Spectrophotometric study of bilirubin and hemoglobin interactions in several hydrogen peroxide generating procedures

The interference effects of bilirubin and hemoglobin have been described for the peroxidase-hydrogen peroxide oxidation of a hydrogen donor and the catalase-hydrogen peroxide oxidation of methanol to formaldehyde. A competition between bilirubin and the intended hydrogen donor is shown for the substitute analyte, hydrogen peroxide, with a resultant diminution of color due to the loss of intended reaction. No inhibition of peroxidase action appears to take place; its action when complexed with hydrogen peroxide is directed toward the competing hydrogen donor, bilirubin. The final color measured appeared to be partially compensatory, that is the sum of intended color plus the color of residual bilirubin. The subtraction of a serum blank representing a static system will result in a lowered value and a larger error. Hemoglobin, with its strong Soret band can, if its concentration is excessive, cause a major interference in reactions such as the Hantzsch reaction which result in overlapping bands at the reaction wavelength. Samples which are both hemolyzed and jaundiced would present as formidable blanking problems. Further studies on bilirubin and its glucuronide and their individual effect on the peroxidase-peroxide reaction are presently in progress.     

溶胶凝胶固定化酶流动注射化学发光法测定人体血清中的胆固醇

采用溶胶凝胶技术分别固定了胆固醇脂酶和胆固醇氧化酶,制成固定化酶柱;人体血清中胆固醇脂在胆固醇脂酶的催化作用下生成胆固醇,胆固醇在胆固醇氧化酶的催化作用下被氧化产生H2O2,将其与鲁米诺发生耦合的化学发光反应,在模拟酶血红蛋白的催化作用下产生较强的化学发光。结合流动注射技术,建立了溶胶凝胶固定化酶流动注射化学发光法测定胆固醇的新方法。实验发现,发光强度与胆固醇的浓度在一定范围内呈良好的线性关系,总胆固醇的线性范围为1.01×10-6～2.02×10-4mol/L(r=0.9975);检出限为7.5×10-7mol/L;游离胆固醇的线性范围为5.0×10-8～2.18×10-5mol/L(r=0.9991);检出限为5.0×10-9mol/L。用生化分析仪(东芝TBA-120FR)与本方法进行对照,两种方法无显著性差异。本方法已应用于临床血清样品中胆固醇的检测。     

Improved measurement of formaldehyde in water-soluble polymers by high-performance liquid chromatography coupled with post-column reaction detection

An improved methodology for the analysis of free formaldehyde in water-soluble polymers used for industrial water treatment is reported. Previously, derivatization prior to HPLC or colorimetric techniques has been used. The data generated by these approaches are suspect in that the derivatizing agent can react with the polymer or other sample components to produce high results. Post-column reaction derivatization is applied after separation of the free formaldehyde from the product interferences. The type of polymer product analyzed influences the choice of column(s). The degree of high bias of the commonly used 2,4-dinitrophenylhydrazine pre-column derivatization is reported and the results are compared to those with the post-column reaction for two polymer products. This method, being more selective, should be applicable to any polymer containing formaldehyde.     

Determination of free formaldehyde in the presence of donators in cosmetics by HPLC and post-column derivation

Summary The determination of free formaldehyde in the presence of its donators in cosmetic samples by a combination of reversed phase chromatography and post column reaction detection is described. The free formaldehyde is separated on a RP column with water as eluent from interfering formaldehyde-containing compounds and consecutively determined by the lutidine method in a reaction detector with knitted open tubes. With detection in the visible (420nm) the minimum detectable quantity is 40 ppb, with fluorimetric detection 15 ppb. The sample clean-up procedure for cosmetic products ranging from mascara to shampoo is by extracting the formaldehyde with water of pH 3, where the decomposition rate of the donators is minimal. Smaller amounts of free formaldehyde are always found compared to the standard lutidine method.     

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5?×?10(-7)?M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.     

Preparation of Hybrid Nanocapsules

Hybrid nanoparticles with a polystyrene core and a hybrid copolymer shell were used to produce hybrid nanocapsules by dissolving the polystyrene core from the previously elaborated core-shell particles. Following previous works, the core-shell particles were prepared by emulsion polymerization of styrene and subsequent addition of γ-methacryloxy propyl trimethoxy silane (MPS) to produce the shell by copolymerization reaction of MPS with the residual styrene. Core extraction was performed by diluting the core-shell particles in an excess of tetrahydrofuran (THF). Two procedures were investigated to separate the dissolved polymer chains from the nanocapsules. In the first procedure, the polymer was isolated by successive centrifugation and redispersion in THF, whereas in the second procedure, the free polymer chains were removed by dialysis. The polymer molecular weight was optimized in order to promote dissolution of the polymer chains and allow them to diffuse through the shell.     

HPLC Determination of Hepatic Cholesterol 7α-Hydroxylase Activity Using Immobilized Cholesterol Oxidase and Chemiluminescence Detection

Abstract

This paper describes an HPLC method for the determination of cholesterol 7α-hydroxylase activity, at high or low activity levels, that is sensitive and specific for 7α-hydroxycholesterol. The method relies on the generation of hydrogen peroxide by oxidation of 7α-hydroxycholesterol using the enzyme cholesterol oxidase which has been immobilized on porous glass beads. The hydrogen peroxide is subsequently detected by chemiluminescence generated by reaction of peroxide with bis-(2, 4, 6-trichlorophenyl)-oxalate (TCPO), a commonly used chemiluminescence reagent specific for peroxides. In the procedure, sample preparation is limited to extraction of the incubation mixture and injection of the concentrated extract.     

Determination of Formaldehyde in the Polymerized Ragweed Antigen by HPLC

Abstract

Formaldehyde is used in the polymerization of ragweed antigen for the desensitization treatment of hay fever. Because the polymerized ragweed antigen is used as a vaccine which stimulates the production of the desired immunological effects upon injecting into the body, trace formaldehyde in the injected material is a health concern. Two procedures for the determination of formaldehyde were developed. In the direct procedure, formaldehyde in the sample was reacted with 2,4-dinitrophenylhydrazine (2,4-DNP) in solution to yield a strongly UV absorbing derivative. In an alternate approach, formaledhyde in the sample in a sealed vial was allowed to diffuse in the gaseous form into a reaction chamber consisted of a culture tube insert in which formaldehyde was derivatized with 2,4-DNP reagent. Following extraction into methylene chloride, the derivative was injected into the liquid chromatograph, separated on a reversed phase column and detected at UV 254 nm.     

Capillary electrophoresis for determination of free and albumin-bound bilirubin and the investigation of drug interaction with bilirubin-bound albumin

Capillary electrophoresis (CE) is a promising technique for assessment of free bilirubin and its interaction with albumin, as it requires only a small sample volume and provides a rapid and efficient separation of free bilirubin from its albumin-bound complex in a one-phase system. In order to maintain the equilibrium without dissociation of bilirubin from the albumin/bilirubin complex as in real clinical conditions, the coupling of CE with frontal analysis (FA) was investigated. A very large sample plug was introduced hydrodynamically into the capillary (36 cm length, 50 microm inner diameter) at 15 psi x s to develop the frontal conditions during CE separation. The working conditions for CE/FA separation of bilirubin and albumin were optimized as follows: +20 kV; running buffer, 10 mmol/L phosphate and 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4. The working range for bilirubin was found to vary from 5 to 206 micromol/L; precision with relative standard deviation (RSD) <2.0% for n = 3 and detection limit (signal to noise, S/N = 2) was 2 micromol/L. The residual binding capacity of a simulated cord blood serum for bilirubin was 26 mg/100 mL at pH 7.4. Bilirubin was shown to be displaced from albumin when aspirin was added. The free bilirubin concentration was found to increase to clinical significant concentrations from 11.9 to 21.1% when increasing aspirin was added in the range of 5-50 mg/100 mL, respectively. Thus, the investigation of aspirin displacement of bilirubin from albumin is clinically important and the CE/ FA method is a suitable procedure for this purpose.     

Spectrophotometric study on minimizing bilirubin interference in an enzyme reagent mediated cholesterol reaction

A spectrophotometric study was carried out which describes the effect of the inclusion of potassium ferrocyanide into an enzyme reagent system as a protective device against the competitive interaction of bilirubin in the indicator reaction. A previously described system for depicting how bilirubin interacts competitively in a modified 4-aminoantipyrene-phenolic peroxidase-peroxide coupled reaction was used as a template to judge the effectiveness of the protective action. The results obtained indicate that the protection is not total, but that the close approximation may make this a worthwhile modification in procedure to consider for the determination of total, free, and/or high density lipoprotein cholesterol. At the same time it might be inferred that the interference of bilirubin can be diminished chemically for other oxidase-peroxidase coupled systems. A search for even better solutions to this interference problem may be possible. For us, this lead has pointed the way in other similar directions.In addition, this study reports on a preliminary investigation as to the site of coupling of phenolic compounds with 4-aminoantipyrene.     

Interference study on precipitating reagents used in a sensitive color reaction for high-density lipoprotein cholesterol determination

A spectrophotometric study was carried out which describes the effect of the inclusion of potassium ferrocyanide into an enzyme reagent system as a protective device against the competitive interaction of bilirubin in the indicator reaction. A previously described system for depicting how bilirubin interacts competitively in a modified 4-aminoantipyrene-phenolic peroxidase-peroxide coupled reaction was used as a template to judge the effectiveness of the protective action. The results obtained indicate that the protection is not total, but that the close approximation may make this a worthwhile modification in procedure to consider for the determination of total, free, and/or high density lipoprotein cholesterol. At the same time it might be inferred that the interference of bilirubin can be diminished chemically for other oxidase-peroxidase coupled systems. A search for even better solutions to this interference problem may be possible. For us, this lead has pointed the way in other similar directions.In addition, this study reports on a preliminary investigation as to the site of coupling of phenolic compounds with 4-aminoantipyrene.     

γ-Methylenation of α,β-Unsaturated Ketones

γ-Methylene derivatives of α,β-unsaturated ketones are obtained readily by a one-pot procedure exploiting the reaction of O-silylated dienolates with commercially available formaldehyde N, N-dimethyliminium chloride in anhydrous N, N-dimethylformamide, in situ oxidation of the Mannich reaction product with hydrogen peroxide, and elimination of N,N-dimethylhydroxylamine from the resultant N-oxide.     

氢醌对胆红素氧化过程的促进作用及其分析意义

研究了氢醌与胆红素的作用过程，发现氢醌在碱性条件下可促使胆红素氧化，且胆红素被氧化成胆绿素，此反应过程被证实为自由基反应，考察了介质条件，抗氧化剂等反应过程的影响及其它酚类物质对胆红素氧化的作用情况。     

Total parenteral nutrition turbidity and clearing of serum for assay purposes

Parenteral nutrition such as Liposyn III when introduced into patients causes their serum to become milky thereby creating potentially aberrant analyses when such a medium is subjected to spectrophotometric investigation during the time period prior to the metabolic removal of the intravenously injected triglycerides. However, it is possible to alter chemical reagents by the introduction of enzymatic clearing materials, which rapidly through hydrolytic action by lipases convert the offending triglycerides to glycerol and non-esterified fatty acids. The latter as potential soap formers with calcium and magnesium can be sequentially and quickly incorporated into transparent guest-host complexes with alpha cyclodextrin serving as the host, thus removing them as possible secondary sources of turbidity. Representative procedures incorporating the lipase-alpha cyclodextrin constituents into their reagents thus allow rapid clearing of the medium followed by spectrophotometric measurement during the clarified window of reaction without any change in the modified procedure. Additionally and importantly there is no interference from the addition of the clarifying ingredients added to the reagents. Commonly needed high volume tests such as hemoglobin, glucose, calcium, bilirubin, cholesterol and triglycerides are described as examples in which the clarifying processes are included in this novel analytical technology. No changes in the original methodologies are involved and no preliminary treatments are necessary for these important tests in which the interfering light-scattering properties of a sample are eliminated when optical measurement takes place even during the peak levels of parenteral nutrition necessarily introduced into any patient either adult or neonatal. These procedures involving enzymatic degradation of the triglycerides causing the primary turbidity sequenced to guest-host complexation of liberated fatty acids, the potential secondary turbidity, are effective for clarification of serum samples during measurement. This technology holds whether the source of the hypertriglyceridemia is parenteral nutrition or the pathological presence of excessive triglycerides as in diabetes.     

Interaction of hemoglobin in elevated serum total bilirubin determinations

A spectrophotometric study was carried out in an effort to elucidate how hemoglobin in serum interferes in the diazo coupling reaction used for the determination of bilirubin. Because it is difficult to see hemoglobin in neonatal specimens in which bilirubin is elevated without obtaining spectra of the mixtures, this can be a real problem when handling these specimens while using this particular reaction. From the spectrophotometric evidence described here, one can infer that hemoglobin can act in two ways. In one way, the formation of ferriprotoporphyin generates an oxidizing agent which can convert bilirubin to an oxidized product precluding it from taking part in a diazo coupling reaction. In the other way, the same ferriprotoporphyrin seems capable of oxidizing the azobilirubin complex after it has reached equilibrium again causing some lowering of the results obtained. It may be reasonable to assume that the quicker the analytical event can occur, the more accurate the result might be. If the matrix of reaction can be changed, and the reaction of diazo coupling also speeded up as a feature of the change, then it may be possible to avoid the interference from hemoglobin almost entirely. High concentrations of hemoglobin were tested here in an attempt to clearly show how the interference might take place.     

Strategy for the isolation of native dehydrogenases with potential for biosensor development from the organism Hyphomicrobium zavarzinii ZV580

Dehydrogenases are interesting candidates for the development of electrochemical biosensors. Most dehydrogenases are characterised by a comparatively broad substrate spectrum, yet highly specific enzymes exist as well. A specific formaldehyde dehydrogenase has, e.g., been described for the organism Hyphomicrobium zavarzinii ZV580. Isolation of enzymes from their natural source instead of a recombinant expression renders the isolation more challenging, as common tools such as affinity tags are no longer available. In this contribution, we develop chromatographic procedures for such isolation tasks. The previously described formaldehyde dehydrogenase was isolated by two procedures, one based on affinity chromatography, the other on hydroxyapatite. Neither procedure yielded an active enzyme. In addition two dehydrogenases, a formaldehyde and a methylamine dehydrogenase, were found in the cell free extract, which had not been described previously. Both enzymes could be isolated to near purity by a sequence of hydroxyapatite and anion exchange chromatography. The new formaldehyde dehydrogenase requires reconstitution with calcium and pyrroloquinoline quinone in order to become active. The enzyme shows no cross-reactivity with methylamine or methanol. The methylamine dehydrogenase catalyses the conversion of methylamine into formaldehyde, hence it could become a technical catalyst for the inverse reaction. This enzyme consists of two types of subunit and may be one of the rare alpha,beta-methylamine dehydrogenases.     

Determination of free, total, and esterified cholesterol by high-performance liquid chromatography

A method is described for measuring free, total, and esterified cholesterol in blood serum in which reversed-phase liquid chromatography is used and the eluate is monitored at 200 nm. The sample for total cholesterol is prepared according to the Abell-Kendall procedure, and for free cholesterol an extract of serum--isopropanol (1:5, v/v) is used. The column is a muBondapak C18, 10 micrometers, and the mobile phase for total cholesterol is isopropanol--acetonitrile (50:50, v/v); for free cholesterol, it is isopropanol--acetonitrile--water (60:30:10). An approximation of the free cholesterol, triglycerides, and individual cholesteryl esters is obtained from single chromatograms of isopropanol extracts of serum if the first mobile phase is used. In a comparison study with the Abell-Kendall method for total cholesterol, the correlation is excellent and the precision is acceptable.     

光导纤维胆固醇生物传感器的研究

将胆固醇酯酶、胆固醇氧化酶和辣根过氧化物酶通过戊二醛交联反应,固定在牛血清蛋白上,制成光纤胆固醇生物传感器的传感膜.此传感器偶合了胆固醇酯水解、胆固醇的氧化和鲁米诺同过氧化氢的化学发光等3种酶催化反应,通过光纤输出的化学发光信号进行检测.测定总胆固醇和自由胆固醇的线性范围均为0.5～20μg/mL,检测下限为0.1μg/mL,响应时间为2min,寿命在2个月以上,适用于血清中总胆固醇和游离胆固醇的测定.     

Determination of direct-bilirubin by a fluorimetric-enzymatic method based on bilirubin oxidase

A method for the determination of direct bilirubin by reaction with bilirubin oxidase (BOx) is reported. The procedure is based on the changes in fluorescence which take place during the enzymatic reaction of BOx with any of the three forms of bilirubin (free, conjugated and with albumin) when the solution is excited at 240 nm and the emission is measured at 440 nm. The change in fluorescence was studied thoroughly. It seems mainly due to the fluorescence of one of the reaction products. A theoretical study was carried out to relate the changes in fluorescence observed to the species taking part in the reaction and to establish some of the enzymatic reaction constants. The optimum reaction conditions were studied for each of the three types of bilirubin together with their analytical characteristics (linear range and precision). Selective determination of direct bilirubin was carried out for various synthetic samples with good results. A linear response up to 7 mg L(-1) of direct bilirubin was obtained. Using optimum conditions, the precision for free and conjugated bilirubin was 3.4% (n = 5) and 3.0% (n = 5), respectively.     

Interference by the Catecholic Cephalosporin BRL-41897-A with Diagnostic Clinical Chemistry Assays

The catecholic cephalosporin BRL-41897-A, was investigated for in vitro interference with commonly used clinical chemistry assays. An important finding was a negative interference with the enzymatic cholesterol assay that incorporates the Trinder reaction, in which hydrogen peroxide produced by an oxidase drives the oxidative coupling of a phenol with 4-aminophenazone to produce a colored quinoneimine. The suspected interference by BRL-41897-A with the Trinder reaction was investigated using a uricase driven system in which the phenol reagent was substituted or combined with BRL-41897-A. The results indicate that BRL-41897-A competes with the phenol component to react preferentially with 4-aminophenazone to produce a quinoneimine of low absorptivity. Further investigations conducted with similar enzymatic assays for creatinine, cholesterol, triglycerides, and uric acid showed the interference varied substantially with analyte and was dependent on analyte concentration.