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PubMed1992

Spectrophotometric determination of total proteins in blood plasma with p-benzoquinone.

1. In the present study we have documented the use of the reagent, p-benzoquinone (PBQ) for the spectrophotometric determination of total proteins in blood plasma. 2. Since the products of reaction are stable for several hours at room temperature after the 20-min boiling step, the time at which absorbance is measured is not a critical factor. 3. Common anticoagulants such as EDTA, citrate, or heparin do not interfere with the PBQ method at concentrations used in clinical laboratories. 4. The products of the reaction between PBQ and either plasma (specific absorbance 2.33 x 10(-3) +/- 0.20 x 10(-3) micrograms cm-2) or purified proteins (specific absorbance 2.61 x 10(-3) +/- 0.31 x 10(-3) micrograms cm-2) show an absorption band at 350 nm, which follows Beer's law, and therefore can be used for analytical purposes. 5. The PBQ method has a lower limit of detection (4 micrograms/ml) than that of the biuret method (45 micrograms/ml) for a final reaction mixture of 5.0 and 4.2 ml, respectively.

Animals↗
PubMed1991

Electrochemical detection of oligopeptides through the precolumn formation of biuret complexes.

The relatively slow kinetics of formation of the electroactive Cu(II)-peptide complexes from larger (greater than 6 amino acids) peptides requires relatively high temperature and long reaction times for a postcolumn reactor. The precolumn incubation of bradykinin, Tyr8-bradykinin and insulin A chain with biuret reagent for 20 min at 60 degrees C leads to the formation of biuret complexes which can be subjected to chromatography in acidic or basic eluents. These complexes are detected electrochemically with a sensitivity similar to the Cu(II)-(ala)3 complex (1 nC/pmol at 1.0 ml/min). The influence of the column-packing material on the electrochemical detector response of the Cu-peptide complexes has also been studied.

Biuret↗
PubMed1981

[Quantitative determination of total urinary protein utilizing the principle of Coomassie Brilliant Blue G250 binding to protein (author's transl)].

Coomassie Brilliant Blue G250 was used for the determination of total urinary protein and compared with the biuret-procedure. In the Coomassie Brilliant Blue G250-assay 0.10 ml urine are added to 5.0 ml Coomassie Brilliant Blue G250 reagent and the sample is read against the reagent blank. The Coomassie Brilliant Blue G250 method seems to be superior to the biuret-procedure because of better reproducibility, higher sensitivity and simpler handling. The upper limit or urinary protein excretion in 49 healthy subjects was 120 mg/24 h. To evaluate the clinical significance of the Coomassie Brilliant Blue G250 method the urinary protein concentration of 134 patients with metabolic, systemic and organ diseases was compared with the biuret-procedure. The regression line was y = 0.827 X + 8.713 mg/l with a correlation coefficient of r = 0.966. The type of proteinuria (mixed, glomerular, tubular) had no influence on the protein value measured with the Coomassie Brilliant Blue G250 method. However in selective proteinuria, like Bence-Jones protein excretion, there is no correlation between the two methods, because the concentration of Bence-Jones protein is underestimated. Lower protein values were also frequently obtained for patients with diabetes mellitus. Some specimens from patients with chronic renal failure treated by haemodialysis showed elevated values. In spite of these limitations the Coomassie Brilliant Blue G250 method might become the method of choice for the determination of total urinary protein.

Bence Jones Protein↗
PubMed1985

Ring cleavage and degradative pathway of cyanuric acid in bacteria.

The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.

Ammonia↗
PubMed1981

The formation of chromophores through amino acid thermolysis and their possible role as prebiotic photoreceptors.

The thermal polymerization of amino-acid mixtures was studied at various temperatures and reaction times with specific emphasis on the formation of fluorescent chromophores. The reaction conditions appeared to have a pronounced effect on the ratio of synthesized chromophores and biuret-positive material. During thermolysis of equimolar mixtures of lysine, alanine and glycine or lysine, aspartic acid and glycine small amounts of pteridines and flavines are formed, which are often covalently linked to the thermal oligomer. These heterocyclic compounds are likely formed by condensation reactions of the amino acid break-down and conversion products. Reaction schemes that describe the processes are proposed. The significance of these chromoproteinoids is discussed in respect to prebiotic redox reactions and photoinduced processes.

Amino Acids↗
PubMed1985

Synthetic polymers as drugs.

Biologically active compounds have been incorporated into synthetic polymers. Bithionol, a well-known and potent antibacterial agent, has been used as bisphenol monomers for polyesters, polyphosphates, and phosphonates; the bischloroformate of bithionol has been converted into copolycarbonates and polyurethanes. The copolycarbonates of bithionol, optimally designed polymer structures with poly(ethylene oxide) glycols as the other comonomer, have been hydrolyzed under physiological conditions at a degradation rate of about 1% per day. Primaquine, an antimalarial, and amantadine, an antiviral agent, when reacted with isocyanates gave polymeric biurets as do other primary aliphatic amines. Primaquine also underwent nucleophilic substitution reactions on polyepichlorohydrin. 3-Vinyl-, 4-vinyl-, and 5-vinylsalicylic acids have been synthesized, polymerized, and copolymerized to polymeric antibacterials. Copolymers of vinylsalicylic acid can be selective in their activity, depending on the comonomer. Selectivity can also be achieved by derivation of the poly(vinylsalicylic acid).

Anti-Bacterial Agents↗
PubMed1997

Reference values of common blood chemistry analytes in healthy population of Rawalpindi-Islamabad area.

The reference values of common blood chemistry analytes in healthy population, aged newborn to 80 years, of Rawalpindi Islamabad area were determined at AFIP, Rawalpindi. A total of 2115 healthy subjects, 1206 males and 909 females, were included in the study. Plasma glucose was analysed by GOD/POD, serum cholesterol by CHOD/PAP, triglycerides by GPO/PAP, urea by urease/GLDH, creatinine by Jaffe' rate reaction, uric acid by uricase, total bilirubin by Jendrassik and Grof, total protein by biuret, alanine transaminase (ALT) by optimized IFCC and alkaline phosphatase (AP) by optimized DGKC method. The between batch CVs of all the parameters were within acceptable quality goals. The reference values were calculated using 2.5 and 97.5 percentiles as lower and upper limits (95% CI). In healthy adult males the reference values were: fasting plasma glucose, 3.6-6.0 mmol/l; serum cholesterol; 3.2-6.6 mmol/l; triglycerides, 0.6-2.3 mmol/l; urea, 2.8-6.4 mmol/l; creatinine, 65-132 umol/l; uric acid, 164-430 umol/l; total bilirubin, 5-18 umol/l; total protein, 57-83 g/l; ALT, 15-45 U/l and AP, 185-620 U/l. The values in adult females, children and elderly subjects were slightly different than adult males. The reference values of our population show mild to moderate differences from the other Asian, European and American populations. It is recommended that reference values of different biochemical investigations should be established in various areas of Pakistan to make appropriate use of such investigations.

Adolescent↗
PubMed1975

Pseudoproteinuria in tolmetin-treated patients.

In a clinical study of the new nonsteroidal anti-inflammatory drug tolmetin, the urines of 5 patients reacted positively when tested for protein by the standard sulfosalicylic acid method. Pseudoproteinuria, due to the precipitation by acid of the major metabolite of tolmetin, was suspected. The urines were subsequently tested for protein by the following alternate methods: solubility of acid precipitate test, heat coagulation test, Upjohn modification of the sulfosalicylic acid test, biuret test, Albustix, and electrophoresis. All were negative for protein. These results confirmed that the reactions with the sulfosalicylic acid test did not indicate true proteinuria but pseudoproteinuria. It is suggested that patients who receive tolmetin be tested for proteinuria by methods other than those involving acid precipitation.

Anti-Inflammatory Agents↗
PubMedDate not supplied

[Estimation of various quantitative methods for the determination of native and formaldehyde-treated proteins].

Quantitative estimation of native and treated with 4% formaldehyde albumin, pepsin, lysozyme, histone, gelatin and other proteins was carried out using four procedures--biurete, Lowry-Folin, ninhydrin and coomassy R-250. Chromogeneity of proteins in corresponding color reactions was expressed as OD per 100 micrograms of nitrogen estimated by means of Kjeldahl micromethod. The proteins were treated with formaldehyde in corresponding buffers at 20 degrees within 7 days, non-bound or loosely bound formaldehyde was dialysed. Native proteins were dissimilar in their chromogeneity; these differences were the highest for Lowry-Folin and coomassy procedures. Formalinization affected the protein chromogenencity depending on a protein nature, conditions of formaldehyde treatment and on the procedure of estimation used. As shown by analysis of the data obtained the alterations of chromogeneity did not reflect the rate of reactive group blocking by formaldehyde in a protein molecule. The quantitative methods should be used very carefully in estimation of formalinized proteins.

Albumins↗
PubMed1992

The products of the reaction between toluene diisocyanate and water contract isolated guinea pig bronchi.

We have investigated the ability of the products of the reaction between toluene diisocyanate (TDI) and water to contract bronchial smooth muscle. The experiments were performed in isolated guinea pig bronchi. TDI, both 2,4- and 2,6-toluenediamine (TDA) and mixtures of 2,4- and 2,6-TDA (ratio 80:20 and 20:80) caused concentration-dependent contraction in the isolated bronchi. The mixture of disubstituted urea and biuret also contracted the bronchi, but not in a concentration-dependent fashion. Our results provide evidence that all products of the reaction between toluene diisocyanate and water have the ability to contract isolated bronchial smooth muscle in guinea pigs. Whatever the role of toluenediamine in the adverse respiratory effects induced by exposure to isocyanates, our findings reveal the necessity of in vivo studies on the metabolism of inhaled toluene diisocyanate in humans to improve our understanding of the mechanism of action of isocyanates.

Animals↗
PubMed1994

Alternative bases in the RNA world: the prebiotic synthesis of urazole and its ribosides.

Urazole is a five-membered heterocyclic compound which is isosteric with uracil's hydrogen-bonding segment. Urazole reacts spontaneoulsy with ribose (and other aldoses) to give a mixture of four ribosides: alpha and beta pyranosides and furanosides. This reaction occurs in aqueous solution at mild temperatures. Thermodynamic and kinetic parameters for the reaction of urazole with ribose were determined. In contrast, uracil is completely unreactive with ribose under these conditions. Urazole's unusual reactivity is ascribed to the hydrazine portion of the molecule. Urazole can be synthesized from biuret and hydrazine under prebiotic conditions. The prebiotic synthesis of guanazole, which is isosteric in part to diaminopyrimidine and cytosine, is accomplished from dicyandiamide and hydrazine. Kinetic parameters for both prebiotic reactions were measured. Urazole and guanazole are transparent in the UV, which would be a favorable property in the absence of an ozone layer on the early Earth. Urazole makes hydrogen bonds with adenine in DMSO similar to those of uracil, as established by H NMR. All of these properties make urazole an attractive potential precursor to uracil and guanazole a potential precursor to cytosine in the RNA or pre-RNA world.

Cytosine↗
PubMed1995

Sensitivity and selectivity of the electrochemical detection of the copper(II) complexes of bioactive peptides, and comparison to model studies by rotating ring-disc electrode.

Post-column reaction of peptides with Cu(II) can be used for the electrochemical detection of peptides as their biuret complexes. Understanding of the behavior (sensitivity at the anode and cathode in the dual-series electrochemical detector) of the system is facilitated through the observation of the rotating ring disc voltammetry of some model compounds. In operation, the anodic signal from the oxidation of the Cu(II)-peptide to the Cu(III) form can be used to detect peptides, or the downstream cathode can be used to detect the Cu(III) form. The signals appear at about 0.4 V (anode) for tetra- and longer peptides, 0.65 V for tripeptides. The anode signal is augmented by tyrosine (oxidation at 0.4-0.5 V) and tryptophan (0.5-0.6 V). If the cathode is used as the detector in a two working electrode cell, the sensitivity depends on the stability of the Cu(III) product. This is peptide dependent, but the signal is significant and useful analytically. Twenty-three bioactive peptides in two groups, naturally electrochemically active and naturally electrochemically silent, and several model compounds have been studied. Both naturally electrochemically active peptides (contain tyrosine and/or tryptophan) and naturally electrochemically silent peptides have been studied. Chromatography with an acetonitrile gradient has been used to separate the peptides in each group. Detection limits are for non-electroactive peptides in the range of 16-100 fmol (10- microliters injection 1.6-10 nM, 100 microliters injection 0.16-1.0 nM), and for electroactive peptides in the range of 6-40 fmol (0.6-4.0 nM for a 10- microliters injection and 60-400 pM for a 100- microliters injection). A tryptic digest of bovine cytochrome c is easily seen at 100 nM.

Amino Acid Sequence↗
PubMed2003

Myocardial infarction in patients aged less than 40 years. Frequency of BclI polymorphism in the fibrinogen beta-chain gene and plasma fibrinogen.

BACKGROUND: Several polymorphisms in the genes encoding for three separate chains of fibrinogen have been described. Some of them (Hae III and B854) are associated with elevated fibrinogen plasma level. AIM: To determine the frequency of BclI polymorphism in the fibrinogen beta-chain gene (BclI betaFb) in young survivors of myocardial infraction (MI) and to assess the relationship between allele status, plasma fibrinogen concentration and the number of affected coronary arteries. METHODS: The study group consisted of 99 male patients (mean age 43.5, range 29-49 years) with premature coronary artery disease (CAD) diagnosed by coronary angiography who had MI in the mean age of 37.4+/-3.2 years. The control group involved 78 age- and gender-matched healthy volunteers. DNA was extracted from peripheral blood leukocytes using standard methods. Fibrinogen blood concentration was determined using biuretic method. The BclI polymorphism in the fibrinogen beta-chain gene was investigated using polymerase chain reaction (PCR). RESULTS: Obesity was found in 15%, smoking - in 89%, hypertension - in 21%, diabetes - in 14% and hyperlipidemia - in 86% of MI patients. A family history of MI was present in 50% of patients. Coronary angiography revealed single-vessel disease in 34%, two-vessel disease in 36%, and three-vessel disease in 16% of patients. In two patients coronary angiography was normal. The frequency of BclI polymorphism of the beta-fibrinogen gene was significantly higher in MI patients than in controls (40.4% vs 29.5%, p<0.01). Moreover, in MI patients carrying the mutant allele a higher concentration of blood fibrinogen was found in comparison to patients without this anomaly (3.87 vs 3.55 g/L, p=0.05). There was no evidence of an association between the number of affected coronary arteries and polymorphism of BclI betaFbg gene status. However, all patients carrying BclI polymorphism of betaFbg gene had abnormal coronary angiography, contrary to patients without any defect. CONCLUSIONS: 1. Polymorphism of BclI betaFbg gene is associated with an increased fibrinogen plasma level. 2. There is no association between BclI polymorphism of betaFbg gene and the number of affected coronary arteries. This may confirm the hypothesis of multi-factorial aetiology of CAD in young patients suffering MI.

Adult↗
PubMed1991

Validation of a non-invasive technique to assess immediate or delayed onset of airway hypersensitivity in guinea-pigs.

Conscious Hartley guinea-pigs were sensitized with trimellitic anhydride (TMA) or trimeric hexamethylene diisocyanate biuret (Des-N) by repeated intradermal injections or by inhalation exposure. Immediate- and delayed-onset pulmonary reactions were recorded during/after challenges with the hapten or protein conjugate of the hapten, respectively. The positive control was sensitized and challenged with ovalbumin (OA) by inhalation. Homocytotropic antibodies of the IgG1 type were determined to correlate antibody titres and pulmonary responses. Immediate-onset pulmonary reactions were apparent in guinea-pigs sensitized by inhalation of TMA and OA. Animals sensitized intradermally with TMA demonstrated more vigorous immediate-onset reactions than animals sensitized by inhalation. The measurement of breathing parameters demonstrated that the ability to detect immediate-onset airway hyperreactivity was best when the breathing rate and tidal and minute volumes were measured. None of the measurements for delayed-onset reactions presented conclusive results, since sensitized as well as naive guinea-pigs demonstrated a delayed increase of breathing frequency. Antigen-specific IgG1 antibodies were several orders of magnitude higher in intradermally sensitized animals compared with animals sensitized by inhalation. Although Des-N-sensitized guinea-pigs experienced high IgG1-antibody titres, no response of pulmonary hypersensitivity could be elicited after hapten or conjugate challenge. In summary, it would appear that pulmonary hypersensitivity depends on factors other than IgG1 antibody titres. For the screening of chemicals, the dermal induction and inhalation challenge protocol was revealed to be more sensitive than the inhalation induction and inhalation challenge protocol.

Administration, Inhalation↗
PubMed2006

Felinine stability in the presence of selected urine compounds.

The stability of felinine, an amino acid present in feline urine, was investigated. Synthetic felinine was unstable in the urine of a selection of mammals. Felinine was found to stable in feline urine in which urea had been degraded. Synthetic felinine was found to react specifically with urea and did not react with urea analogues such as biuret or thiourea or other nucleophilic compounds such as ammonia which is more nucleophilic or acetamide and water which are less nucleophilic than urea. The reaction of urea and felinine was independent of pH over the range of 3-10. Urea did not react with N-acetyl-felinine suggesting a felinine N-terminal interaction with urea. Mass spectral analysis of the reaction products showed the presence of carbamylated felinine and fragmentation ions derived from carbamyl-felinine. The physiological relevance of felinine carbamylation is yet to be determined.

Animals↗
PubMed1980

Relationship between exposure to heavy metals and prevalence of renal dysfunction.

Total proteinuria (biuret method) and the urinary excretion of specific proteins (albumin, transferrin, orosomucoid and IgG measured by an automated immunoprecipitin reaction and beta 2-microglobulin determined by radioimmunoassay) have been assessed in four groups of workers: a control group (n = 88) and a group exposed to cadmium (n = 148), mercury vapour (n = 63) and lead (n = 25) respectively. The results demonstrate that a moderate exposure to lead (lead concentration in blood < 62 micrograms/100 ml) does not change the prevalence of renal dysfunction, whereas mercury vapour exposure increases the prevalence of subjects with excessive excretion of albumin (> 12 mg/g creatinine), orosomucoid (> 4.35 mg/g creat.) and IgG (> 3.5 mg/g creat.). Besides an increased excretion of beta 2-microglobulin (> 200 micrograms/g creat.) workers exposed to cadmium excrete a greater amount of high molecular weight proteins. An increased excretion of urinary proteins occurs when mercury and cadmium level in urine exceeds 50 and 10 micrograms/g creatinine respectively.

Cadmium Poisoning↗
PubMed1980

Automated multiple flow-injection analysis in clinical chemistry: determination of total protein with Biuret reagent.

We have examined the feasibility of the automated multiple flow-injection technique for application to clinical chemistry by adapting to this system the biuret method for the determination of total protein. Samples were discretely and rapidly introduced into a continuously flowing, nonsegmented reagent stream by means of an automatic sampler and high-pressure injection valve. Pumps operating at 1380-2070 kPa (200-300 psi) were utilized to introduce the biuret reagent and saline diluent into the system separately at flow rates of 72 and 47 microL/s, respectively. Use of 20-microL sample and a 3.0-s reaction-delay coil was adequately sensitive for analysis for total protein by this method. Samples were analyzed at a rate of 150/h with no detectable between-sample carryover. Within-run precision studies yielded relative standard deviations of 2.5% and less. Total protein values obtained by this method correlated well with those obtained by centrifugal analyzer and bubble-segmented continuous-flow biuret methods.

Autoanalysis↗
PubMed2001

A study on grafting and characterization of HMDI-modified calcium hydrogenphosphate.

It is known that the organic molecules can provide an effective means to manipulate the surface properties of the biodegradable ceramic. There are two ways to modify the surface of the biodegradable ceramic by organic molecules. The first one is through surface adsorption but organic molecules will easily be washed out in the physiological environment. The second approach is to graft organic molecules through covalent bond to the hydroxyl groups that are available on the surface of the ceramics. Isocyanate group has been reported as a coupling agent for hydroxyapatite and organic molecule. The studies showed that the isocyanate could react with hydroxyl groups of hydroxyapatite and form a covalent bond between isocyanate and hydroxyapatite. In the study, hexamethylene diisocyanate (HMDI) was used as coupling agent and calcium hydrogenphosphate (CaHPO4, CHP) was the candidate ceramic. CHP will react with HMDI at the temperature of 20 degrees C, 30 degrees C, 40 degrees C, 50 degrees C, 60 degrees C, and 70 degrees C for 4h. Dibutyltin dilaurate and hydroquinone were used as catalyst and inhibitor, respectively. The effect of reaction temperature on the grafted yield will be described. The linkage between CHP and HMDI will be characterized by DTA, TGA, FTIR, XRD, and 31P, 13C liquid state NMR. From the results, we successfully modified the surface of CHP with coupling agent of HMDI. The grafted yield of HMDI on CHP was increasing with the reaction temperature. The best temperature for CHP modified by HMDI is around 50 degrees C. The linkage between HMDI and the surface of CHP is a urethane linkage as CHP-O-CO-NH-(CH2)6-N=C=O. After further treatment, the terminal group of CHP treated with HMDI (MCHP) will be converted into a primary amine group as the formula of CHP-O-CO-NH-(CH2)6-NH2. If reaction temperature is 60 degrees C, long extension chain will occur with a urea linkage between the isocyanate groups as the formula of CHP-O-CO-NH-(CH2)6-(NH-CO-NH-(CH2)6)n-NH2. At reaction temperature higher than 60 degrees C, the HMDI will become prepolymerized forms in solution. The prepolymerized forms such as allophanate, biuret, uretidione and urea linkage will turn the solution into gel type mixture, which will lead to low grafted yield of HMDI on CHP. When MCHP prepared at the temperature 20 degrees C, there is no evidence of long extension but the grafted yield is the lowest only 0.9 wt% around.

Calcium Phosphates↗