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。     

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.     

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.     

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.     

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%.     

Micro-quantification of cyclosporine and its metabolites and determination of their spectral absorptivities.

We report a micromethod for the analysis of cyclosporine (CsA), based on quantification of its constituent amino acids. The amino acids were released by gas-phase hydrolysis, derivatized with fluorenylmethyl chloroformate, and separated and analyzed in a reversed-phase HPLC system. The imprecision (CV) of the amino acid analysis was less than 4%, and several determinations of the amount of standard CsA were within 1% of the weighed material. The detection limits (signal-to-noise ratio = 2) were 500 fmol for ultraviolet detection and 100 fmol for fluorescence detection. We also used this method to determine the ultraviolet absorptivities of CsA and five metabolites at 210, 214, and 230 nm. The molar absorptivity of most metabolites was about 10% higher than that of CsA, although the metabolite that was oxidized to a carboxyl group on the terminal carbon of N-methyl-butenyl-methyl-threonine (AM1A) had a molar absorptivity about 40% higher than that of CsA.     

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.     

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.     

A sensitive, direct colorimetric assay of serum copper using 5-Br-PSAA

A direct colorimetric assay of serum copper in 0.2-ml sized samples, using a new, water-soluble reagent, 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino) aniline Na salt (5-Br-PSAA epsilon = 6.5 X 10(4) l/mol per cm at 580 nm), is presented. Protein-bound serum copper is dissociated with guanidine HCl which acts also as a protein denaturant. Metal ions, except for cobalt and palladium, do not affect the determination even when present at levels exceeding those normally found in serum. Although bilirubin does interfere with the Cu-5-Br-PSAA complex formation, the copper values in the icteric sera with total bilirubin concentrations of less than 137 mumol/l (80 mg/l) can be corrected by a correction factor which varies with the bilirubin content. Within-run and between-run precisions were in the ranges of 0.8-2.3% and 1.1-3.0% coefficient of variation (CV). Good correlation was obtained between this method and atomic absorption spectrometry.     

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.     

A highly sensitive colorimetric determination of serum zinc using water-soluble pyridylazo dye

A colorimetric method for precise and accurate determination of zinc in serum is presented. Only 0.3 ml of sample is necessary, because of the use of a new, highly sensitive reagent, 2-(5-bromo-2-pyridylazo)-5-(N-n-propyl-N-3-sulfopropylamino)-phenol ($\text{?}{}_{\mathrm{<mtext>554</mtext><mtext>nm</mtext>}}\text{= 1.3 × 10}{}^5\text{1 ·}\text{mol}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$), which is water-soluble and has long-term stability. Interference of iron and copper in serum can be removed by co-precipitation of the iron fluoride complex with trichloroacetic acid precipitated proteins and the copper dithiocarboxy sarcosine complex, respectively. Within-run and day-to-day precision (CV) are in the range of 0.3–3.5% and 1.9–3.1%, respectively, depending on the serum zinc content. A good correlation (r = 0.98, p < 0.05) was obtained between this method and atomic absorption spectrometry. In contrast to previous colorimetric methods, the present method does not involve heating, extraction with organic solvents, or a cyanide masking system.     

Production and certification of an enzyme reference material for gamma-glutamyltransferase (CRM 319). Part 2: Certification campaign

We describe the process of certification for a gamma-glutamyltransferase reference material (CRM no. 319). Fifteen laboratories participated to this interlaboratory evaluation. All steps of the measurements were controlled in an effort to locate potential sources of variations. In particular, the exclusion of some data was strictly documented or justified by the non-observance of the IFCC method and (or) discrepancies in instrumentation, reconstitution of the lyophilized samples, or measurement technique. Inaccuracy in the reconstitution of the lyophilized material was +/- 0.68%, and the molar absorptivity of the 5-amino-2-nitrobenzoate reported by each laboratory was within +/- 2% limits of the value reported by the IFCC. Calculated from the sets of accepted results, the total CV among samples was 2.6% and the overall CV was 3.2%. Within-day and between-day CVs were 1.1% and 1.4%, respectively. The greatest variation for a single component was the between-laboratory variability (CV 3.1%); the within-laboratory CV, including the day effect, was 1.8%. Finally, the certified value for the catalytic concentration of this enzyme in the reconstituted lyophilized reference material was 86.8 U/L with an uncertainty of +/- 2.1 U/L (0.95 confidence interval). The uncertainty appeared to be compatible with the end-use of this reference material.     

Simple, rapid spectrophotometry of urinary N-acetyl-beta-D-glucosaminidase, with use of a new chromogenic substrate

We have developed a new spectrophotometric assay for urinary N-acetyl-beta-D-glucosaminidase (NAGase) with use of sodio m-cresolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide (MCP-NAG). MCP-NAG was synthesized from acetochloro-glucosamine and m-cresolsulfonphthalein (MCP) in four steps. MCP-NAG reacts well with NAGase (Km = 0.41 mmol/L) and is highly water soluble. The absorption maximum and molar absorptivity of the aglycone MCP are 580 nm and 40 670, respectively. Spectral overlap of interfering substances at 580 nm is almost negligible, so that the urine blank can be omitted from the assay procedure. The high molar absorptivity of MCP gives sufficient analytical sensitivity at a reaction time of 15 min. The correlation between the MCP-NAG method (y) and the fluorimetric method (x) involving 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide is represented by the equation y = 0.995x - 0.669 (r = 0.991). Thus, the present method provides practical advantages over conventional methods, for use in the routine laboratory.     

A sensitive, direct colorimetric assay of serum iron using the chromogen, nitro-PAPS

A direct colorimetric method is presented for the determination of serum iron in 0.1-ml sized samples, using a new, water-soluble, reagent, 2-(5-Nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol Na salt (epsilon 585 nm = 9.4 X 10(4) l/mol per cm). Interference of copper and zinc in sera can be eliminated entirely by forming copper- and zinc-thioglycollate complexes immediately upon the dissociation of the protein-bound iron, copper and zinc by thioglycollate and sodium dodecyl sulfate. The serum blank was minimized by the use of sodium dodecyl sulfate as a protein denaturant. Within-run and between-run precision (CV) were in the range of 0.7-2.9% and 1.1-3.6%, respectively, depending on the serum iron content. A good correlation (r = 0.995) was obtained between this method and the reference method proposed by the International Committee for Standardization in Hematology.     

The use of infrared spectrophotometry for measuring body water spaces.

BACKGROUND: The conventional method of measuring total body water by the deuterium isotope dilution method uses gas isotope ratio mass spectrometry (IRMS), which is both expensive and time-consuming. We investigated an alternative method, using Fourier transform infrared spectrophotometry (FTIR), which uses less expensive instrumentation and requires little sample preparation. METHOD: Total body water measurements in human subjects were made by obtaining plasma, saliva, and urine samples before and after oral dosing with 1.5 mol of deuterium oxide. The enrichments of the body fluids were determined from the FTIR spectra in the range 1800-2800 cm-1, using a novel algorithm for estimation of instrumental response, and by IRMS for comparison. RESULTS: The CV (n = 5) for repeat determinations of deuterium oxide in biological fluids and calibrator solutions (400-1000 micromol/mol) was found to be in the range 0.1-0.9%. The use of the novel algorithm instead of the integration routines supplied with the instrument gave at least a threefold increase in precision, and there was no significant difference between the results obtained with FTIR and those obtained with IRMS. CONCLUSION: This improved infrared method for measuring deuterium enrichment in plasma and saliva requires no sample preparation, is rapid, and has potential value to the clinician.     

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.     

一种复方消毒乳液对新型冠状病毒的杀灭效果

试验观察了一种含1700～1900mg/L醋酸氯己定、1000mg/L纳米氧化锌的复方消毒乳液,对引起重症急性呼吸综合症(SARS) 的新型冠状病毒的杀灭效果。按照我国《消毒技术规范》中的病毒灭活试验方法进行测试。结果证明,该产品80％浓度的原液直接作用1min,即可完全杀灭试验室培养的新型冠状病毒,平均病毒灭活对数值>5.00。该产品可用于非典型肺炎流行区人员的手、皮肤消毒。     

5-Aminolaevulinic acid peptide prodrugs enhance photosensitization for photodynamic therapy

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-L-Phe-ALA-Me (compound 1) and Ac-L-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD(50) (2.5 J/cm(2)) of 25 micromol/L for ALA, 6 micromol/L for 5-aminolaevulinic hexyl ester, and 2.6 micromol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding D-enantiomer, Ac-D-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH.     

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.