New values for the molar extinction coefficients of NADH and NADPH for the use in routine laboratories (author's transl)]

Extensive re-investigations with regard to the molar extinction coefficients of NADH and NADPH proved that in future, calculations in routine work can be performed with the following much more accurate epsilon-values: 6.15 x 10(3) 1 x mol-1 x cm-1 at Hg 334 nm (NADH and NADPH), 6.3 X 10(3) 1 X mol-1 x cm-1 at 340 nm (NADH and NADPH), 3.4 X 10(3) 1 X mol-1 X Cm-1 (NADH) and 3.5 x 10(3) 1 x mol-1 x cm-1 (NADPH) at Hg 365 nm, respectively. The safest measurement is performed at Hg 334 nm, because here epsilon is identical for both coenzymes and deviations of the epsilon-value caused by temperature, pH and ionic strength are less than 0.5%.     

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.     

Candidate reference method for determination of total bilirubin in serum: development and validation

This candidate Reference Method for measuring total bilirubin in serum is based on the Jendrassik-Gróf principle (Clin Chem 29: 297-301, 1983). Standard Reference Material no. 916 bilirubin (National Bureau of Standards) is used as the standard. Bilirubin standard solutions may be prepared either in human serum or in 40 g/L albumin solution (human or bovine), because we found the molar absorptivity of the azopigment at 598 nm to be identical in these media. The absorptivities of the unconjugated and conjugated azopigments appear to be identical, but the conjugated azopigment is completely hydrolyzed in the final reaction mixture. Bilirubin added to serum from adults or neonates was quantitatively accounted for. Interference by hemoglobin (up to 2 g/L), ascorbic acid (up to 20 mg/L), or zinc (at physiological concentrations) is negligible. Of the therapeutic drugs we tested, only L-dopa and alpha-methyldopa interfere. We established normal adult reference values for total bilirubin and examined the intraindividual variation in 19 subjects.     

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.     

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.     

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。     

Methods for determination of conjugated bilirubin in rat faeces

Conjugated bilirubin was prepared from the faeces of germ-free (GF) rats by three different preparative methods. The bilirubin conjugate preparations were coupled with diazotized ethyl anthranilate and the formed ethyl anthranilate azopigments were quantified spectrophotometrically and separated by thin-layer chromatography (tlc). The most polar azopigment was purified by tlc and subjected to ammonolysis followed by tlc of the released saccaride. As a result of this procedure, only glucuronic acid was detected as the conjugating saccaride thus indicating that the most polar azopigment prepared from GF rat faeces was the delta ethyl anthranilate azopigment. Reference azopigments were prepared from GF rat small intestinal contents and subjected to separation by tlc. The azopigment pattern was very similar to the pattern obtained with the faecal azopigment preparations and a maximum of ten separated azopigment spots were detected. The findings indicated that, in addition to bilirubin glucuronides, other bilirubin conjugates with unknown structure are excreted with the faeces of GF rats. One of the preparative methods used for the preparation of conjugated bilirubin from GF rat faeces was tested on faeces from conventional (CONV) rats. From these preparations, no ethyl anthranilate azopigments were formed, thus indicating that faeces from CONV rats is devoid of conjugated bilirubin.     

Investigation of protoporphyrin IX standard materials used in acid-extraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX

Erythrocyte protoporphyrin (EP) has been used for more than 30 years as an indicator of lead intoxication, iron deficiency, and porphyrias. Recently, numerous analytical problems associated with various EP methods have been reported, including a lack of consensus among investigators regarding the best calibration material or analytical procedure. We investigated commercially available protoporphyrin IX (PPIX) standard materials and measured the millimolar absorptivity (m epsilon) of these materials, focusing on variables affecting the determination of their absorptivities. Among the five forms of PPIX available, PPIX dimethyl ester, when hydrolyzed to PPIX free acid, gave the most consistent and reproducible results. This work confirmed our earlier observations, made on more than 600 separate occasions during 12 years, that the m epsilon of PPIX free acid in 1.5 mol/L HCl at the Soret maximum is 297 +/- 1.3 L.mmol-1.cm-1, 19% higher than the arbitrary value of 241 L.mmol-1.cm-1 generally accepted by most investigators but based on unpublished data. We propose that the m epsilon of 297 L.mmol-1.cm-1 for PPIX be adopted and that PPIX dimethyl ester be used for the calibration of acid-extraction methods. A detailed protocol for the preparation and verification of PPIX from the dimethyl ester is available upon request.     

Delta bilirubin: absorption spectra, molar absorptivity, and reactivity in the diazo reaction

Delta bilirubin (B delta), isolated from serum, has an absorption maximum near 440 nm and a molar absorptivity of 72,000 L mol-1cm-1 in either Tris HCl (0.1 mol/L, pH 8.5) or phosphate (0.13 mol/L, pH 7.4) buffer. This absorptivity exceeds by approximately 50% and 59%, respectively, that of unconjugated bilirubin in the same buffers. This finding suggests that substantial errors can be incurred in direct spectrophotometry of bilirubins in serum. In the total diazo (TBIL) assay (Clin Chem 1985;31:1779-89), the color yield from B delta increases by 10% as the final diazo concentration is increased from 0.27 to 0.81 mmol/L. In the direct (DBIL) assay, if done in HCl (50 mmol/L), B delta yields approximately 15% more color as the diazo concentration is increased from 0.51 to 1.53 mmol/L, whereas in acetate buffer (0.4 mol/L, pH 4.7) the corresponding color yield is 25% greater. However, the absolute color yield for the reaction in HCl exceeds that in acetate buffer. In both the TBIL and the DBIL assay, B delta reacts slowly, nearly complete reaction requiring 10 min. Thus, B delta may be seriously underestimated in diazo (especially DBIL) methods in which short reaction times (20 s to 1 min) are used.     

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 beta-NADH and beta-NAD at 260 nm.

To determine the molar absorptivities of beta-NADH and beta-NAD at 260 nm, we purified the reduced form of the coenzyme by repeated anion-exchange chromatography. A NADH preparation was so obtained for which the 260 nm/340 nm absorbance ratio was 2.265. When calculated with epsilon 340 beta-NADH = 6.22 times 10-3 for beta-NADH at 260 nm and 25 degrees C, a molar absorptivity of 14.1 times 10-3 liter - mol minus 1 - cm minus 1 resulted from this quotient. By use of the lactate dehydrogenase or glycerol-3-phosphate dehydrogenase assay, respectively, and referring to the new absorption coefficient for NADH at 260 nm, the molar absorptivity of beta-NAD at 260 nm and 25 degrees C was established to be 17.4 times 10-3 liter - mol minus 1 - cm minus 1. The values observed are lower than those reported thus far.     

Fluorimetric quantitation of citalopram and escitalopram in plasma: developing an express method to monitor compliance in clinical trials.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) in general, and citalopram/escitalopram in particular, are widely used to treat clinical depression. However, SSRI bioavailability and non-compliance represent major issues, especially in the clinical trials setting. In this context, frequent drug-level measurements for compliance monitoring would be a desirable tool to improve clinical outcomes with SSRIs. However, the liquid chromatography techniques available are expensive, requiring excessive sample preparation, and suffer from high complexity. We sought to develop a rapid method for the measurement of citalopram/escitalopram levels in human plasma by fluorimetry. METHODS: A total of 34 frozen human plasma samples were thawed at room temperature and repeatedly centrifuged in cellulose to remove aggregates, proteins and solids. Fluorescence spectra were measured in the range 270-450 nm with excitation at 240 nm on a FluoroMax 3 spectrofluorimeter. Control samples contained known concentrations of SSRIs. RESULTS: SSRI absorbance spectra were recorded in the range 230-320 nm. The shape of the spectra and the absorbance of citalopram and escitalopram were very similar, with UV maximum absorbance at 239 nm. The maximum extinction coefficient was epsilon239=15,930 M-1 cm-1 for citalopram and epsilon239=13,630 M-1 cm-1 for escitalopram. The fluorescence spectra of SSRIs are unique and are characterized by the presence of two well-defined conjugated spectra with maxima at 300 and 382 nm. CONCLUSIONS: Fluorimetry is very suitable for assessment of plasma SSRI levels. This inexpensive and efficient technique can objectively and reliably quantify drug levels in biological fluids, thereby directly determining the level of patient adherence to the prescribed drug regimen. This method will be useful in a broad spectrum of applications, from compliance/bioavailability assessments in animal and human experiments to utilization in large-scale clinical trials.     

Purification and characterization of agrocin 84.

A procedure for the rapid purification of milligram quantities of agrocin 84, a bacteriocin-like compound produced by Agrobacterium radiobacter strain K-84, has been developed. This procedure, which employs charcoal adsorption, ion-exchange, sieving chromatography, and continuous-flow electrophoresis, can yield agrocin 84 which is 65% pure on a dry weight basis. The purest preparations were strongly ultraviolet absorbing, with a maximum at 264 nm (epsilon 7.0/264 = 22,675 cm2 - M-1) and a minimum at 227 nm (ratio of 264 to 227 nm, 6.00). As has been reported, agrocin 84 contains an unusual phosphoramidate or 6-N-acyl linkage to adenine. Adenine, glucose, and phosphate are present in a 1:1:2 molar ratio. The molecular weight was estimated to be 1,350.     

The determination of total haemoglobin as haemiglobin

This study describes a new method for routine total haemoglobin determination by conversion to haemiglobin (Hi), and compares it with the haemiglobin cyanide (HiCN) method. Experiments were performed in two phases. Firstly, the absorbance coefficient (epsilon Hi,500mm) at pH = 6.8, determined from 24 blood samples, was found to be 998 +/- 31 m2.mol-1. Secondly, using this value of epsilon, the range of haemoglobin concentration measured was 3.7 to 19.2 mmol.l-1 (6 to 31 g.dl-1). The correlation data shows an excellent correlation (r = 0.982, p less than 0.001) with the reference procedure. Reproducibility and accuracy were shown to be good.     

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.     

One-step protocol for assays of total and direct bilirubin with stable combined reagents

We describe an improved colorimetric method for assays of total and direct bilirubin in serum. Bilirubin reacts with diazotized sulfanilic acid in an acidic medium to form a blue azopigment. Total bilirubin is assayed in the presence of reaction accelerators (caffeine, urea, and citric acid), direct bilirubin in their absence. The azo compound so formed is read at the same wavelength (570 nm) in both assays. A sample blank is run in parallel. Standard curves are linear for total and direct bilirubin concentrations up to 513.0 and 256.5 mumol/L, respectively. The method is characterized by (a) use of the same protocol for both assays, i.e., a one-step procedure with short reaction time (5 min at room temperature), and (b) use of a single working solution, which, refrigerated, is stable for one month. The method is reliable, yields results that compare closely with those of the classical Jendrassik--Gróf method, is suitable for routine use, and lends itself to automation.     

Chemiluminescence of UV-irradiated linolenic acid and squalene

An emission spectral analysis was carried out on ultraweak chemiluminescence emitted from UVB-irradiated linolenic acid and squalene. The main emission species produced by the transition of (1 delta g) (1 delta g) dimer and an additional weak band near 477.5 nm (0, 0) by the transition of (1 delta g (1 epsilon g+) to (3 epsilon g-) (3 epsilon g-) were found by spectroscopic analysis of chemiluminescence in both cases of irradiated linolenic acid and of squalene. A distinct peak around 410-420 nm was observed in irradiated squalene and the emitter seems to be due to the excited carbonyl compound.     

氧合、碳氧及高铁血红蛋白拉曼光谱的测定

目的测定比较氧合、碳氧和脱氧血红蛋白的拉曼光谱。方法分别向氧合血红蛋白(HbO2)中通入一氧化碳(CO)、加入高铁氰化钾(K3Fe(CN)6)制备得到碳氧血红蛋白(COHb)及高铁血红蛋白(MetHb);在785 nm波长激发光下,测定HbO2、COHb及MetHb的拉曼光谱。结果 HbO2、COHb及MetHb分别在1 210～1 230、1 375～1 379和1 550～1 650 cm-1处的差异可在拉曼光谱中分辨。COHb相较于HbO2在1 550～1 650 cm-1处特征峰(与自旋态及卟啉环中心孔径大小相关)发生变化,即1 559 cm-1特征峰峰强增高,1 589和1 645 cm-1特征峰强度降低;MetHb相较于HbO2在1 210～1 230 cm-1(与自旋态及次甲基键变形振动相关)、1 375～1 379 cm-1(与血红蛋白氧化态相关)和1 550～1 650 cm-1处特征峰变化,即1 228 cm-1特征峰移至1 215 cm-1处,1 375～1 379 cm-1处特征峰峰强增高,1 550 cm-1特征峰峰强增高,1 589和1 645 cm-1特征峰强度降低。结论用785 nm激发的拉曼光谱可以鉴别HbO2、COHb及MetHb,可作为1种针对血红蛋白样品快速、准确、无损的在线监测方法。