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PubMed2003

Effects of organic acids on adsorption of lead onto montmorillonite, goethite and humic acid.

Adsorption isotherms for Pb onto six soil components (quartz, feldspar, kaolinite, montmorillonite, goethite and humic acid) were studied. The influence of pH, EDTA and citric acid on the adsorption of Pb onto montmorillonite, goethite and humic acid were considered. Results indicate that the experimental data fit the Langmuir Adsorption Isotherm. The adsorption capacity for Pb at pH 6 was found to be in the order: humic acid (22.7 mg g(-1)) > goethite (11.04 mg g(-1)) > montmorillonite (10.4 mg g(-1)) > kaolinite (0.91 mg g(-1)) > feldspar (0.503 mg g(-1)) > quartz (0.148 mg g(-1)). Generally, the amount of Pb adsorbed onto montmorillonite, goethite and humic acid decreased with increasing concentrations of EDTA and citric acid and with increases in alkality. However, there were two exceptions: (1) addition of citric acid increased the amount of Pb adsorbed onto humic acid; and (2) the amount of Pb adsorbed onto goethite decreased with increasing pH in the presence of EDTA. Some mechanisms involved in the adsorption reactions are discussed.

Adsorption↗
PubMedDate not supplied

Redox cycling and lipid peroxidation: the central role of iron chelates.

Toxicities associated with redox cycling, including lipid peroxidation, are often attributed to the hydroxyl radical through a superoxide-driven, iron-catalyzed Haber-Weiss reaction. However, other cellular reducing agents more prevalent than superoxide, i.e., glutathione, ascorbate, cysteine and certain enzymes, can also reduce chelated iron and thereby initiate lipid peroxidation which is not inhibited by superoxide dismutase. The autoxidation of ferrous chelates yields partially reduced oxygen intermediates similar to those produced during the iron-catalyzed Haber-Weiss reaction. The mechanism of their formation, and the nature of the strong oxidant responsible for the initiation of lipid peroxidation are poorly understood, but are apparently influenced by the chelation of the iron. These conclusions stem from experiments which demonstrated variable amounts of inhibition of lipid peroxidation, dependent on the autoxidation of various ferrous chelates, by added superoxide dismutase or catalase. In addition, certain ferrous chelates are unable to initiate peroxidation of lipids in a liposomal configuration, yet capable of initiating peroxidation of lipids which were dispersed with a detergent. The results of these studies point out the need to isolate and identify the predominant physiological iron chelates for assessment of their ability to participate in redox related toxicities within the cell.

Adenosine Diphosphate↗
PubMed1980

Fate of the light chains in the course of proteolytic digestion of rabbit fast skeletal myosin.

During proteolytic digestion of myosin to prepare HMM or HMM-S-1 subfragments, myosin light chains are affected variously according to experimental conditions. In the presence of Ca2+ at low ionic strength trypsin rapidly degrades the DTNB light chain to a 18 K peptide. This new DTNB light chain is compared to a DTNB (17K) light chain obtained by chymotryptic digestion under similar conditions as shown here and in parallel studies. (Weeds and Pope (1977), J. Mol. Biol, 111, 129--157). Whereas the chymotryptic DTNB (17K) has lost its phosphorylation site (Ser-15), tryptic DTNB (18K) has lost only a strongly basic N-terminal peptide. A transitory (ca 14K) fragment is formed when digestion occurs in the presence of EDTA. A-1 light chain (20.7K) is cut to form a 20K species when myosin (of (CT)-HMM obtained ina high ionic strength medium) is digested with trypsin whether Me2+ is present or not. The new formed species has also lost its strongly basic N-terminal peptide and assumes a primary structure closer to that of A-2. Chymotrypsin was shown to have no effect on the A-1 light chain under the present conditions, whereas A-2 is not affected by chymotrypsin or trypsin under any of the conditions described in the present study.

Animals↗
PubMed2001

Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography.

Estimation of lipid peroxidation through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products separated by HPLC remains the method of choice to study the development of oxidative stress in blood plasma. In this report we describe the influence of citrate and EDTA anticoagulants used for blood collection on estimation of MDA concentrations using HPLC analysis of MDA-TBA adducts. We analyzed a group of 40 blood donors (21 men and 19 women), median age 27 years, range 19-48 years. The mean MDA concentration in citrate plasma was 1.43+/-0.51 micromol/l (range: 0.61-2.57 micromol/l) and in EDTA plasma 0.36+/-0.10 micromol/l (range: 0.13-0.63 micromol/l). There was a significant difference in MDA mean concentration that we attribute to different antioxidant properties of anticoagulants used for blood collection. Consistency in the choice of anticoagulant is clearly extremely important.

Adult↗
PubMed1987

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis.

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.

Alkaptonuria↗
PubMed1998

Determination of the ascorbate free radical concentration in mixtures of ascorbate and dehydroascorbate.

Ascorbate free radical is considered to be a substrate for a plasma membrane redox system of eukaryotic cells, and might be involved in stimulation of cell proliferation. It can be generated by transition metal-dependent oxidation of ascorbate or by an equilibrium reaction of ascorbate with dehydroascorbic acid. Using ESR spectroscopic measurements at pH 7.4, we show that when ascorbate and dehydroascorbic acid are mixed at concentrations lower than 2.5 mM, the ascorbate free radical concentration was determined by metal-dependent reactions and not by the equilibrium reaction. We conclude that, for studies under physiological conditions, the ascorbate free radical concentration cannot simply be calculated from the equilibrium constant and the ascorbate and dehydroascorbic acid concentration, but has to be determined experimentally.

Ascorbic Acid↗
PubMed1996

Effects on renal function of a continuous infusion of nifedipine during cardiopulmonary bypass.

OBJECTIVES: To evaluate the effects on renal function of continuously administered nifedipine during cardiopulmonary bypass (CPB) in patients undergoing cardiac surgery. DESIGN: The study was prospective, randomized, and controlled. SETTING: The study was performed in the Department of Anesthesia and intensive care unit of a regional hospital in Italy. PARTICIPANTS: Thirty-four patients scheduled for elective coronary artery surgery; all patients had preoperative renal and hemodynamic function within normal limits. INTERVENTIONS: The patients were randomly divided into two equal groups: nifedipine and control. Twenty patients were included in the study: 10 patients in the nifedipine group (group A) and 10 patients in the control group (group B). In group A, nifedipine was continuously administered during CPB at an infusion rate ranging from 0.24 to 0.59 micrograms/kg/min to maintain the mean systemic arterial pressure (MAP) between 60 and 70 mmHg. In group B, increases of MAP above 70 mmHg were treated with IV boluses of urapidil (5 mg). MEASUREMENTS AND MAIN RESULTS: Renal function was studied using creatinine clearance (CRCL), determined before, during, and after the operation, and the glomerular filtration rate (GFR) was measured the day before and after the operation by plasma and urine clearance of 51-chromium edetic acid (51Cr-EDTA). Hemodynamic monitoring was performed using a pulmonary artery catheter. In comparison with preoperative determinations, CRCL and GFR values increased significantly after CPB (p < 0.001) and after the operation (p < 0.01) in the patients treated with nifedipine, whereas the two parameters showed a small and not significant reduction at the same times in the control patients. Hemodynamic function was well maintained in all patients throughout the study. CONCLUSIONS: It is concluded that, besides the maintenance of adequate hemodynamics, a continuous infusion of nifedipine during CPB can be an additional therapeutic tool to protect renal function in cardiac surgical patients.

Aged↗
PubMed2001

Standardisation of platelet counting accuracy in blood banks by reference to an automated immunoplatelet procedure: comparative evaluation of Cell-Dyn CD4000 impedance and optical platelet counts.

Prophylactic and therapeutic platelet transfusions are increasingly used for patients with conditions associated with thrombocytopenia in order to prevent the development of potentially life threatening bleeding. These clinical strategies have led to a significant expansion in platelet unit manufacture, and this now represents a major resource and cost commitment for blood banks. As part of the manufacturing process, blood banks are required to implement control procedures, and the determination of platelet counts in particular is necessary to confirm that the quality of platelet unit production meets the standards defined by national or international guidelines. Apart from linearity analysis and comparisons of platelet counts given by different instruments, there has been no systematic standardisation of platelet counting methods in blood bank practice because to date there has been no suitable reference method for counting platelets in citrate anticoagulants. The recent introduction of an automated immunoplatelet procedure on the Cell-Dyn CD4000 provides a means of determining a true platelet count that is unaffected by changes induced either by storage or anticoagulant. The CD4000 in its routine configuration also provides simultaneous impedance and optical platelet counts and this study was therefore undertaken in order to compare all three different platelet counting methods in parallel with a representative series of platelet units. Platelet counts determined after sub-sampling of platelet units into EDTA vs plain non-anticoagulated tubes revealed no differences in impedance or immunoplatelet counts but generally lower optical counts when aliquoted into tubes that did not contain EDTA. This study therefore routinely used EDTA for platelet unit sub-samples. Comparative results of platelet counts for buffy coat platelet units (n = 36) aliquoted into EDTA indicated that the impedance count was higher than the reference immunoplatelet count by a mean factor of 1.25 while the optical count was lower by a mean factor of 0.87. The degree of impedance count overestimation was particularly consistent while the optical count underestimation was more variable. Linearity studies of 10 fresh platelet units showed no deviation in the range 0-2305 x 10(9) l(-1) for impedance and 0 to 1420 x 10(9) l(-1) for the optical counts, and the relative numerical relationships between impedance and optical counts were conserved throughout the range of dilutions tested. In the CD4000 optical analysis, blood samples anticoagulated with EDTA showed a distinctive elliptical population distribution that fell within the system thresholds. In contrast, the optical pattern observed for platelet units (in CPD) and ACD-anticoagulated venous blood showed a wider 90 degrees scatter with a population of platelet events above the upper parallel discriminator. As these were excluded from the optical count (but were still identified as platelets by the immunoplatelet method) it meant that the optical counts of samples in citrate-based anticoagulants were systematically lower than immunoplatelet counts. Platelet units (n = 15) analysed daily over a seven day period of storage revealed that the greatest decline in platelet counts was with the optical measurement while the most stable value was obtained by impedance analysis. The results of the immunoplatelet analysis further suggested a progressive increase in small platelets with increasing storage time. The use in this study of a standardised immunoplatelet reference method to examine the question of analyser suitability for determining platelet counts/yields of platelet units thus provided a number of important findings. An impedance platelet counting method is utilised by the great majority of haematology instruments in current use, and in common with the CD4000 analyser, a correction factor is employed to take account of RBC/platelet coincidence. This study found that when analysed samples such as platelet units were RBC-free, that an inappropriate correction factor was applied. Consequently, the CD4000 impedance platelet count will provide reliable platelet counts, irrespective of the day of platelet unit storage, when a factor of 1.25 is applied to the system-reported result. By comparison, optical methods are more likely to be affected by subtle morphological changes that may result from anticoagulants or progressive storage time. The method limitations documented by this study may well affect many other analysers and mean that the implementation of process control statistics related to platelet counts may be less reliable than previously assumed. It is suggested that standardisation could be much better achieved if there was some form of system cross-calibration that was referenced to an independent method such as an immunoplatelet assay. It is proposed that studies of this type should be extended to a wide assessment of platelet count accuracy of blood bank instruments in order to standardise data within national organisations. If consistent inter-instrument correction factors such as those documented here can be identified, it would considerably increase the relevance of determining platelet counts in production control processes.

Adult↗
PubMed1993

Ascorbate and dehydroascorbate measurements in aqueous solutions and plasma determined by gas chromatography-mass spectrometry.

The tert-butyldlmethylsllyl derivatives of ascorbic acid and dehydroascorbic acid were characterized by gas chromatography-mass spectrometry, and an isotope dilution assay for ascorbate and dehydroascorbate was developed using [13C6]ascorbic acid and [13C6]- and [6,6-2H2]dehydroascorbate. This assay was used to monitor ascorbic acid loss and the resulting rise of dehydroascorbic acid in aqueous solutions and plasma. Ascorbic acid was shown to rapidly decompose in aqueous solutions containing transition metal ions or when exposed to oxygen. Ethylenedlaminetetraacetic acid chelation did not prevent ascorbic acid degradation in aqueous solution, and ascorbate in ethylenedlaminetetraacetic acid chelated plasma was converted to dehydroascorbate on freezing. Gas chromatography-mass spectrometry appears to be a satisfactory method for determining the ascorbate and dehydroascorbate content of solutions including human blood plasma, whether or not there is ongoing oxidation of ascorbate in those solutions.

Ascorbic Acid↗
PubMedDate not supplied

Hepatobiliary delivery of polyaminopolycarboxylate chelates: synthesis and characterization of a cholic acid conjugate of EDTA and biodistribution and imaging studies with its indium-111 chelate.

A conjugate in which the steroid nucleus of cholic acid was linked to EDTA via an 11-atom spacer was obtained by reacting the succinimidyl ester of cholic acid with the amine formed by reaction of a benzyl isothiocyanate derivative of EDTA with N-(tert-butoxycarbonyl)ethylenediamine and subsequent deprotection. Potentiometric titration studies with model complexes showed that the EDTA moiety retained the ability to form 1:1 chelates of high thermodynamic stability, although formation constants were some 3-4 log K units lower for complexes of the conjugate than for the analogous chelates with underivatized EDTA. A complex formed between the cholic acid-EDTA conjugate and 111InIII was clearly rapidly into the liver when injected iv into mice, with subsequent excretion from the liver into the gastrointestinal tract being complete within 1 h of injection. Radioscintigraphic imaging studies conducted in a rabbit given the 111In-labeled conjugate also showed early liver uptake followed by rapid clearance from the liver into the intestine, with good visualization of the gallbladder in images obtained at 20-25 min postinjection. It is concluded that conjugation to cholic acid provides a useful means for the hepatobiliary delivery of EDTA chelates that otherwise exhibit predominantly extracellular distribution and renal clearance.

Animals↗
PubMed1993

Disruption of the cysteine-75 and zinc ion coordination is not sufficient to activate the precursor of human matrix metalloproteinase 3 (stromelysin 1).

Matrix metalloproteinases (MMPs) are activated in vitro from their precursors (proMMPs) by multiple means such as treatment with proteinases, mercurial compounds, chaotropic agents, sodium dodecyl sulfate, HOCl, and heat. The latency of proMMPs is stabilized by intramolecular interaction of the single cysteine residue in the conserved sequence PRCG(V/N)PD of the propeptide and the zinc atom at the active site. The activation of proMMP-1 (interstitial procollagenase) by multiple treatments has been explained by the "cysteine switch" model, in which the disruption of the Cys-Zn interaction is considered to be critical for activation [Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., & VanWart, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 364-368]. To further test this hypothesis we dissociated the Cys-Zn interaction by specifically modifying Cys-75 of proMMP-3 (prostromelysin 1) with iodoacetamide, (4-aminophenyl)mercuric acetate (APMA), or 5,5'-dithiobis(2-nitrobenzoate) and examined the expression of enzymic activity. The enzymic assays of the modified proMMP-3s against protein and synthetic substrates did not reveal any significant activity. The modified 57-kDa proMMP-3s were stable and did not show spontaneous activation. Activation of the modified proMMP-3s required further treatment with APMA or a proteinase and was accompanied by conversion of the proMMP-3 to a 45-kDa species. Circular dichroism studies of proMMP-3 treated with HgCl2 demonstrated time-dependent conformational changes in proMMP-3 prior to the expression of proteolytic activity and processing of the zymogen to lower molecular weight species. These results indicate that the disruption of the Cys-Zn coordination alone is not sufficient to activate proMMP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1982

Mechanism of light-induced reduction of biological redox centers by amino acids. A flash photolysis study of flavin photoreduction by ethylenediaminetetraacetate and nitrilotriacetate.

The mechanism of flavin photoreduction by the amino acids, EDTA, and nitrilotriacetate is shown to be due to light-induced charge separation, which is irreversible in the dark. The irreversibility originates from the decarboxylation of the amino acid radical. This fast process changes the redox properties of the radical and makes a further donation of an electron equivalent possible. In the case of EDTA the electron acceptor of the second electron is flavin, which was left unexcited by the flash or is formed by dismutation from the flavosemiquinone, generated in the primary one-electron transfer process. In contrast to this, a mechanism for the flavin photoreduction by nitrilotriacetate is proposed, in which the decarboxylated nitrilotriacetate radical adds to the flavosemiquinone to yield an alkylated flavohydroquinone. The latter decays to free reduced or oxidized flavin, depending on the position of addition at the flavin chromophore. The difference in reaction mechanism between the nitrite anion, EDTA, and nitrilotriacetate is discussed in terms of differences in molecular structure.

Acetates↗
PubMed1979

Subunit association and side-chain reactivities of bovine erythrocyte superoxide dismutase in denaturing solvents.

The copper- and zinc-containing superoxide dismutase of bovine erythrocytes retains its native molecular weight of 32 000 in 8.0 M urea for at least 72 h at 25 degrees C, as evidenced by sedimentation equilibrium analysis. Subsequent to prolonged exposure to urea, the dimeric enzyme could be dissociated by sodium dodecyl sulfate in the absence of reductants, indicating the absence of unnatural disulfide cross-links. The sulfhydryl group of cysteine-6 was unreactive toward 5,5'-dithiobis(2-nitrobenzoic acid) or bromoacetic acid in both neutral buffer and 8.0 M urea. The histidine residues of the enzyme were resistant to carboxymethylation in neutral buffer and 8.0 M urea. However, when the enzyme was exposed to bromoacetic acid in the presence of 6.0 M guanidinium chloride and 1 mM (ethylenedinitriol)tetraacetic acid (EDTA), both sulfhydryl and histidine alkylation were observed. Guanidinium chloride (6.0 M) increased the reactivity of the sulfhydryl group of cysteine-6 and allowed the oxidative formation of disulfide-bridged dimers. This was prevented by 1 mM EDTA. It follows that 8.0 M urea neither dissociates the native enzyme into subunits nor produces a conformation detectably different than that possessed under native conditions.

Animals↗
PubMed1977

On the location of the divalent metal binding sites and the light chain subunits of vertebrate myosin.

The divalent metal ion binding sites of skeletal myosin were investigated by electron paramagnetic resonance (EPR) spectroscopy using the paramagnetic (Mn(II) ion as a probe. Myosin possesses two high affinity sites (K less than 1 muM) for Mn(II), which are located on the 5,5'-dithiobis(2-nitrobenzoate) (DTNB) light chains. Mn(II) bound to the isolated DTNB light chain gives rise to an EPR spectrum similar to that of Mn(II) bound to myosin and this indicates that the metal binding site comprises ligands from the DTNB light chain alone. Myosin preparations in which the DTNB light chain content is reduced by treatment with 5,5'-dithiobis(2-nitrobenzoate) show a corresponding reduction in the stoichiometry of Mn(II) binding, but the stoichiometry is recovered on reassociation of the DTNB light chain. Chymotryptic digestion of myosin filaments in the presence of ethylenediaminetetraacetic acid yields subfragment 1, but digestion in the presence of divalent metal ions produces heavy meromyosin. Myosin with a depleted DTNB light chain content gives rise to subfragment 1 on proteolysis, even in the presence of divalent metal ions. It is proposed that saturation of the DTNB light chain site with divalent ions protects this subunit against proteolysis, which, in turn, inhibits the cleavage of the subfragment 1-subfragment 2 link. Either the DTNB light chain is located near the region of the link and sterically blocks chymotryptic attack, or it is bound to the subfragment 1 moiety and affects the conformation of the link region. When the product heavy meromyosin was examined by sodium dodecyl sulfate gel electrophoresis, an apparent anomaly arose in that there was no trace of the 19 000-dalton band corresponding to the DTNB light chain. This was resolved by following the time course of chymotryptic digestion of the myosin heavy chain, the DTNB light chain, and the divalent metal binding site. The 19 000-dalton DTNB light chain is rapidly degraded to a 17 000-dalton fragment which comigrates with the alkali 2 light chain. The divalent metal site remains intact, despite this degradation, and the 17 000 fragment continues to protect the subfragment 1-subfragment 2 link. In the absence of divalent metal ions, the 17 000-dalton fragment is further degraded and attack of the subfragment 1 link ensues. Mn(II) bound to cardiac myosin gives an EPR spectrum basically similar to that of skeletal myosin, suggesting that their 19 000-dalton light chains are analogous with respect to their divalent metal binding sites, despite their chemical differences. The potential of EPR spectroscopy for characterizing the metal binding sites of myosin from different sources and of intact muscle fibers is discussed.

Animals↗
PubMed1976

Sialic acid uptake by fibroblasts.

The existence of surface sialytransferases that use cytidine monophosphate (CMP)-sialic acid as substrate has been postulated in previous studies. This is based on the assumption that if whole, viable cells can catalyze the transfer of sialic acid from CMP-sialic acid to endogenous acceptors, then the transferases carrying out the reaction must be on the cell surface, provided that (1) CMP-sialic acid does not enter the cells, and (2) CMP-sialic acid does not break down outside the cells, yielding free sialic acid which then may enter the cells, in amounts large enough to explain the incorporation. We now report evidence showing that after incubation of intact NIL, BHK, and 3T3 fibroblasts with CMP-sialic acid, at least 78% of the sialic acid incorporated by these cells is the result of free sialic acid uptake. When cells growing in a monolayer were incubated with a mixture of CMP-[14C]sialic acid and [3H]CMP-sialic acid with a ratio of 3H/14C=0.60, this ratio was found to be markedly increased in whole cells. Chemical analyses of the radioactive species in the incubation medium showed that a considerable portion of the radiolabeled sugar nucleotide had broken down to cytidine, phosphoric acid, and sialic acid. Upon incubation of cells with doubly labeled sugar nucleotide in the presence of a large excess of both nonradiolabeled cytidine and sialic acid, the cells incorporated less than 6% of both isotopes. Incubation of cells with a mixture of CMP-[14C]sialic acid and [3H]sialic acid resulted in only 20-40% of the radioactivity within the cells being membrane bound, and 70-90% of this incorporation could be inhibited by the addition of 10 mM azide to the incubation medium. The possibility that a small fraction of the total incorporation of sialic acid by these cells is due to surface sialytransferases cannot be completely ruled out. The uptake of free sialic acid by these fibroblasts is concentration dependent and a portion of it is incorporated into glycoproteins and glycolipids. Considerable loss of cell integrity was observed when fibroblasts grown on plates were removed by (ethylenedinitrilo)-tetraacetic acid or trypsin and subsequently incubated in buffer, indicating that these preparations are not suitable for intact cell studies.

Azides↗