Analysis of nitrate,nitrite, and [15N]nitrate in biological fluids

A new automated system for the analysis of nitrate via reduction with a high-pressure cadmium column is described. Samples of urine, saliva, deproteinized plasma, gastric juice, and milk can be analyzed for nitrate, nitrite, or both with a lower limit of detection of 1.0 nmol NO₃^? or NO₂^?/ml. The system allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in urine and other biological fluids. Analysis rate is 30 samples per hour, with preparation for most samples limited to simple dilution with distilled water. The application of gas chromatography/mass spectrometry for the analysis of ¹⁵NO₃^? in urine after derivatization to ¹⁵NO₂-benzene is also described.     

Spontaneous calcification in F344/Slc and F344/JCL rats

F344/Slc and F344/JCL rats 2 years of age were histologically examined for the incidence and distribution of calcification. In the male rats of both strains, calcification was observed in the testis, lung, brain, kidney, heart, aorta, cornea, prostate and seminal vesicle respectively. In the female F344/JCL rats, calcification appeared in the kidney, lung, cornea, brain, stomach, ovary and heart. Among these of both sexes, the lung was one of the most affected organs for calcification. On the other hand, calcification in the kidney was more severe and frequent in the females than in the males, suggesting that the sex may be one of enhancing factors for calcification.     

高效液相色谱法测定体液中硝酸盐及亚硝酸盐

目的和方法 :应用高效液相色谱技术建立一种灵敏的检测不同体液中硝酸盐和亚硝酸盐的方法。结果 :唾液、血清及尿液经过不同方法处理后应用高效液相色谱ODS反相柱分离 ,紫外检测器于 2 10nm检测其中的硝酸盐和亚硝酸盐的含量。整个分离过程少于 7min ,硝酸盐和亚硝酸盐的测定线性范围分别为 0 .7～ 10 0ng、5～ 10 0ng ,最低检测极限分别为 0 .3ng和 2ng。硝酸盐回收率为 99%～ 10 2 % ,亚硝酸盐回收率为 99%～ 10 4 %。测定硝酸盐及亚硝酸盐的精密度分别为 0 .8%和 1.7%。结论 :本法测定硝酸盐和亚硝酸盐简便、灵敏度高、特异性好     

Osteosclerosis in F344/DuCrj rats

Osteosclerosis was observed in the tibia and sternum in F344/DuCrj rats of both sexes at 6, 18 and 30 months of age. The lesion first seen was a proliferation of osteogenic tissues on the marrow surface of the cortical bone and bone trabeculae, resulting in replacement of the marrow cavity by lamellar bone. Most of the affected rats had associated degenerative osteoarthrosis and regressive changes of the growth plate. Osteosclerosis was considered to be an aging change, lesions were observed at 6 months and increased in frequency with age.     

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

ReviewMethods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

Measurement of the NO metabolites, nitrite and nitrate, in human biological fluids by GC-MS

In this article we critically review the development and application of gas chromatography-mass spectrometry (GC-MS) techniques to the measurement of the nitric oxide (NO) metabolites, nitrite and nitrate, in human biological fluids. Our focus is on the issue of the fitness of any analytical strategy to its intended purpose and the validity of the analytical results generated. The accuracy, precision, recovery, selectivity and sensitivity of the various methods are evaluated and the potential pitfalls, both specific to the methods, and general to the area, are considered. Several examples of the applications of these techniques to clinical investigations of NO physiology are also critically evaluated.     

Cumene hydroperoxide-supported denitrification of 2-nitropropane in uninduced mouse liver microsomes

Cumene hydroperoxide supported oxidative denitrification of 2-nitropropane was investigated in uninduced mouse liver microsomes. The cytochrome P-450 peroxygenase catalyzed reaction resulted in the production of nitrite and acetone. Several lines of evidence suggested the involvement of multiple forms of cytochrome P-450. Acetone production was at least two times greater than nitrite release possibly due to sequestration of nitrite in the reaction mixtures.     

Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens

A pure culture of Pseudomonas fluorescens was used as a model system to study the kinetics of denitrification. An exponentially growing culture was harvested and resuspended in an anoxic acetate solution buffered with K/Na phosphate at pH values of 6.6, 7.0, 7.4, and 7.8. The temperature was kept at 28 degrees C in all assays. Nitrate pulses of approximately 0.2 mg N/L caused nitrite to accumulate due to a faster rate of nitrate reduction over nitrite reduction. The rate of nitrate reduction was observed to depend on its concentration as predicted by the Michaelis-Menten equation. At nonlimiting nitrate concentrations, nitrite reduction was described by the same equation. Otherwise, nitrite reduction also depended on nitrate concentration. Consequently, nitrate and nitrite reductions compete with each other for the oxidation of common electron donors. A kinetic model for nitrate competitive inhibition of nitrite reduction is proposed. The model was used to interpret the nitrate and nitrite profiles observed at the four pH values: the optimum pH value was 7.0 in both cases; the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(3) ) = 0.04 mg N/L); the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(2) ) = 0.06 mg N/L), but it decreased sharply for the pH value of 7.8. Although the ratio between the two maximum reduction rates (V(max NO(2) )/V(max NO(3) )) is constant, nitrite accumulation depends on the medium pH value. Therefore, the regulation mechanism that shifts the electron flow between the two terminal reductases is readily reversible and does not change their relative maximum reduction rates. (c) 1995 John Wiley & Sons, Inc.     

Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography

Various analytical techniques have been developed to determine nitrite and nitrate, oxidation metabolites of nitric oxide (NO), in biological samples. HPLC is a widely used method to quantify these two anions in plasma, serum, urine, saliva, cerebrospinal fluid, tissue extracts, and fetal fluids, as well as meats and cell culture medium. The detection principles include UV and VIS absorbance, electrochemistry, chemiluminescence, and fluorescence. UV or VIS absorbance and electrochemistry allow simultaneous detection of nitrite and nitrate but are vulnerable to the severe interference from chloride present in biological samples. Chemiluminescence and fluorescence detection improve the assay sensitivity and are unaffected by chloride but cannot be applied to a simultaneous analysis of nitrite and nitrate. The choice of a detection method largely depends on sample type and facility availability. The recently developed fluorometric HPLC method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) and the enzymatic conversion of nitrate into nitrite, offers the advantages of easy sample preparation, simple derivatization, stable fluorescent derivatives, rapid analysis, high sensitivity and specificity, lack of interferences, and easy automation for determining nitrite and nitrate in all biological samples including cell culture medium. To ensure accurate analysis, care should be taken in sample collection, processing, and derivatization as well as preparation of reagent solutions and mobile phases, to prevent environmental contamination. HPLC methods provide a useful research tool for studying NO biochemistry, physiology and pharmacology.     

Influence of nitrate starvation on nitrite accumulation during denitrification byPseudomonas stutzeri

Anaerobically denitrifyingPseudomonas stutzeri may transiently accumulate from 0% to 100% of nitrate as nitrite, depending on the nitrate availability during the preceding 24 h. The variations of transient nitrite level were related to the length of previous nitrate starvation. Cells harvested in a late anaerobic growth phase did not accumulate any nitrite during denitrification. Five hours of nitrate starvation caused about 40% (mol/mol) nitrite formation from the further added nitrate and 16 h nearly 80%. Concomitant with this, whereas the nitrate reduction capacity was not significantly affected, the initial nitrite reduction capacity was decreased. These results suggested that nitrate starvation caused a rapid loss of the originally produced nitrite-reducing capacity in the preculture. The lost capacity could be regenerated when nitrate or nitrite was resupplied to the cells. To investigate the nature of the restoration of nitrite-reducing capacity, chloramphenicol was used. The presence of chloramphenicol at 83 μg/ml entirely stopped this restoration, which was otherwise observed in all instances. This indicated that the recovery of nitrite-reducing activity required de novo protein synthesis, which was further confirmed by Western immunoblot assay of cd₁ nitrite reductase.     

Sex and dietary fat modulate hepatic prostaglandin F2 alpha in F344/N rats.

The study was designed to determine whether sex and fat calories altered hepatic prostaglandin (PG) F2 alpha status; a factor which may reflect susceptibility to cancer development. For 4 weeks, groups of 8 male and 8 female F344/N rats were fed diets with 9% of energy (en%) from linoleate and 15.5, 20, 30 or 40 en% fat. Females had greater hepatic stearate, arachidonate and PGF2 alpha whereas males had greater hepatic myristate, palmitate and oleate. Females also had greater plasma stearate levels. Greater hepatic arachidonate may have stimulated PG production in females. Hepatic oleate increased and hepatic palmitate decreased with increasing en% fat (p < 0.05). Hepatic stearate was greater and hepatic linoleate less when 40 en% fat was fed compared with other levels of dietary fat (p < 0.05). Plasma oleate was greater at 30 or 40 en% fat than at lower levels of fat, whereas plasma linoleate was less at 40 en% than at 15.5% en% fat. The ability of a 30 en% fat diet, containing equal proportions of linoleate and oleate, to suppress hepatic PG production may be related to the effects of dietary fat content and composition on plasma fatty acid profiles. Because suppressed PG production has been linked with suppression of cancer development, dietary recommendations to consume 30 en% fat with a P:M ratio of 1:1 may be cancer-protective.     

Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes

Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.     

Populations of follicles in F344/N rats during aging

Follicular populations were investigated in female F344/N rats to better understand the aging process of the rat ovary. Ovaries dissected at various ages (spanning 1–36 months old) were submitted for histological examination. The total number of primordial, growing (primary and secondary), tertiary, and atretic follicles as well as corpora lutea (CL) were counted in hematoxylin–eosin- and azocarmine–aniline-blue-stained ovarian sections. The number of healthy follicles including primordial, growing and tertiary follicles decreased rapidly between the first and third months and gradually thereafter. CL were found in 3-month-old rats, and their number remained unchanged until 18 months of age, at which point it decreased. The number of atretic follicles started to increase in rats older than 18 months, which corresponded to the cessation of estrous cyclicity. Several healthy follicles and CL were observed even in 36-month-old rats.     

Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the L-arginine/nitric oxide area of research

In the Griess reaction, first reported by Johann Peter Griess in 1879 as a method of analysis of nitrite (NO(2)(-)), nitrite reacts under acidic conditions with sulfanilic acid (HO(3)SC(6)H(4)NH(2)) to form a diazonium cation (HO(3)SC(6)H(4)-N[triple bond]N(+)) which subsequently couples to the aromatic amine 1-naphthylamine (C(10)H(7)NH(2)) to produce a red-violet coloured (lambda(max) approximately 540 nm), water-soluble azo dye (HO(3)SC(6)H(4)-NN-C(10)H(6)NH(2)). The identification of nitrite in saliva has been the first analytical application of this diazotization reaction in 1879. For a century, the Griess reaction has been exclusively used to identify analytically bacterial infection in the urogenital tract, i.e. to identify nitrite produced by bacterial reduction of nitrate (NO(3)(-)), the major nitrogen oxide anion in human urine. Since the discovery of the l-arginine/nitric oxide (l-Arg/NO) pathway in 1987, however, the Griess reaction is the most frequently used analytical approach to quantitate the major metabolites of NO, i.e. nitrite and nitrate, in a variety of biological fluids, notably blood and urine. The Griess reaction is specific for nitrite. Analysis of nitrate by this reaction requires chemical or enzymatic reduction of nitrate to nitrite prior to the diazotization reaction. The simplicity of the Griess reaction and its easy and inexpensive analytical feasibility has attracted the attention of scientists from wide a spectrum of disciplines dedicated to the complex and challenging L-Arg/NO pathway. Today, we know dozens of assays based on the Griess reaction. In principle, every laboratory in this area uses its own Griess assay. The simplest Griess assay is performed in batch commonly as originally reported by Griess. Because of the recognition of numerous interferences in the analysis of nitrite and nitrate in biological fluids and of the desire to analyze these anions simultaneously, the Griess reaction has been repeatedly modified and automated. In recent years, the Griess reaction has been coupled to HPLC, i.e. is used for post-column derivatization of chromatographically separated nitrite and nitrate. Such a HPLC-Griess system is even commercially available. The present article gives an overview of the currently available assays of nitrite and nitrate in biological fluids based on the Griess reaction. Special emphasis is given to human plasma and urine, to quantitative aspects, as well as to particular analytical and pre-analytical factors and problems that may be associated with and affect the quantitative analysis of nitrite and nitrate in these matrices by assays based on the Griess reaction. The significance of the Griess reaction in the L-Arg/NO pathway is appraised.     

Experimental investigation of nitrogen and oxygen isotope fractionation in nitrate and nitrite during denitrification

In batch experiments, we studied the isotope fractionation of nitrogen and oxygen during denitrification of two bacterial strains (Azoarcus sp. strain DSM 9056 and Pseudomonas pseudoalcaligenes strain F10). Denitrification experiments were conducted with succinate and toluene as electron donor in three waters with a distinct oxygen isotope composition. Nitrate consumption was observed in all batch experiments. Reaction rates for succinate experiments were more than six times higher than those for toluene experiments. Nitrogen and oxygen isotopes became progressively enriched in the remaining nitrate pool in the course of the experiments; the nitrogen and oxygen isotope fractionation varied between 8.6–16.2 and 4.0–7.3‰, respectively. Within this range, neither electron donors nor the oxygen isotope composition of the medium affected the isotope fractionation process. The experimental results provide evidence that the oxygen isotope fractionation during nitrate reduction is controlled by a kinetic isotope effect which can be quantified using the Rayleigh model. The isotopic examination of nitrite released upon denitrification revealed that nitrogen isotope fractionation largely follows the fractionation of the nitrate pool. However, the oxygen isotope values of nitrite are clearly influenced by a rapid isotope equilibration with the oxygen of the ambient water. Even though this equilibration may in part be due to storage, it shows that under certain natural conditions (re-oxidation of nitrite) the nitrate pool may also be indirectly affected by an isotope equilibration.     

Differences in survivability among F344 rats.

Important parameters to identify and develop appropriate animal models for longevity science include survivability, age-related disorders, and easy handling of aged individuals. It is found that F334/Du and F344/N have distinctive strain difference in these parameters. The finding suggests F334/Du and F344/N, even though they are historically siblings, need clearly separate identification when used as animal models for aging science, in particular, longevity science.     

Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high-performance anion-exchange liquid chromatography for their determination

Measurements of nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO(2)(-) and NO(3)(-) in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO(2)(-)/NO(3)(-) contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO(2)(-) and NO(3)(-) in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO(2)(-) and NO(3)(-) content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO(2)(-) and NO(3)(-) in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO(2)(-) and NO(3)(-) in standards was 3 pmol. Recoveries of NO(2)(-) and NO(3)(-) from spiked plasma (1-100 microM KNO(2)/KNO(3)) and from extracted standards (1-250 microM) were approximately 100%. Intra-assay and inter-assay RSDs for NO(2)(-) and NO(3)(-) in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO(2)(-) and NO(3)(-) in various biological fluids, e.g., plasma.