Presence Of Whole Blood Research Articles

Whole blood assay of glucose-6-phosphate dehydrogenase: potential for simplified immunoassay

Enzyme labels combined with electrochemical product detection have considerable advantages in the development of non isotopic immunoassays. In particular, there is the possibility of signal detection in whole blood samples using cheap, robust and ultimately portable instrumentation. In this study the amperometric measurement, in whole blood, of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme commonly employed in homogeneous immunoassays, has been investigated using a simple membrane covered electrode. On comparison of several compounds for electrochemical mediation of the enzyme cofactor NADH, in whole blood, napthoquinone sulphonic acid has been found to be optimal giving lower background responses (<1 nA vs. Ag/AgCl) whilst mediating effectively NADH detection at low overpotentials (≤0.15 V vs. Ag/AgCl). Employing this mediator it is possible to measure electrochemically NADH and subsequently G6PDH at concentrations down to 0.6 U ml −1 in the presence of whole blood. This simplified G6PDH detection system will facilitate greatly the development of dehydrogenase based amperometric immunoassays in whole blood.

111Indium-F(ab)′ 2-NCA 102 monoclonal antibody: In vitro study of a specific agent for the detection of inflammatory foci

A new antigranulocyte antibody was evaluated in vitro for the detection of inflammatory foci in man. The specificity for polymorphonuclear cells (PMN) of NCA 102, an anti-NCA 95 monoclonal IgG1, was determined with immunohistochemical and cytofluorometrical tests. Its affinity, assessed by Scatchard analysis, was 1.1 × 10 9L/mol and the number of epitopes per granulocyte reached about 10 5. The biological properties of PMNs incubated with NCA 102 were not inhibited even when coupled with DTPA. A F(ab)′ 2 fragment was radiolabelled with 111Indium and incubated in the presence of whole blood. More than 65% radioactivity was selectively taken up by the PMN population. These findings indicated that NCA 102 antibody is suitable for sepsis detection.

Metabolites in Plasma after Oral Administration of Dopamine ProdrugTA-870 and Dopamine to Dogs and Metabolism of TA-870 in in vitro.

TA-870のドーパミンプロドラッグとしての有効性を明らかにするために，TA-870およびドーパミン塩酸塩(DA)をイヌに経口投与し，血漿中の代謝物濃度をHPLCを用いて測定した．また，TA-870の各種動物でのin vitroにおける代謝について検討した．活性体である遊離型DAの最高血漿中濃度はTA-870投与時(33．5mg，71μmol/kg)で252ng/ml，DA投与時(13.5mg，71μmol/kg)で29ng/mlであり，TA-870投与時の方が約9倍高かった．逆にDA抱合体およびDOPACの濃度はDA投与時に高かった．これらの結果から，TA-870のカテコールおよびアミノ基に結合した保護基は，初回通過代謝による不活性化を軽減させているものと思われ，ドーパミンプロドラッグとしての有効性が示唆された．TA-870の血漿中エステラーゼによる加水分解はラット>モルモット≈家兎>ヒト>イヌの順に速かった．また，本薬物は血漿中ではDAに変換しないが，ラットの全血小腸壁および肝臓ホモジネートではDAに変換した．DAへの変換速度ぱ肝臓>小腸壁>血液の順に速かった．

Selection and Evaluation of an Appropriate Model for Screening Adenosine Analogs for Chromosome Aberrations

As part of a preclinical safety assessment profile, the adenosine analog SC-46267, a candidate antihypertensive drug, was tested for potential genotoxicity in a series of in vitro and in vivo assays. Negative results were obtained in the Salmonella/microsome and CHO/HGPRT point mutation assays and in an acute mouse micronucleus test. However, in the presence of exogenous metabolism, positive results in the mouse lymphoma L5178Y assay and an in vitro chromosome aberration assay with purified rat lymphocytes indicated that the analog was a potential clastogen. Both adenosine and the analog were subsequently tested in rat lymphocytes using whole-blood versus purified lymphocyte cultures. High concentrations of adenosine and the adenosine analog induced chromosome aberrations in the purified lymphocytes. However, neither compound induced chromosome damage when cultured in the presence of whole blood, regardless of the presence or absence of the metabolic activation system. These results were similar to those reported in the literature that suggested that high concentrations of adenosine and other naturally occurring nucleotides can produce nonphysiological perturbations of nucleotide pools that are clastogenic in purified cultures of lymphocytes but not in whole-blood cultures. Our studies appear to support the hypothesis that adenosine deaminase, present at normal physiological levels in whole blood, is capable of detoxifying excessive levels of adenosine. Both compounds were also negative in a 10-day repeated dose mouse micronucleus test, which further ensures that the positive in vitro findings are not biologically significant. An in vitro clastogenicity assay using whole blood appears to be more appropriate than use of purified lymphocytes for screening adenosine analogs. The results from this series of experiments demonstrate the importance of considering culture conditions when designing in vitro test protocols. Furthermore, this study exemplifies the need to explore the mechanisms of activity as well as in vivo relevance of in vitro results.

Arginase Immobilization on Poly(hydroxyethyl acrylate) Matrix Beads

A study was made on the preparation and behaviour of polymer matrix im mobilized arginase, an enzyme involved in the urea cycle. Immobilization of purified calf liver arginase was obtained by a physical entrapment method based on the low temperature polymerization induced by radiation of a glass forming monomer mixed with an aqueous solution of the enzyme. Enzymatic activity was found to be retained in an acceptable range (30-35%) in a bead shaped matrix only when the monomer was hydroxyethylacrylate (HEA) purified by alkaline extraction and a particular sample preparation procedure was followed. The poly(hydroxyethyl acrylate) (PHEA) immobilized arginase was characterized by evaluating its chemical and enzymological properties such as Km, activation energy, stability to proteolytic enzymes, and activity de pendence on pH. Performance runs of continuous biocatalytic conversion of ar ginine to ornithine were carried out utilizing a reactor consisting of a column with immobilized arginase beads. After weeks of operation the substrate con version remained almost constant, indicating a practically unaltered enzy matic activity. PHEA immobilized arginase beads also retained their activity for days either in the presence of whole blood or when implanted intraperi toneally or subcutaneously into rats. No significant inflammation or other adverse reactions were observed after one week of residence of such implants. This seems promising for lowering the arginine blood level in specific diseases.

Degradation of atrial natriuretic factor in the rat.

The biologically active circulating form of atrial natriuretic factor (ANF) in the rat is the 28-amino-acid peptide ANF-(Ser-99-Tyr-126). Degradation of this peptide in vivo as well as in vitro, in whole blood, in plasma and by the isolated mesenteric artery was investigated. Studies in vivo in the rat demonstrated that the elimination and degradation of ANF was extremely fast: within 3 min more than 95% of the injected immunoreactive material was eliminated from circulation. The production of a short C-terminal peptide was detected on injection of 125I-ANF-(Ser-99-Tyr-126) into the rat. This peptide increased proportionately with incubation time. Experiments in vitro in the presence of whole blood or plasma did not cause any major destruction of ANF even after incubation for 60 min. After this prolonged incubation in plasma, ANF-(Ser-99-Tyr-126) was partially converted into ANF-(Ser-103-Tyr-126), a less potent peptide. Isolated mesenteric-artery preparation appeared to degrade ANF in a manner very similar to the system in vivo. These results suggest that degradation of ANF may occur either after internalization in the vascular cells or by a membrane-bound enzyme in the vasculature.

Cytotoxic effect of unilamellar detergent dialysis liposomes on Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro

Liposomes from egg yolk lecithin and egg yolk lecithin/ganglioside are cytotoxic for Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro. The trypanocidal effect is influenced by the liposome age and concentration. This effect is diminished in the presence of whole blood in vitro and could not be observed in vivo. Freeze-fractured parasite membrane showed an intramembranous particle aggregation after incubation with liposomes. Liposomes from egg yolk lecithin kill trypanosomes more rapidly than do liposomes from egg yolk lecithin/cholesterol.

SODIUM NITROPRUSSIDE AND CYANIDE RELEASE: REASONS FOR RE-APPRAISAL

The standard method for estimating cyanide liberated from sodium nitroprusside (SNP) has been colorimetric. Using a cyanide ion-selective electrode technique, it was found that SNP remained unchanged in the presence of whole blood, plasma and washed erythrocytes, no cyanide being detected. Previously reported releases of cyanide from SNP in vivo and in vitro are probably inaccurate. Values obtained could have arisen not from reaction with blood, but from photo-decomposition during the lengthy analysis required by the colorimetric method or during infusion, if the solution was left uncovered. Because of the supposed cyanide hazard, low doses of SNP have been recommended; it may now be possible to revise these.

A Chemical Investigation of the Shroud of Turin

Michrochemical testing of materials recovered on “sticky” tape samples taken from the Shroud of Turin was undertaken. The Shroud is a linen cloth, bearing the image of what appears to be a crucified man with the classical stigmata of Christ's crucifixion. The presence of whole blood was established by detection of heme derivatives, bile pigments, and proteins. Although iron in several forms is found over the whole cloth its distribution is shown to be accounted for by natural processes rather than as an added pigment. There is no chemical evidence for the application of any pigments, stains, or dyes on the cloth to produce the image found thereon. The chemical differences between image and non-image areas of the cloth indicate that the image was produced by some dehydrative oxidative process of the cellulose structure of the linen to yield a conjugated carbonyl group as the chromophore. However, a detailed mechanism for the production of this image, accounting for all of its properties, remains undetermined.

Cholesterol content of small unilamellar liposomes controls phospholipid loss to high density lipoproteins in the presence of serum

[ 1,2] requires control of the extent of drug retention by its carrier in the biological milieu. Thus, although in many instances drugs must be transported quantita- tively to where they are needed,in others their gradual release may be preferable, especially when membrane barriers prevent liposomes from reaching target sites [ 11. However, work on the in vivo fate of a number of liposome-entrapped drugs [2,3] indicates that, after intravenous injection, there is an almost imme- diate release of drug in the blood circulation, The rate at which this occurs is much higher than that expected from solute diffusion through intact bilayers [4,5] and it has been attributed, at least in part, to the loss of the structural integrity of liposomes as a result of phospholipid removal by high density lipoproteins [6-81. We have shown [4,.5,9] that in the blood of injected animals or in vitro in the presence of whole blood, plasma or serum, stability (in terms of bilayer permeability to solutes) of small multilamellar [4,9] or small unilamellar [S] liposomes can be controlled by adjusting their cholesterol content. Greatest liposomal stability with total retention of entrapped solutes both in vivo and in vitro was achieved by using small unilamellar liposomes composed of equimolar amounts of phospholipid and cholesterol [.5]. Here we show that cholesterol stabilises liposomes in the presence of serum by reducing the loss of their phos- pholipid component to high density lipoproteins. 2.

Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro.

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4 degrees C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4 degrees C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.

Stability of liposomes in vivo and in vitro is promoted by their cholesterol content and the presence of blood cells.

Abstract The stability of liposomes containing 6-carboxyfluorescein or β-fructofuranosidase was measured in the blood of injected animals and also in vitro in the presence of whole blood or serum. Stability, monitored by measuring changes in the permeability of liposomes to 6-carboxyfluorescein and sucrose, was found to depend on the cholesterol content of liposomes and it was promoted by the presence of blood cells.

Rapid [formula omitted] sulfoxidation of chlorpromazine by human blood: Inhibition by an endogenous plasma protein factor

Sulfoxidation of chlorpromazine is a major metabolic pathway in humans and is clinically significant insofar as chlorpromazine-sulfoxide is thought to be pharmacologically inactive. Sulfoxidizing capacities of hepatic and extrahepatic tissues were examined in vitro . Liver, lung, and gut, but not brain, were found to have differential activities (liver>lung>gut) which were NADPH dependent and had characteristics of enzymatic reactions. NADPH independent sulfoxidation of chlorpromazine is seen in the presence of whole blood. This reaction reaches completion in less than 5 sec. and is considerably more active than that seen with other tissues; the total sulfoxidizing capacity of whole blood can be attributed to hemoglobin. Plasma contains a protein-like factor that actively inhibits blood-catalyzed chlorpromazine sulfoxidation. This factor has a molecular weight of 72,000±6000 daltons. The possible roles of this protein in regulating phenothiazine metabolism are discussed.

Kinetic analysis of cholinesterases using a choline ester selective electrode

A new electrochemical method for the determination of the cholinesterase activity of blood-derived fractions is presented. The rate of change of concentration of several choline esters in the presence of whole blood, serum and erythrocytes can be continuously monitored by an acetylcholine-selective electrode. Acetylcholine, butyrylcholine and acetyl β-methylcholine were employed as substrates. The assay of 0.05 units of enzyme activity can be readily conducted. The reproducibility of the procedure compares favorably to established colorimetric and pH-stat techniques.

Plasma levels of viomycin in man after single intramuscular injection.

Twenty patients with pulmonary tuberculosis received single intramuscular injections of Viomycin sulfate, 10 receiving 1.0 Gm. and 10, 0.5 Gm. Blood samples were withdrawn immediately before the injection of the drug and at intervals of 1, 2, 4 and 8 hours thereafter. Specimens were collected in oxalate and centrifuged in the same tubes. Although sterile needles, tubes and syringes were used throughout, no other precaution to prevent contamination of the plasma was attempted. The agar diffusion assay procedure employed was essentially the same as that of Ehrlich, Iverson and Kohberger. Medium. Streptomycin assay-agar (beef extract, 1.5 Gm.; yeast extract, 3.0 Gm.; peptone, 6.0 Gm.; agar, 15 Gm.; and distilled water, 1000 ml.) adjusted to pH 7.4-7.6; autoclaved 15 minutes at 15-pounds pressure. Inoculum. Bacillus subtilis I strain, obtained from the Army Medical Department Research and Graduate School, Walter Reed Army Hospital, Washington, D. C., was used. A suspension was prepared by washing well-sporulated growth from nutrient agar slants, killing the vegetative cells by pasteurizing for 15 minutes at 80 C , and titrating the number of viable spores by plating serial dilutions in Streptomycin assay-agar. The growth of this organism is not appreciably affected by the presence of whole blood or serum. Preparation of the assay plates. Sufficient Bacillus subtilis spore suspension was added to the melted cooled agar (45 to 50 C.) to make a final concentration of 100,000 viable spores per milliliter of medium. Ten milliliters of this seeded agar was pipetted into sterile flat-bottom petri dishes and allowed to harden, and the plates were stored in the refrigerator (4 C.) until used. Stock solutions. Eight 2-fold serial dilutions of Viomycin sulfate in M/15 phosphate buffer pH 7.4 were prepared in concentrations ranging from 100 to 0.78 jug. per ml. Method of performing the assay. All determinations were made in triplicate. Using flamed forceps, filter paper disks (Schleicher and Schuell No. 740-E, 3^-inch diameter) were saturated in the stock solutions or in the supernatant

Metabolic relationship between acetoacetate and glucose.

Summary1. A metabolic relationship between glucose and acetoacetate has been shown to exist in the living body. 2. Increased concentration of acetoacetate (Na salt) distinctly inhibits normal glucose utilization when incubated at 37 ° C at a pH of 7.3 in the presence of whole blood (rabbit). 3. The condensation product of glucose and ethylacetoacetate has no effect on the normal ketonemic level and blood sugar tolerance curve of a rabbit and has neither any abnormal effect on the urine constituents of the treated animals.

The amperometric titration of plasma chloride and urine chloride

It has been shown that the method of Laitinen, Jennings, and Parks for the amperometric titration of chloride can be easily adapted to analyses of plasma (serum) and urine chlorides. For the procedure 1.0-ml. samples are diluted so that the mixture being titrated is 50% in acetone and 0.8 N in nitric acid. Silver nitrate, 0.0100 N, is used as the titrant, and the rotating platinum electrode is used as the indicator electrode. The method is simple, rapid, and as accurate as existing methods. When 0.1-ml. samples are analyzed, the accuracy is decreased a little over that obtained when 1.0-ml. samples are used. The method is not satisfactory for the analysis of chloride in the presence of whole blood or muscle tissue.