E. Duque-Guimarães ◽

Thomas P. Ong ◽

Lucas C. Pantaleão ◽

Asha A. Carpenter ◽

...

Keyword(s):

Adipose Tissue ◽

Glucose Tolerance ◽

Er Stress ◽

Maternal Obesity ◽

Luciferase Assay ◽

Whole Body ◽

Mrna Levels ◽

Protein Levels ◽

Novel Targets

Abstract Aims/hypothesis Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. Methods miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic–hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. Results The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. Conclusions/interpretation Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target. Graphical abstract

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Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism

Parasitology ◽

10.1017/s0031182000047740 ◽

1978 ◽

Vol 76 (2) ◽

pp. 159-176 ◽

Cited By ~ 56

Author(s):

W. E. Gutteridge ◽

B. Cover ◽

Maria Gaborak

Keyword(s):

Trypanosoma Cruzi ◽

Blood Cells ◽

Light And Electron Microscopy ◽

Deae Cellulose ◽

Whole Body ◽

Differential Centrifugation ◽

Γ Irradiation ◽

Hind Limb Muscle ◽

Radio Tracer

SummaryIsolation of blood and intracellular forms of Trypanosoma cruzi was made mainly from rats (90–110 g) which had received 580 rad of whole-body γ-irradiation not more than 24 h before subcutaneous inoculation with 107 trypomastigotes of the Sonya strain of T. cruzi. Unirradiated chinchillas (250–350 g) were, however, used for some experiments. Blood forms were isolated using a technique involving differential centrifugation to remove most of the erythrocytes and DEAE–cellulose chromatography to remove the remaining blood cells. Overall recoveries were usually in the range 30–70%. Parasites were mainly (approximately 98%) broad forms and were motile, metabolically active (as judged by respiratory and radio-tracer incorporation studies) and had lost none of their infectivity for mice. Intracellular forms were isolated from hind-limb muscle tissue. This was disrupted in an MSE tissue homogenizer and the homogenate incubated with DNase, collagenase and trypsin. Parasites, contaminated only by a few blood cells, were then obtained by differential centrifugation. For purer preparations, a terminal sucrose gradient step was used. Recoveries ranged between 40 and 70%. About 1–3% of the parasites isolated were epimastigotes and trypomastigotes; the remainder are probably best collectively termed ‘amastigotes’, though they were pointed and most had a short, free flagellum. They were undamaged as judged by light and electron microscopy and metabolically active as judged by respiratory and radio-tracer incorporation studies. However, the infectivity for mice of both these purified preparations and the initial cell homogenates could be accounted for by the epimastigotes and trypomastigotes present in them. Preliminary biochemical studies with isolated parasites have shown that blood, intracellular and culture forms of T. cruzi have a respiratory system which is in part sensitive to CN- and that all forms synthesize nucleic acids and proteins when incubated in vitro. There appears, however, to be a lack of DNA synthesis in blood stages, and thus it is not surprising that these forms do not divide.

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