Luminometric quantitation of photinus pyralis firefly luciferase and Escherichia coli beta-galactosidase in blood-contaminated organ lysates

Firefly luciferase and Escherichia coli beta-galactosidase chemiluminescent reporter gene assays are rapid and sensitive means of detecting reporter enzyme activities in cell lysates of both eukaryotic and prokaryotic systems. In these assays, expression vectors containing the luciferase or beta-galactosidase genes are transferred to cells in culture or animal tissues in vivo. Crude cell or organ lysates are then prepared and submitted to enzyme assays. The level of enzyme activity is proportional to the efficiency of gene delivery and expression. When used with modified substrates that emit light when cleaved by the appropriate enzyme, luciferase and beta-galactosidase activity can be detected luminometrically. Attempts to apply these assays to cell lysates contaminated with blood, as from any whole organ lysate, have had questionable results thus far because of light absorption by hemoglobin in the ranges of light emission by both of these assays. We have made several adjustments to standard chemiluminescent reporter gene assay protocols to minimize errors in quantitation contributed by hemoglobin. To this end, we have developed a method for quantitating the protein due to blood and due to the organ itself in a blood-contaminated organ lysate. We have also found that the use of a colorimetric protein assay that is unaffected by hemoglobin absorbance is preferred for protein quantitation. In conclusion, luciferase and beta-galactosidase assays can be applied to blood-contaminated organ lysates; however, the luciferase assay proved to be superior due to minimal endogenous activity and lower absorption by hemoglobin of light emitted by the enzyme product.     

The use of cis-parinaric acid to determine lipid peroxidation in human erythrocyte membranes. Comparison of normal and sickle erythrocyte membranes

The recently developed parinaric acid assay is shown to offer possibilities for studying peroxidation processes in biological membrane systems. Taking the human erythrocyte membrane as a model, several initiating systems were investigated, as well as the effect of residual hemoglobin in ghost membrane preparations. The effectivity of a radical generating system appeared to be strongly dependent upon whether radicals are generated at the membrane level or in the water phase. Thus, cumene hydroperoxide at concentrations of 1.0-1.5 mM was found to be a very efficient initiator of peroxidation in combination with submicromolar levels of hemin-Fe3+ as membrane-bound cofactor. In combination with cumene hydroperoxide, membrane-bound hemoglobin appeared to be about 6-times more effective in promoting peroxidation than hemoglobin in the water phase. Results comparing the behaviour of normal and sickle erythrocyte ghost suspensions in the peroxidation assay suggest that the increased oxidative stress on sickle erythrocyte membranes could be due to enhanced membrane binding of sickle hemoglobin, but also partly to a characteristically higher capability of sickle hemoglobin to promote peroxidation. The order of peroxidation-promoting capabilities that could be derived from the experiments was hemin greater than sickle hemoglobin greater than normal hemoglobin.     

An improved method excluding hemoglobin interferences for lysosomal hydrolase assays using colorimetric synthetic substrates, 2-(N-hexadecanoylamino)-4-nitrophenol derivatives

Artificial chromogenic substrates, derived from 2-(N-hexadecanoylamino)-4-nitrophenol, can be used to assay sphingomyelin phosphodiesterase, glucosylceramidase, or galactosylceramidase. Nevertheless, these enzymatic spectrophotometric assays cannot be realized on tissue preparations containing hemoglobin which interferes in the measurement. We present a selective extraction method of 2-(N-hexadecanoylamino)-4-nitrophenol which allows to avoid hemoglobin interference in this spectrophotometric assay of 2-(N-hexadecanoylamino)-4-nitrophenol. The solvents used have been tested to obtain on the one hand maximal absorbance and organic extraction of 2-(N-hexadecanoylamino)-4-nitrophenol, and on the other hand the minimal hemoglobin interference. None of the pure solvents studied being suitable, two solvent mixtures were selected: ethyl acetate/2-propanol (5/1) and 2-ethyl-1-hexanol/4-methyl-2-pentanone (1/1). These methods were tested to determine sphingomyelinase activity in enzymatic preparations and prove that they are available for lysosomal hydrolase assays using these colorimetric substrates.     

Verdohemoglobin as a substrate for proteases is applicable to assays over a wide pH range.

Verdohemoglobin, a heme-modified derivative readily obtained by ascorbic acid-coupled oxidation of oxyhemoglobin, was found to be a suitable substrate for protease with which assay can be carried out at all pH values. The advantage of verdohemoglobin over such popular substrates as alkali-and-urea-denatured hemoglobin and casein was demonstrated by a pH-profile study with Pronase E.     

A haptoglobin radioassay based on binding to solid-phase hemoglobin.

A specific and sensitive assay for haptoglobin based on binding to an easily prepared Sepharose-bound hemoglobin reagent is described. The assay is suitable for directly determining radiolabeled amino acid incorporation into haptoglobin in several liver cell systems in vitro and can be adapted to measure unlabeled free haptoglobin in plasma samples regardless of the presence of the haptoglobin-hemoglobin complex.     

A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid.

An assay for the detection of hydroperoxy derivatives of linoleic acid formed by the action of 15-lipoxygenase is described. The assay developed is based on a method first reported by Ohishi et al. (1985) Biochem. Int. 10, 205-211) with some important modifications. The assay described herein takes advantage of the ability of (9Z,11E)-13-hydroperoxyoctadecadienoic acid (13-HPODE), the product of the action of 15-lipoxygenase on linoleic acid, to oxidize N-benzoyl leucomethylene blue to methylene blue in the presence of hemoglobin. The resultant blue color is stable to light and air and can be quantified spectrophometrically at 660 nm. The linear range of the assay is 1.6-32 nmol (0.5-10 micrograms) of 13-HPODE. The utility of the assay can be extended to detect other peroxides as well as inhibitors of 15-lipoxygenase. The assay is a rapid, reliable method for the detection of lipid hydroperoxide production.     

The surface activity of PVP and other polymers and their antihemolytic capacity

The polymeric cryoprotective agents polyvinylpyrrolidone, dextran, and hydroxyethyl starch do not penetrate the cell membrane and are not present in high osmotic concentrations. Thus, they can exert little of the "antifreeze" behavior generally attributed to glycerol or dimethyl sulfoxide, and must protect cells from freezing injury by some action external to the cell surface. Surface energy measurements of droplets of hemoglobin solution immersed in solutions of cryoprotective polymers indicate that these polymers lower the surface energy of the solution below that of the hemoglobin droplets and form a stable interface. In injured cells, these polymers will therefore hide membrane defects by forming an interface across which hemoglobin cannot easily pass. When freezing is slow, the polymers have little if any true cryoprotective effect but interfere with hemoglobin release as an assay of injury.     

Characterization of hemoglobin binding to Actinobacillus actinomycetemcomitans

In this study, binding of hemoglobin to Actinobacillus actinomycetemcomitans was characterized. The ability of A. actinomycetemcomitans to utilize hemoglobin as an iron source was examined by growth studies. Although the bacterial growth was limited almost completely by adding 400 microM 2, 2'-dipyridyl to culture medium, addition of hemoglobin recovered the growth in a dose-dependent manner, revealing that hemoglobin can be utilized effectively as an iron source by A. actinomycetemcomitans. Binding of A. actinomycetemcomitans to hemoglobin was examined by dot-blot assay. Optimal hemoglobin-binding activity occurred at pH 6 and activity under acidic conditions was found to be higher than that under alkaline conditions. Hemoglobin-binding activity was higher under anaerobic conditions than under aerobic conditions, and iron restriction in culture medium decreased the activity by 55%. Heat and trypsin treatments of the bacterial components reduced the activity by 28% and 60%, respectively. Globin inhibited the activity by 49%, while transferrin, lactoferrin and tested amino acids and sugars had little or no inhibitory effects. These results indicate that proteinaceous components of the bacterial cells may be involved in hemoglobin binding and that globin moiety of the hemoglobin molecule may be essential for the binding. In order to identify hemoglobin-binding proteins, the bacterial cell components extracted with n-octyl-beta-D-thioglucoside were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with hemoglobin and bound hemoglobin was detected with anti-hemoglobin antibodies. About 40- and 65-kDa proteins from A. actinomycetemcomitans reacted with hemoglobin. The 65-kDa protein was detected despite the iron concentration in culture medium, whereas expression of the 40-kDa protein was enhanced only when the organism was grown in iron-restricted culture medium. From these results, it is suggested that 40- and 65-kDa proteins of A. actinomycetemcomitans may be involved in hemoglobin binding.     

Quantifying adductive modification of hemoglobin from mice exposed to benzo[a]pyrene

The present work describes a method for the detection of minute amounts of benzo[a]pyrene, as the diolepoxide metabolite, bound covalently to the hemoglobin of erythrocytes isolated from mice previously exposed to the carcinogen. The technique consists of the acid-induced removal of the pyrenyl moiety from the hemoglobin as the strongly fluorescent free tetrols and their isolation by bonded-phase extraction methods and subsequent quantitation by fluorescence/HPLC. With this procedure as little as 5 pg of tetrol can be detected. The assay was used to determine the amount of benzo[a]pyrene-hemoglobin adduct formation in mice bearing a carcinogen-induced fibrosarcoma.     

A radioimmunoassay specific for opsin

A radioimmunoassay is developed for bovine opsin using a rabbit antiserum against bovine rod outer segment membranes. The assay is specific for opsin. Rhodopsin, bacteriorhodopsin and hemoglobin do not show cross-reaction. It can be carried out rapidly, has a sensitivity of 0.01 pmol bovine opsin and gives accurate results, even in the presence of a large excess of rhodopsin. Under the conditions described, the assay can be used to measure bovine opsin and rhodopsin in each other's presence by running a sample before and after illumination, with a sensitivity 2000-times higher than with spectrophotometric methods. The opsin content of rather crude preparations such as bovine retina homogenates can be accurately determined. Rabbit and mouse opsin can also be assayed with a reasonable degree of accuracy using the same rabbit antiserum.     

Diagnostic Accuracy of the HemoCue Hb 301, STAT-Site MHgb and URIT-12 Point-of-Care Hemoglobin Meters in a Central Laboratory and a Community Based Clinic in Durban,South Africa

In South Africa, various point-of-care hemoglobin meters are used. However, the regulatory framework for approval, implementation and oversight of use of point-of-care hemoglobin meters is suboptimal. We assessed the diagnostic accuracy of the HemoCue Hb 301, STAT-Site M^Hgb and URIT-12 point-of-care hemoglobin meters, compared to a central laboratory based reference assay, in a central laboratory and a community based clinic in Durban, South Africa. Differences in performance of the point-of-care assays, compared to the reference assay, were more pronounced in the community based clinic. Results were reasonable for the HemoCue Hb 301, but poor for the STAT-Site M^Hgb and the URIT-12. Poor test performance of point-of-care hemoglobin meters, and inadequate evaluations and oversight in South Africa, leads to suboptimal clinical care and clinical research, and increased costs. There is a need for proper evaluation and quality assurance of point-of-care tests, the results of which should be made widely available to key stakeholders.     

A rapid spectrophotometric assay for ferrochelatase activity in preparations containing much endogenous hemoglobin and its application to soybean root-nodule preparations.

A simple and rapid spectrophotometric assay for ferrochelatase (EC 4.99.1.1) activity is described which is suitable for use with enzyme preparations containing large amounts of hemoglobin. In this method mesoporphyrin IX is used as substrate and the product, mesoheme IX, is measured by recording the reduced minus the oxidized pyridine hemochrome spectrum. Pyridine mesohemochrome has an α peak at 547 nm and a trough at 531 nm while the α peak and trough of pyridine protohemochrome (from hemoglobin) are at 557 and 541 mm, respectively. Thus the contribution of the protohemochrome to E_1cm^547-531nm, which can be allowed for, is small, and so the method gives very reliable determinations of the amounts of mesoheme IX formed.By means of this assay, it was shown that in excess of 90% of the ferrochelatase activity of soybean root-nodules is present in the bacteroid cells and less than 10% in the plant mitochondria: No ferrochelatase activity could be detected in the soluble plant fraction.     

Hemoglobin: normal values

Hematologists are not in agreement as to the "normal" amount of hemoglobin in the blood, nor is there agreement as to what amount of hemoglobin can be considered "a hemoglobin value of 100 per cent." Different hospitals base reports of hemoglobin on different standards, which obviously can be misleading. By biometric study of the great mass of data on hemoglobin content that has become available as a result of the blood procurement program, it should be possible to determine what "normal" values are and to provide a basis for uniformity in reporting.     

Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin.

Hemoglobin complexed to the plasma protein haptoglobin can be used by Neisseria meningitidis as a source of iron to support growth in vitro. An N meningitidis mutant, DNM2E4, was generated by insertion of the mini-Tn3erm transposon into the gene coding for an 85-kDa iron-regulated outer membrane protein. Membrane proteins prepared from DNM2E4 were identical to those of the wild-type strain except that the 85-kDa protein was not produced. This mutant was unable to use hemoglobin-haptoglobin complexes as an iron source to support growth and was also impaired in the utilization of free hemoglobin. The mutant failed to bind free hemoglobin, hemoglobin-haptoglobin complexes, or apo-haptoglobin in a solid-phase dot blot assay. The 85-kDa protein was affinity purified when hemoglobin-haptoglobin complexes were used as a ligand but was not purified when free hemoglobin was used. We hypothesize that the 85-kDa iron-regulated protein is the hemoglobin-haptoglobin receptor and designate this protein Hpu (for hemoglobin-haptoglobin utilization).     

Hemoglobin effects in the Saville assay.

There is a great need to establish accurate, sensitive methods for measuring the concentration of nitrosothiols. Although some progress may have been made recently, differing methodologies have lead to reports of basal levels of nitrosothiols in human plasma that differ by three orders of magnitude. The Saville assay has been widely accepted as an accurate method for measuring nitrosothiols, but one that suffers from sensitivity below that of some other methods. Recently, it has been suggested that when hemoglobin is included in reaction mixtures used for the Saville assay, the sensitivity can be increased by an order of magnitude. Here we show that, on the contrary, the presence of sufficient hemoglobin in the Saville assay decreases its sensitivity.     

A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.

Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.     

Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment

The tri-iodide-based chemiluminescence assay is the most widely used methodology for the detection of S-nitrosothiols (RSNOs) in biological samples. Because of the low RSNO levels detected in a number of biological compartments using this assay, criticism has been raised that this method underestimates the true values in biological samples. This claim is based on the beliefs that (i) acidified sulfanilamide pretreatment, required to remove nitrite, leads to RSNO degradation and (ii) that there is auto-capture of released NO by heme in the reaction vessel. Because our laboratories have used this assay extensively without ever encountering evidence that corroborated these claims, we sought to experimentally address these issues using several independent techniques. We find that RSNOs of glutathione, cysteine, albumin, and hemoglobin are stable in acidified sulfanilamide as determined by the tri-iodide method, copper/cysteine assay, Griess-Saville assay and spectrophotometric analysis. Quantitatively there was no difference in S-nitroso-hemoglobin (SNOHb) or S-nitroso-albumin (SNOAlb) using the tri-iodide method and a recently described modified assay using a ferricyanide-enhanced reaction mix at biologically relevant NO:heme ratios. Levels of SNOHb detected in human blood ranged from 20-100 nM with no arterial-venous gradient. We further find that 90% of the total NO-related signal in blood is caused by erythrocytic nitrite, which may partly be bound to hemoglobin. We conclude that all claims made thus far that the tri-iodide assay underestimates RSNO levels are unsubstantiated and that this assay remains the "gold standard" for sensitive and specific measurement of RSNOs in biological matrices.     

HEMOGLOBIN: NORMAL VALUES

Hematologists are not in agreement as to the “normal” amount of hemoglobin in the blood, nor is there agreement as to what amount of hemoglobin can be considered “a hemoglobin value of 100 per cent.” Different hospitals base reports of hemoglobin on different standards, which obviously can be misleading.By biometric study of the great mass of data on hemoglobin content that has become available as a result of the blood procurement program, it should be possible to determine what “normal” values are and to provide a basis for uniformity in reporting.     

A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo

Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2,000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins.