Molar absorptivities of beta-NADH and beta-NADPH.

Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.     

Determination of the molar absorptivity of NADH.

The molar absorptivity of NADH at 340 nm has been determined by an indirect procedure in which high-purity glucose is phosphorylated by ATP in the presence of hexokinase, coupled to oxidation of the glucose-6-phosphate by NAD+ in the presence of glucose-6-phosphate dehydrogenase. The average value from 85 independent determinations is 6317 liter mol-1 cm-1 at 25 degrees C and pH 7.8. The overall uncertainty is -4.0 to +5.5 ppt (6292 to 6352 liter mol-1 cm-1), based on a standard error of the mean of 0.48 ppt and an estimate of systematic error of -2.6 to +4.1 ppt. Effects of pH, buffer, and temperature on the molar absorptivity are also reported.     

A highly sensitive, simple determination of serum iron using chromazurol B.

A highly sensitive, simple determination of serum iron and binding capacity is described. FeIII/FeII reacts with chromazurol B (CAB) and cetyltrimethyl ammonium bromide (CTMA), the resulting substance being a highly coloured ternary complex. Maximal absorbance of the complex occurs at pH 4.6--5.5 at 630 nm. Lambert Beer's law holds between 0 and 80 mumol Fe/l. Molar absorptivity is 1.68 X 10(5) 1 . mol-1 . cm-1 at 630 nm. Interference by other serum components is negligible even at high concentrations.     

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.     

Comparisons of 17 lots of 2-oxoglutarate, and specifications for use of this substrate in reference methods

We examined 17 lots of 2-oxoglutarate (seven acid forms, three K salt forms, and seven Na salt forms), obtained from eight commercial suppliers, for suitability for measuring aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in human serum. Measurements of the catalytic activity concentrations of these two aminotransferases with each of these 17 preparations were not sufficiently sensitive to distinguish good from poor-quality material. Thus, we ranked these lots for purity, by specific analysis with glutamate dehydrogenase and by liquid chromatography, and determined the water content, acid content, and spectral characteristics of each. On the basis of a 2-oxoglutarate assay value by glutamate dehydrogenase of 98% or greater, we considered seven of the preparations acceptable and 10 unacceptable. The molar absorptivities (L X mol-1 X cm-1, mean +/- SD) of the seven acceptable lots in 1 mol/L HCl were: epsilon 325 nm = 9.12 +/- 0.02 (CV = 0.2%), epsilon 279 nm = 2.63 +/- 0.23 (CV = 9.9%), and epsilon 245 nm = 37.9 +/- 4.1 (CV = 10.9%). Use of these spectrophotometric limits alone unambiguously distinguished the inferior lots of 2-oxoglutarate. We urge the inclusion of detailed spectrophotometric specifications for 2-oxoglutarate in Reference Methods for aminotransferase measurements.     

Sensitive, direct colorimetric assay for copper in serum

We have developed a sensitive procedure for determination of serum copper by use of the color reagent 4-(3,5-dibromo-2-pyridylazo)-N-ethyl-N-sulfopropylaniline. After mixing serum sample and reagent, and incubating at 37 degrees C for 5 min, we measure the absorbance of the resulting chelate complex at 580 nm (molar absorptivity, 80,000 L.mol-1.cm-1). Results of the method varied linearly with copper concentration to at least 5 mg/L; the lower limit of detection was 0.1 mg/L. Within-run CVs were 1.6% and 3.3% for copper concentrations of 1.03 and 0.72 mg/L, respectively (n = 10 each). Between-run CV was 2.8% at 1.22 mg/L (n = 14). Results of the proposed method (y) correlated well with those determined by standard atomic absorption spectrophotometric techniques (x): y = 0.99x - 0.02 mg/L; Syx = 0.08; r = 0.977; n = 56. Iron, zinc, cadmium, cobalt, and lead do not interfere.     

5-Br-PADAP直接光度法测定血清锌

目的建立一种快速、简便、灵敏的分光光度法测定血清锌。方法在表面活性剂TritonX-100存在下,用2-(5-溴-2-吡啶偶氮)-5-二乙氨基酚(5-Br-PADAP)作显色剂,不去蛋白直接光度法测定血清锌。结果该方法显色络合物最大吸收波长为558nm,线性范围达51.0μmol/L,表观摩尔吸光数为1.05×105L*mol-1*cm-1。回收率为98.1%～103.2%,批内和批间变异系数(CV)分别为2.1%与3.6%,与原子吸收分光光度法比较相关良好,Y=1.02X-0.41,r=0.9862,P＞0.05。48名健康人血清锌含量为(9.5～24.3)μmol/L(±2s)。结论该法血清用量少,不必去蛋白,具有操作快速、简便、结果灵敏可靠等优点,适合临床应用。     

Spectrophotometric investigation of sensitive complexing agents for the determination of zinc in serum

Of two sensitive complexometric reagents for the colorimetry of serum zinc that we investigated, one, 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP), was found to be a potentially useful compound for trace-metal determinations. It has a high molar absorptivity (120 000 L mol-1 cm-1) but is not convenient to use because it is not very soluble in water. The other reagent, a related pyridylazo compound, is 2-(5-bromo-2-pyridylazo)-5-(N-n-propyl-N-3-sulfopropylamino)phenol (5-BR-PAPS). It seems better suited for use in routine zinc determinations because, besides being water soluble, it has a higher molar absorptivity, 130 000 L mol-1 cm-1. Results by the proposed method developed with 5-Br-PAPS correlated well with those by atomic absorption spectrophotometry. The between-run CV for control sera was less than 5%; the within-run CV (same controls) was less than 4%.     

Kinetic assay of gamma-glutamyltransferase with use of bilirubin oxidase as a coupled enzyme

We studied the kinetic measurement of gamma-glutamyltransferase (EC.2.3.2.2), coupling the reaction with that catalyzed by bilirubin oxidase (EC 1.3.3.5), which oxidizes and combines a phenylenediamine derivative with an aniline derivative to produce a green pigment. We measured the formation of the pigment kinetically (at lambda max745 nm, epsilon = 75 000 L mol-1 cm-1), with L-gamma-glutamyl-N-hydroxyethylaminoanilide as substrate and N-ethyl-N-hydroxy-3-sulfopropyl)-m-toluidine as a the coupling derivative. The within-run CV for measuring this reaction in samples of normal sera was 2.4%. A calibration plot of the change in absorbance per minute vs enzyme activity concentration showed good proportionality in the range of 0-1300 U/L. The results of this assay correlated well (r = 0.995) with those of the Boehringer method, in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the substrate. This new, highly sensitive procedure may be adapted to other assays involving phenylenediamine derivates as synthetic substrates.     

Mechanism of inhibition of erythrocyte glutathione reductase by mitomycin-C

Mitomycin-C (MT-C) inhibits human erythrocyte glutathione reductase (EGR). We found that Km values of EGR for GSSG, NADPH, and FAD were 1.3 X 10(-4) mol/l, 2.3 X 10(-5) mol/l, and 3.9 X 10(-7) mol/l respectively, and that EGR was inhibited by MT-C non-competitively with respect to both the substrate GSSG (Ki = 4.8 X 10(-5) mol/l) and the cofactor NADPH (Ki = 2.2 X 10(-4) mol/l), and competitively with respect to FAD (Ki = 4.2 X 10(-5) mol/l). On the other hand, we demonstrated that FAD makes a complex with MT-C, the dissociation constant (K = 6.5 X 10(-5) mol/l) of which was obtained from fluorescence quenching of FAD with MT-C. It also became clear that spectra wf FAD at around 375 nm changes with increasing MT-C concentration. So, the data strongly suggest that the mechanism of inhibition of EGR by MT-C is mainly due to complex formation of the co-enzyme FAD with MT-C.     

An equilibrium model of the oxygen association curve of normal human erythrocytes under standardized conditions

Hemoglobin-oxygen association curves of human erythrocytes were measured in metabolically stable cells under the standardized conditions of extracellular pH 7.400, carbon dioxide tension 38 mmHg (approximately 5 kPa) and temperature 37 degrees C. A model of the oxygen association curve of normal erythrocytes was subsequently developed, based on assumed equilibrium reactions between hemoglobins (Hb), oxygen, and 2,3-diphosphoglycerate (DPG). Conditional equilibrium constants were determined for reactions leading to the formation of species designated Hb-O2 (K = 0.14 mmHg-1), Hb-(O2)4 (K = 0.44 X 10(-4) mmHg-4), Hb-DPG (K = 0.30 X 10(4) L mol-1) and Hb-O2-DPG (K = 0.61 X 10(2) L mol-1 mmHg-1) for unmodified hemoglobin, and Hb-O2 (K = 0.14 mmHg-1) and Hb-(O2)4 (K = 0.44 X 10(-4) mmHg-4) for glycosylated hemoglobin. This model provides a practical means of calculating the expected oxygen association curve for a sample of erythrocytes under standardized conditions, given the intracellular concentrations of hemoglobins and 2,3-diphosphoglycerate and the assumption that the hemoglobins in those cells function normally.     

Stability of NADPH: effect of various factors on the kinetics of degradation

Seeking to minimize degradation of NADPH during storage, reagent preparation, and assays, we investigated the effects of pH, temperature, and ionic strength as well as the effects of phosphate and acetate. NADH was also included for comparison. Our results indicate that the rate of degradation of NADPH is proportional most importantly to temperature and concentrations of hydronium ion, but also to concentrations of phosphate and acetate. The degradation rate decreased with increasing ionic strength at neutral pH, but increased slightly at lower pH. NADPH generally is less stable than NADH under the same conditions. The reaction orders with respect to hydronium ion and anions were near 1 for NADH degradation reactions, about 0.5 for NADPH. Rate constants for NADH and NADPH differed more at higher pH and lower phosphate and acetate concentrations.     

Correlated,simultaneous, multiple-wavelength optical monitoring in vivo of localized cerebrocortical NADH and brain microvessel hemoglobin oxygen saturation

Current forms of brain monitoring, such as electroencephalography (EEG), have had limited clinical utility. The EEG records spontaneous cerebrocortical activity and thus is an indirect indicator of metabolic demand and, to a lesser extent, an indicator of mismatch of supply versus demand. Ischemia modulates EEG activity in ways that can usually be detected, but EEG patterns can be similarly modulated by many other factors, including temperature and pharmacologic manipulation. This in vivo study in physiologically monitored animals evaluated the use of correlated optical spectroscopy, performed with an instrument having a fiberoptic light-guide bundle in contact with the cerebral cortex, for the simultaneous monitoring of cerebrovascular oxygen availability and intracellular oxygen delivery. A highly specific monitor of cerebral intracellular oxygen supply, the cerebrocortical intramitochondrial NADH redox state, was monitored in vivo with a fluorescence technique. Absorption spectroscopy was used concurrently to monitor hemoglobin content (blood volume) and oxygen saturation in the microcirculation. Correlated changes in optical signals from cerebrocortical NADH and hemoglobin were studied in a swine model (n=7) of nitrogen hypoxia. Measurements were made at four wavelengths with a time-division, multiplexed fluorometer/reflectometer. Because the NADH fluorescence signal at 450 nm is affected by local changes in blood volume, a “corrected” fluorescence signal is usually calculated. In previous studies, where only two wave lengths have been measured, attempts at correction were based on reflectance at the excitation wavelength (366 nm). We compared estimators of changes in microcirculatory blood volume using reflection at two wavelengths: 366 nm and 585 nm, the wavelengths for maximum and isobestic absorption. The results of the studies were as follows: (1) during transient hypoxia, NADH and local hemoglobin saturation signals changed in concert with arterial pulse oximetry, with changes in NADH lagging behind changes in saturation by an average of 5.3 seconds; (2) after hypocapnic ventilation to a mean Paco ₂ of 20.2 ± 0.8 mm Hg, NADH increased by 11.5 ± 8.7% (as compared with maximal change during anoxia), local hemoglobin saturation decreased by 7.7 ± 6.4%, and local blood volume decreased by 12.5 ± 13%, while arterial SpO₂ was unchanged; (3) our two measures of local blood volume were closely correlated during carbon dioxide perturbations, but poorly correlated during hypoxic perturbation; and (4) NADH fluorescence provided a more rapid, sensitive indicator of oxygen deprivation than did the EEG. During transient hypoxia, EEG changes occurred 57.4 ± 10.4 seconds after the onset of decline in local hemoglobin saturation, after NADH had completed 50% of its maximal increase. This work was supported in part by research grants from the NIH (GM34767), the Academic Senate of the University of California, and the UCSF Anesthesia Research Foundation.     

NADH and NADPH in human skeletal muscle at rest and during ischaemia

Summary. A method for determining the content of NADH and NADPH in biopsy specimens from human skeletal muscle is described. It is based on the bioluminescent technique, utilizing oxidoreductases specific for NADH and NADPH, respectively. Muscle samples were taken from the lateral portion of the quadriceps muscle in the basal state and following local circulatory occlusion. In resting human skeletal muscle, the content of NADH was 91±6 μmol/kg dry muscle (mean ± SE, n = 11) and the NAD/NADH ratio was 18·8±1·3. The content of NADPH determined in four subjects was 108±2 μmol/kg dry muscle. After 5 min of circulatory occlusion, NADH had risen about 100% and a further increase was found after 10 and 20 min, to a plateau about 150% above the basal value. The muscle content of NADPH also increased but to a minor extent (about 30% above the basal value). The changes in muscle lactate after 5 and 10 min occlusion were not uniform between subjects but an increase was obtained in all subjects after 20 min occlusion. No relation was found between the ratios pyruvate/lactate and NAD/NADH and the latter ratio was smaller than the estimated value, calculated from the LDH equilibrium in the cytoplasm. The results indicate that the major part of NADH in muscle tissue is confined to the mitochondrial compartment. It is concluded that measurement of NADH provides information primarily about the mitochondrial redox state rather than the cytosolic and that changes in NADH precede lactate formation and thus are a more sensitive index of tissue hypoxia than increases in lactate.     

Automated enzymatic assay for plasma ammonia.

An enzymatic method for determining plasma ammonia with the Du Pont Automatic Clinical Analyzer (aca) is described. The assay requires a sample volume of 500 muL for a kinetic ammonia measurement. The reaction is initiated with glutamate dehydrogenase and the rate of depletion of NADPH is monitored with two measurements, 17 s apart, at 340 nm. Reaction conditions have been optimized for maximum sensitivity through both one-factor-at-a-time and multiple variable response surface optimization techniques. Linearity to 1000 mumol of ammonia per liter of plasma has been achieved. No significant interferences were observed from anticoagulants or endogenous blood components, including pyruvate and oxalacetate. Use of the coenzyme NADPH (instead of NADH) in this aca procedure eliminates the lengthy pre-incubation otherwise required for endogenous dehydrogenase reactions.     

Radiocompetitive assay of sulfamido-3-chloro-4-benzoic acid with carbonic anhydrase as binding reagent (author's transl)]

The control of patients treated by diuretic sulfonamides can be carried out by a radiocompetitive assay using their binding properties to carbonic anhydrase (CA). In this paper we have studied the assay of sulfamido-3-chloro-4-benzoic acid (SD3) using dialysis equilibrium as separation procedure. With (CA) 2 X 10(-6) M and 14C-SD3 0.5 X 10(-6) M (specific activity: 2 muCi/mg), can be detected 0.5 X 10(-6) M of (SD3) in the assay medium. 6.5 mg protein present in serum lower the assay sensitivity twenty times, owing to an elevated value of the affinity constant, Ka, of albumin-(SD3) complex (10(3) mol-1). On the other hand, the molecules with sulfamidobenzoic group cannot be differentiated in this procedure.     

Wall stress and patterns of hypertrophy in the human left ventricle.

It is generally recognized that chronic left ventricular (LV) pressure overload results primarily in wall thickening and concentric hypertrophy, while chronic LV volume overload is characterized by chamber enlargement and an eccentric pattern of hypertrophy. To assess the potential role of the hemodynamic factors which might account for these different patterns of hypertrophy, we measured LV wall stresses throughout the cardiac cycle in 30 patients studied at the time of cardiac catheterization. The study group consisted of 6 subjects with LV pressure overload, 18 with LV volume overload, and 6 with no evidence of heart disease (control). LV pressure, meridional wall stress (sigman), wall thickness (h), and radius (R) were measured in each patient throughout the cardiac cycle. For patients with pressure overload, LV peak systolic and end diastolic pressures were significantly increased (220 plus or minus 6/23 plus or minus 3 mm Hg) compared to control (117 plus or minus 7/10 plus or minus 1 mm Hg, P less than 0.01 for each). However, peak systolic and end diastolic (sigman) were normal (161 plus or minus 24/23 plus or minus 3 times 10-3 dyn/cm-2) compared to control (151 plus or minus 14/17 plus or minus 2 times 10-3 dyn/cm-2, NS), reflecting the fact that the pressure overload was exactly counterbalanced by increased wall thickness (1.5 plus or minus 0.1 cm for pressure overload vs. 0.8 plus or minus 0.1 cm for control, P less than 0.01). For patients with volume overload, peak systolic (sigman) was not significantly different from control, but end diastolic (sigmam) was consistently higher than normal (41 plus or minus 3 times 10-3 dyn/cm-2 for volume overload, 17 plus or minus 2 times 10-3 dyn/cm-2 for control, P less than 0.01). LV pressure overload was associated with concentric hypertrophy, and an increased value for the ratio of wall thickness to radius (h/R ratio). In contrast, LV volume overload was associated with eccentric hypertrophy, and a normal h/R ratio. These data suggest the hypothesis that hypertrophy develops to normalize systolic but not diastolic wall stress. We propose that increased systolic tension development by myocardial fibers results in fiber thickening just sufficient to return the systolic stress (force per unit cross-sectional area) to normal. In contrast, increased resting or diastolic tension appears to result in gradual fiber elongation or lengthening which improves efficiency of the ventricular chamber but cannot normalize the diastolic wall stress.     

A convenient enzymatic method for the determination of 4-methyl-2-oxopentanoate in plasma: comparison with high performance liquid chromatographic analysis

A simple and rapid spectrophotometric method for the estimation of 4-methyl-2-oxopentanoate in plasma samples by use of NAD+-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum is described. It is based on the kinetic measurement of the decrease of NADH absorbance at 334 nm. Applicability is demonstrated by comparative measurement of 4-methyl-2-oxopentanoate content in plasma of patients with maple syrup urine disease by the enzymatic and a reversed phase high performance liquid chromatographic method.     

Evaluation of elastase and alpha 1-proteinase inhibitor-elastase uptake by polymorphonuclear leukocytes and evidence of an elastase-specific receptor

Neither resting nor stimulated isolated human polymorphonuclear leukocytes did bind or ingest preformed complexes of alpha 1-proteinase inhibitor and unlabeled/125I-labeled human leukocyte elastase. In contrast, granulocytes bound unlabeled/125I-labeled elastase and the extent of binding was reduced in the presence of respiratory burst stimulators, such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, E. coli endotoxin, and N-formyl-L-methionyl-L-leucyl-L-phenylalanine. In association/dissociation and competition inhibition experiments it was demonstrated that granulocyte-elastase binding was specific and saturable. From Scatchard and non-linear regression analysis there was evidence of a two-class receptor model with independent binding sites. Calculated by the non-linear regression method assuming a two-class receptor model the characteristics of the high affinity/low capacity binding site were K1 = 216 +/- 129 X 10(6) l X mol-1 (means +/- s; n = 3) and R1 = 1.38 +/- 0.95 nmol X l-1 corresponding to 0.083 X 10(6) receptors per cell, whereas the low affinity/high capacity binding site had the characteristics K2 = 0.50 +/- 0.09 X 10(6) l X mol-1 and R2 = 237 +/- 103 nmol X l-1 corresponding to 14.3 +/- 6.2 X 10(6) receptors per cell.     

Correlated, simultaneous, multiple-wavelength optical monitoring in vivo of localized cerebrocortical NADH and brain microvessel hemoglobin oxygen saturation

Current forms of brain monitoring, such as electroencephalography (EEG), have had limited clinical utility. The EEG records spontaneous cerebrocortical activity and thus is an indirect indicator of metabolic demand and, to a lesser extent, an indicator of mismatch of supply versus demand. Ischemia modulates EEG activity in ways that can usually be detected, but EEG patterns can be similarly modulated by many other factors, including temperature and pharmacologic manipulation. This in vivo study in physiologically monitored animals evaluated the use of correlated optical spectroscopy, performed with an instrument having a fiberoptic light-guide bundle in contact with the cerebral cortex, for the simultaneous monitoring of cerebrovascular oxygen availability and intracellular oxygen delivery. A highly specific monitor of cerebral intracellular oxygen supply, the cerebrocortical intramitochondrial NADH redox state, was monitored in vivo with a fluorescence technique. Absorption spectroscopy was used concurrently to monitor hemoglobin content (blood volume) and oxygen saturation in the microcirculation. Correlated changes in optical signals from cerebrocortical NADH and hemoglobin were studied in a swine model (n=7) of nitrogen hypoxia. Measurements were made at four wavelengths with a time-division, multiplexed fluorometer/reflectometer. Because the NADH fluorescence signal at 450 nm is affected by local changes in blood volume, a corrected fluorescence signal is usually calculated. In previous studies, where only two wave lengths have been measured, attempts at correction were based on reflectance at the excitation wavelength (366 nm). We compared estimators of changes in microcirculatory blood volume using reflection at two wavelengths: 366 nm and 585 nm, the wavelengths for maximum and isobestic absorption. The results of the studies were as follows: (1) during transient hypoxia, NADH and local hemoglobin saturation signals changed in concert with arterial pulse oximetry, with changes in NADH lagging behind changes in saturation by an average of 5.3 seconds; (2) after hypocapnic ventilation to a mean Paco ₂ of 20.2 ± 0.8 mm Hg, NADH increased by 11.5 ± 8.7% (as compared with maximal change during anoxia), local hemoglobin saturation decreased by 7.7 ± 6.4%, and local blood volume decreased by 12.5 ± 13%, while arterial SpO₂ was unchanged; (3) our two measures of local blood volume were closely correlated during carbon dioxide perturbations, but poorly correlated during hypoxic perturbation; and (4) NADH fluorescence provided a more rapid, sensitive indicator of oxygen deprivation than did the EEG. During transient hypoxia, EEG changes occurred 57.4 ± 10.4 seconds after the onset of decline in local hemoglobin saturation, after NADH had completed 50% of its maximal increase.This work was supported in part by research grants from the NIH (GM34767), the Academic Senate of the University of California, and the UCSF Anesthesia Research Foundation.