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PubMed2001

Use of EDTA samples for prothrombin time measurement in patients receiving oral anticoagulants.

BACKGROUND AND OBJECTIVES: Oral anticoagulant therapy is commonly called for in health care. Hitherto sampling for prothrombin time (PT) has been measured on blood collected into a coagulation tube and diluted in citrate solution. Blood samples anticoagulated with EDTA are used for hematologic tests and the same sample could also be available for PT. DESIGN AND METHODS: We studied 107 patients on oral anticoagulant therapy. Samples were taken by from both coagulation tubes (citrate) and EDTA tubes. The PT from both samples was measured with an ACL 7000 analyzer and reported in seconds and as an international normalized ratio (INR). The regression equation between citrate and EDTA samples was calculated in both units. We studied the clinical significance of INR results from both sample types and compared the effect of different combined thromboplastin reagents on the correlation equation between citrate and EDTA samples. RESULTS: The regression equation for PT by Owren's PT reagent from citrate (y) and EDTA (x) plasma was y = 1.11 x -0.24 INR, R(2)= 0.99. We observed no clinically significant difference between INR results from citrate and EDTA samples using the regression equation for INR calculation from EDTA samples. ISI depends on sample type (dilution, anticoagulant) and the difference is 0.117, 10%. We calculated ISI for EDTA samples and no clinically significant difference was seen between citrate and EDTA INR results. INTERPRETATION AND CONCLUSIONS: A good correlation was observed between INR results with citrate and EDTA samples from patients receiving oral anticoagulants using Owren's PT reagent with the same citrate calibration. Using the regression equation (INR or sec) for analysis of INR results from EDTA samples is clinically acceptable and offers the possibility of using EDTA samples for PT measurement with citrate calibrators. Reagent international sensitivity index (ISI) values for citrate and EDTA samples differ from each other. ISI determination for EDTA samples requires mathematical calculation of EDTA ISI as in the present study or EDTA-based ISI calibrators. The regression equation for INR from citrate and EDTA samples depended on the reagent used, not only on sample dilution or anticoagulant.

Administration, Oral↗
PubMed1979

Retinal detachment from hyperosmotic intravitreal injection.

Hyperosmotic solutions were injected into the rabbit vitreous to study their effects upon the retina. Injection of 0.05 ml of a 1000 mOsm solution caused rapid whitening of the posterior retina followed by the development of a large detachment and permanent retinal degeneration. The weakest solutions which produced ophthalmoscopically visible changes in the retina (after an injection of 0.05 ml) were near 500 mOsm. Sodium chloride, sodium aspartate, EDTA, mannitol, sucrose, and penicillin were effective at similar osmolarities. An osmotic load in the vitreous caused immediate loss of the c-wave of the electroretinogram (ERG), and a slower decline of the a- and b-waves. The reported intravitreal toxicity of some drugs may relate to osmotic rather than pharmacologic effects. Osmolarity should be accounted for in planning the amount and location of any vitreous injection.

Animals↗
PubMed1976

Latency of inosine-5'-diphosphatase in microsomes isolated from rat liver.

The latency of inosine-5'-diphosphatase has been studied in microsomes isolated from rat liver. The appearance of latent activity was the result of an increase in the Vmax of the enzyme. This was observed when assays were carried out in the presence of sodium deoxycholate, after microsomes were treated wtih phospholipase C, or at pH 10.3 and after microsomes were subjected to nitrogen cavitation. The apparent Km of inosine-5'-diphosphatase for IDP was unchanged when microsomes were treated with phospholipase C or at pH 10.3 after both these treatments approximately 85% of the enzyme remained bound to the membrane. In contrast, when microsomes were treated with phospholipase C or at pH 10.3 after both these treatments approximately 85% of the enzyme remained bound to the membrane. In contrast, when microsomes were treated with sodium deoxycholate or subjected to nitrogen cavitation, approximately 75% of the inosine-5'-diphosphatase activity was released from the membrane, and the apparent Km of the enzyme for IDP increased 4- and 2-fold, respectively. Microsomal cisternae were loaded with lead phosphate by incubation with glucose-6-P and Pb2+, and the release of this lead phosphate following the addition of EDTA to the medium was determined to estimate the permeability of the microsomal membrane. When microsomes were treated with sodium deoxycholate, phospholipase C, or at alkaline pH, the microsomal membrane became almost completely permeable to EDTA under conditions where there was little or no increase in the activity of inosine-5'-diphosphatase. Microsomes were treated at pH 10.3 and then adjusted slowly to pH 7.5. The activity of inosine-5'-diphosphatase decreased to the same activity observed in untreated preparations. The results seem of exclude the possibility that latent inosine-5'-diphosphatase activity is the result of an increased permeability of the membrane to IDP. They are, however, consistent with the presence of a noncompetitive inhibitor of the enzyme in the microsomal membrane.

Animals↗
PubMedDate not supplied

Comparison of the ammonium oxalate extraction of fertilizer for K2O, method 983.02, with the ammonium citrate/EDTA extraction, method 993.31.

The direct available method for phosphorus in fertilizers, Method 960.03, is an Official Method for extraction of both P2O5 and K2O. Because K2O is much more soluble and easier to extract than P2O5, the conditions of the extraction procedure are set for efficient extraction of P2O5, and the extraction of K2O is, in effect, a bonus. In 1993, a modified extraction procedure, using an extractant of ammonium citrate/ethylenediaminetetraacetic acid (EDTA) solution, was evaluated for P2O5 and then collaboratively studied. This now-modified extraction procedure for phosphorus is Official Method 993.31, which is used extensively as an alternative to Method 960.03 for phosphorus because it is less labor-intensive. Method 993.31 was also evaluated in our laboratory for K2O extraction, but it was never formally collaboratively studied for K2O. To demonstrate the suitability of Method 993.31 as an extraction procedure for K2O, as well as P2O5 in fertilizers, Method 993.31 extraction for K2O was compared with Method 983.02, the traditional ammonium oxalate extraction procedure for K2O. A wide range of fertilizer samples was extracted by using both procedures. The t-test for samples containing < 30% K2O had a t-value of 1.23 and a probability of 0.22, which indicates there was no significant difference in the results obtained by the 2 extraction methods. The t-test for samples containing > 30% K2O had a t-value of 0.30 and a probability of 0.77, also indicating no significant difference. It is recommended that Method 983.02 be modified to include the ammonium citrate/EDTA extraction procedure as an additional option for extraction of K2O from commercial fertilizers.

Citric Acid↗
PubMed1992

The influence of age on nephrotoxicity following chemotherapy in children.

Nephrotoxicity is an important adverse effect of chemotherapy in children. Renal function after treatment with either ifosfamide or cisplatinum in children aged 5 years or less ('younger children') was compared with that in those over 5 years ('older children'). Eighteen children (six younger, 12 older) given ifosfamide were studied after completion of chemotherapy, and 28 patients (16 younger, 12 older) were evaluated after cisplatinum. Glomerular filtration rate was measured from the plasma clearance of 51chromium-labelled edetic acid. Proximal tubular function was assessed by determination of plasma and urine calcium, phosphate, magnesium and glucose concentration; calculations of their fractional excretions, and of the renal threshold for phosphate; and measurement of urinary excretion of beta 2-microglobulin. Distal tubular function was evaluated by measurement of the early morning urine osmolality. Younger children had more severe proximal tubular toxicity than older children treated with ifosfamide, with significantly lower plasma phosphate concentrations and higher fractional excretions of glucose. However, there was no evidence of any such difference in glomerular or distal tubular damage after ifosfamide, and no difference in any aspect of renal function between younger and older children treated with cisplatinum. Increased proximal tubular toxicity after ifosfamide in younger children may have serious implications for future growth and development.

Age Factors↗
PubMed1992

Recognition and prevention of two cases of erroneous haemocytometry counts due to platelet and white blood cell aggregation. The use of acid citrate dextrose as an auxiliary anticoagulant.

Two cases of anticoagulant-induced platelet-white blood cell aggregation are described, which resulted in erroneous haemocytometry counts. Aggregation was avoided by the use of acid citrate dextrose (ACD) as an auxiliary anticoagulant, thus enabling quantification of platelets and white blood cells.

Anticoagulants↗
PubMed1992

Oxidative inactivation of carbamoyl phosphate synthetase (ammonia). Mechanism and sites of oxidation, degradation of the oxidized enzyme, and inactivation by glycerol, EDTA, and thiol protecting agents.

Acetylglutamate and ATP accelerate the oxidative inactivation of carbamoyl phosphate synthetase I by mixtures of Fe3+, ascorbate, and O2, but the mechanism of the inactivation differs with each ligand. In the presence of acetylglutamate, MgATP prevents, Mg2+, Mn2+, and catalase have no effect, and EDTA increases the inactivation, and the two phosphorylation steps of the enzyme reaction are lost simultaneously. The inactivation appears to be mediated by dehydroascorbate and is associated with the reversible oxidation of the highly reactive cysteines 1327 and 1337 and with oxidation of non-thiolic groups in the second 40-kDa domain (the enzyme consists of 4 domains of 40, 40, 60, and 20 kDa, from the amino terminus). The data are consistent with oxidation of groups at or near the site for ATPA (ATPA yields Pi; ATPB yields carbamoyl phosphate), and with the location of this site at the interphase between the second 40-kDa and the COOH-terminal domains. The oxidative inactivation promoted by ATP is inhibited by Mg2+, Mn2+, catalase, and EDTA, is not mediated by dehydroascorbate, and is not associated with oxidation of cysteines 1327 and 1337. Groups in the 60-kDa domain are oxidized. The phosphorylation step involving ATPB is lost preferentially, and the inactivation and the binding of ATPB exhibit the same dependency on the concentration of ATP. The results indicate that the oxidation is catalyzed by FeATP bound at the site for ATPB and support the binding of ATPB in the 60-kDa domain. We also demonstrate that mercaptoethanol, reducing impurities in glycerol, and dithioerythritol, in the presence of EDTA, replace ascorbate in the oxidative system. In addition, we study the influence of the oxidation on the degradation of the enzyme by rat liver lysosomes, mitochondria, and cytosol.

Adenosine Triphosphate↗
PubMed1991

Alpha 2-macroglobulin binds and inhibits activated protein C.

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine-Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed1991

[Pseudothrombocytopenia: incidence, causes and methods of detection].

A prospective study of 20,761 routine clinical blood specimens was conducted in order to evaluate incidence and causes of pseudothrombocytopenia. All specimens were drawn in outpatient regimen and were analyzed by using the Coulter STKR autoanalyzer. The incidence of pseudothrombocytopenia was 0.15%, values clearly lower than others previously reported in hospitalized patients (1.9%). Of these patients, 72% had EDTA-induced platelet clumping sometimes associated to sodium citrate and 28% had spurious low platelet counts due to large platelets. No other causes of pseudothrombocytopenia were appreciated. It is important to remark that 49% of low platelet counts detected by the Coulter STKR have been due to pseudothrombocytopenia, and this instrument showed normal platelet and leukocyte histogram in 10% of these cases. Although time consuming these results confirm the necessity of preparation and examination of all blood smears presenting thrombocytopenia by automated cell-counting despite normal histograms. This approach would avoid erroneous diagnosis and dangerous medical or surgical therapy. A correlation of pseudothrombocytopenia with age, sex or clinical diagnosis were not found.

Adolescent↗
PubMed1990

[The allosteric nature of substrate inhibition of rabbit skeletal muscle fructose-1,6-diphosphatase].

The mechanism of substrate inhibition of rabbit skeletal muscle fructose-1.6-bisphosphatase was examined. Analysis of substrate saturation curves obtained at different concentrations of Mg2+ revealed that the inhibiting effect of the substrate is manifested only within the complex with Mg2+, whereas the free form of the substrate causes no inhibition. Evidence for the allosteric nature of substrate inhibition was obtained by partial desensitization of the enzyme in the presence of salicylates. It was shown that fructose-1.6-bisphosphatase inhibition by the substrate obeys positively cooperative kinetics and is noncompetitive with respect to the substrate involved in the catalytic process. Studies on enzyme modification in the presence of DTNB and pyridoxal-5'-phosphate demonstrated that the inhibiting concentrations of the substrate are bound to the center which differs from the allosteric site for AMP. It is suggested that the antagonism of simultaneous action of AMP and high substrate concentrations may be due to the competition of the phosphate groups of these ligands for binding to the common lysine residue located in the overlapping region of two allosteric sites.

Adenosine Monophosphate↗
PubMed1990

A technique for microfilarial detection in preserved blood using nuclepore filters.

Nuclepore filtration is now the most widely used method of detecting microfilaremia, particularly if microfilariae are few in number. However, this system requires the blood sample to be processed promptly after collection. Using blood from Wuchereria bancrofti-infected patients, 3 solutions were tested to determine whether blood could be held for delayed processing. Of these, we identified 1, 2% formalin/10% Teepol, in which microfilaremic blood can be held for at least 9 mo without deterioration of microfilarial structure or decrease in microfilarial numbers. In addition, this mixture passed easily through a 5-microns filter at all times tested. Examination of more than 300 blood samples held in 2% formalin/10% Teepol showed that this solution can utilize the convenience and sensitivity of membrane filtration while eliminating the need to perform testing immediately after the blood is collected.

Animals↗