Synchrotron Radiation Fourier Transform Infrared (SR-FTIR) spectroscopy in exploring ancient human hair from Roman period Juliopolis: Preservation status and alterations of organic compounds.

We explore the preservation status and alterations of organic compounds in Roman period human hairstrandsfrom a specific individual (M196) excavated at Juliopolis (JP). How do these organic compounds present in this c. 2000-year-old human hair compare to those present in modern hair? Alterations to organic compounds in archaeological human hair are caused by biological degradative processes dependent on multifactorial processes acting on the hair since the deposition of a body in a mortuary context. We investigate the type of organic compounds present using Synchrotron Radiation Fourier Transform Infrared (SR-FTIR). Juliopolis (Iuliopolis) is an ancient multiperiod city, located in the Çayirhan district of Nallihan, northwest of Ankara. The Juliopolis necropolis from which M196 was recovered was in use throughout the Hellenistic, Roman, and Byzantine periods, and yielded over 700 tombs with numerous human remains. One tomb (M196) contained human remains of exceptional preservation status, including substantial amounts of hair. Human hair from archaeological contexts is not only extremely rare, but importantly, has high analytical value, with potential for analysis of diet, geographical origins, ancient DNA, metal exposure, and other aspects of life in a time-resolved manner. These data make significant contributions to the life history of the individual (osteobiography), as well as contribute towards key archaeological questions. As these analyses are in their majority destructive, prior evaluation of the preservation of sufficient amounts of the organic compounds on which many such analyses rely upon is crucial, to avoid unnecessary loss of precious ancient samples. The results of our SR-FTIR analyses at SESAME synchrotron show that keratin in the JP M196 is more degraded in comparison to the modern reference sample. However, the results also point to clear potential for further analyses with techniques relying on organic compound preservation, such as C and N isotopic analyses for diet, and aDNA.

Continuous monitoring of arylesterase in human serum.

Arylesterase (EC 3.1.1.2) activity in serum was specifically measured using thiophenyl acetate in a mechanized assay at 37 degrees C with 4-bromophenylboronic acid as inhibitor of cholinesterase and hexacyanoferrate-III as indicator. The systematic development of a routine method, apparent limitations of thiophenyl compounds as arylesterase substrates, some kinetic constants of the enzyme, analytical variables such as precision (within-run <2% and between-run <2.5% relative standard deviation) and a preliminary reference interval (19.5-52.4 kU/l) for adults are described.

Routine alpha-amylase assay using protected 4-nitrophenyl-1, 4-alpha-D-maltoheptaoside and a novel alpha-glucosidase.

BACKGROUND: In contrast to numerous methods for measuring alpha-amylase activity, the approved IFCC reference method offers an invariable time-independent constant product pattern, thus avoiding possibly changing stoichiometric calculations. However, reference methods do not lend themselves to routine use, so that such methods need to be modified. METHODS: Ethylidene-blocked 4-nitrophenylmaltoheptaoside (EPS-G7) is degraded to glucose and 4-nitrophenol in a coupled assay with a bacterial alpha-glucosidase under the following measurement conditions: 3.5 mmol/L EPS-G7, 7.1 kU/L alpha-glucosidase, 70 mmol/L sodium chloride, 1 mmol/L calcium chloride, 50 mmol/L HEPES, pH 7.15, at 37 degrees C. The increase of absorbance is continuously monitored for 3 min at 405 nm after a lag phase of 2 min. RESULTS: Catalytic concentrations up to 15-fold higher than the upper reference limit can be determined without dilution. Precision studies in manual performance show CVs of 1.4-2. 6% (within-run) and 1.9-2.8% (day-to-day). There was no interference from 100 mmol/L glucose, 30 mmol/L triacylglycerols, 610 micromol/L bilirubin, and 2.95 g/L hemoglobin. The method closely correlates with other chromogenic assays. The preliminary 0.95 reference interval for adults, not dependent on age and sex, is 33.6-96.2 U/L. CONCLUSION: The procedure is a robust adaptation of the reference method to routine use at 37 degrees C with increased sensitivity, fewer interferences, and reduced cost.

Evaluation of a direct alpha-amylase assay using 2-chloro-4-nitrophenyl-alpha-D-maltotrioside.

We present the adaptation of an IFCC method for alpha-amylase using 2-chloro-4-nitro-phenyl-alpha-D-maltotrio-side as substrate (1) suited for routine work at 37 degrees C. In the assay, a constant proportion of substrate, i. e. 92%, is directly converted to 2-chloro-4-nitrophenol and maltotriose. The method is based on multi- and univariate optimization leading to following measurement conditions: substrate, 2.25 mmol/l; chloride, 310 mmol/l; calcium 5.0 mmol/l; 4-morpholinoethanesulphonic acid, 50 mmol/l; pH 6.28. The assay may be carried out manually or by mechanized procedures, with substrate or sample start, and it shows these analytical properties in measuring amylase activity of sera: no lag phase, detection limit 2.9 U/l, linear range < or = 820 U/l (for 300 s) or < or = 1450 U/l (for 120 s of measurement), and total manual imprecision 3.2% (CV) at 46 U/l. Bilirubin < or = 630 micromol/l, haemoglobin < or =6 g/l, triacylglycerols < or =30 mmol/l, heparin < or =100 kU/l, and glucose < or =120 mmol/l do not interfere. For adults, we established a preliminary 0.95-reference interval of 30-90 U/l not dependent on sex or age. A close association with the IFCC method demonstrates the reliable transfer of its measurement conditions to a robust routine method with minimal changes.

Production and certification of an enzyme reference material for creatine kinase isoenzyme 2 (CRM 608).

We describe the preparation of a lyophilized material containing purified human creatine kinase 2 (CK-MB), and the certification of its catalytic concentration. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control, or for calibration of the creatine kinase 2 catalytic concentration measurements. The enzyme was purified from human heart by ethanol precipitation and chromatography successively on DEAE-Sephacel and Blue-Sepharose. The purified enzyme had a specific activity of 998.4 U/mg and was > 99% pure on polyacrylamide gel electrophoresis. The material was examined for several possible contaminating enzymes, which were found to be absent. The purified creatine kinase 2 had two subunits (B and M) with molecular masses of 43,650 and 41,700 g/mol, respectively, and an isoelectric point at pH 5.8. The material was prepared by diluting the purified creatine kinase 2 in a matrix containing 25 mmol/L PIPES buffer, pH 7.2, 2 mmol/L ADP, 5 mmol/L 2-mercaptoethanol, 154 mmol/L sodium chloride and 50 g/L human serum albumin, dispensing it into vials and freeze-drying. The batch was shown to be homogeneous. The loss of enzyme activity on storage at -20 degrees C is predicted to be less than 0.18% per annum on the basis of accelerated degradation studies. The catalytic concentration of creatine kinase in samples of the reconstituted material is certified to be 67.2+/-1.8 U/L (1.12+/-0.03 microkat/L) when measured, at 30 degrees C, by the Recommended Method of the International Federation of Clinical Chemistry.

Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).

We describe the preparation of a lyophilized material containing purified human pancreatic alpha-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-amylase had a molar mass of 57,500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.

New method for the determination of pancreatic amylase evaluated.

We have carried out a multicenter evaluation of a new reagent carrier for Reflotron, specific for pancreatic amylase where the salivary isoenzyme is inhibited by two specific monoclonal antibodies. This new procedure combines easy handling with low imprecision (median CV less than 3%) in control material, serum, heparinized blood, and plasma) and close correlation (r = 0.991 to 0.999) with established manual and automated methods. The same close correlation was found with values obtained from either venous or capillary finger-stick blood. Salivary amylase up to 54 kU/L (37 degrees C) was inhibited to about 97%. Endogenous interference by hemoglobin, bilirubin, triglycerides, cholesterol, or hematocrit was found to be negligible within a wide range of interferent concentrations. Out of a panel of 28 commonly used drugs it was shown that only two (ascorbic acid and paracetamol), and then only at toxic concentrations, caused a deviation in amylase activity of greater than 10%. From the results of this study we conclude that this new method is suitable for highly precise and accurate measurements of pancreatic amylase in emergency and routine laboratories.

[Studies on the reaction of wheat germ agglutinin with serum glycoproteins. Lectins as reagents. III]. / Untersuchungen zur Reaktion von Wiezenkeimlektin mit Glykoproteinen des Serums. Lektine als Reagentien, III. Mitteilung.

Solutions of wheat germ agglutinin exclusively but incompletely react with serum glycoproteins containing N-acetylneuraminic acid, viz. alpha 2-macroglobulin, haptoglobin, haemopexin, immunoglobulin A, alpha 1-acid glycoprotein, ceruloplasmin, immunoglobulin M (in decreasing order) and others. The precipitation does not proceed stoichiometrically and depends on lectin and polyethyleneglycol concentration, temperature, pH-value, ionic strength, and matrix effects. Presumedly, the reaction is initiated by specific and electrostatic interactions of wheat germ agglutinin with sialic acid residues of the glycoprotein and followed by binding of N-acetylglucosamine residues. A minimal precipitation of albumin is due to its complex formation with glycoproteins via disulphide bonds. Although wheat germ lectin precipitation sensitively detects serum sialoproteins, its intensity does not reflect the amount of N-acetylneuraminic acid in serum glycoproteins, thus calling in question the analytical use of this lectin for the measurement of sialoglycoconjugates.

Isoamylase measurements with monoclonal antibody test strips.

A test strip with fixed monoclonal antibodies binding human salivary amylase (5) was used for the measurement of pancreatic amylase in human sera. The amount of salivary isoamylase adsorbed by the test strip increased with the amount of enzyme in the test solution, reaching a maximum absorption capacity of 620 mU at a test quantity of 900 mU. At about 160 U/l of pancreatic isoamylase (in a mixture containing 214 U/l of total alpha-amylase, both enzymes with 4-nitrophenyl-alpha-D-maltoheptaoside at 25 degrees C) imprecision within-run (and day-to-day) was 2.8% (and 6.8%) at this level of clinical importance. The preliminary 95%-reference interval from 33 healthy adults was calculated to be 16.6-44.2 U/l. The method proved valuable for the interpretation of slightly elevated alpha-amylase levels (up to twice the upper reference limit) and unusual lipase/amylase ratios.

[Protein determination in urine--a critical review]. / Proteinbestimmung im Urin--Eine kritische Ubersicht.

Seven methods for protein determination in urine were systematically checked, viz. turbidimetric assay with benzethonium chloride, turbidimetry with trichloroacetic acid of various concentrations, micellar complex formation with Coomassie Brilliant Blue G-250, formation and gel filtration of protein complexes with copper, reaction of protein tannin compounds with iron, and biuret reaction after different precipitation procedures. The evaluation was mainly based on the following criteria: identical reaction with various proteins, stable and linear absorbance with concentration, complete recovery, and greatest possible freedom from interferences. On this basis, the biuret reaction must now be regarded as the method of choice. Albumin and gamma-globulins are reliably estimated after precipitation by perchloric acid or by an ethanol-diethylether mixture, whereas all proteins, including uromucoid, may be measured after precipitation by Tsuchiya's reagent. Interference from urinary pigments can be avoided by reading versus a sample blank.

Sialic acid in human serum and cerebrospinal fluid. Comparison of methods and reference values.

Sialic acid was estimated simultaneously by three methods: chemical determination based on Warren's method (Meth. Enzymol. 6, 463-464 (1963) with slight modifications, enzymatic measurement with a commercially available test kit, and high performance liquid chromatography (HPLC) according to Silver et al. (J. Chromatogr. 224, 381-388 (1981). These methods showed closely correlated (r greater than 0.930) results and displayed similar precision data. Interference studies demonstrated sufficient specificity for the chemical assay, which was 5-6 times more sensitive than the enzymatic test and hence chosen for the establishment of reference values. From 249 sera from healthy people between 16 and 63 years the 0.025-0.975-reference intervals were calculated to be 1.57-2.63 mmol/l for 127 men, and 1.69-2.64 mmol/l for 122 women with no significant dependence on age and sex. From 43 cerebrospinal fluids from healthy adults the respective values were 17.3-50.4 mumol/l. These data correspond to those of the literature. Some chemical assays employing thiobarbituric acid were compared. They proved reliable in contrast to the reaction of serum with 4-dimethylaminobenzaldehyde.

Evaluation of alpha-amylase assays with 4-nitrophenyl-alpha-oligosaccharides as substrates.

Measurements of alpha-amylase with 4-nitrophenyl glucosides offer the following advantages over methods that rely on the formation of NADH: a short lag phase, no apparent interference by metabolites and enzymes of the sample and extremely stable substrates with low blank values. The intrinsic sensitivity of nitrophenol formation was equal to that of hydrolysis of maltotetraose, but was less than that of glucose-producing methods using oligosaccharides. In contrast to starch, the chromogenic substrates are more rapidly hydrolysed by salivary than by pancreatic amylase. Disadvantages of these substrates are: higher turnover by animal than by human amylases, and a marked susceptibility of the chromophore to small changes of pH and protein concentration. Some analytical qualities such as specificity, accuracy, precision, stability of the substrate and linear range are described in detail and compared with those of other methods.

Properties of human alpha-amylases from urine, pancreas, and saliva.

alpha-Amylases from human urine, pancreas, and saliva were purified to homogeneity. Their molecular and catalytic properties were similar with respect to relative molecular masses, stability, and absorbance in neutral solution, but their isoelectric points differed clearly. Salivary amylase was more sensitive than the other two to inhibition by iodoacetate and EDTA, suggesting a less compact structure. The intermediate qualities of the urinary activity were ascribed to the fact that this enzyme originates from other two without major modifications by metabolism. Human alpha-amylase should be considered as a sole enzyme with multiple forms originating from glycosylation and deamidation. There was no evidence for real isoenzymes.

[Catalytic concentration, multiple forms, and lectin affinity of microsomal enzymes from human tissues: lectins as reagents, II (author's transl)]. / Katalytische Konzentration, multiple Formen und Lektinaffinität von mikrosomalen Enzymen menschlicher Gewebe. Lektine als Reagentien, II. Mitteilung.

We estimated the concentrations, multiple forms, and lectin binding of five microsomal enzymes in particle free extracts from human kidney, pancreas, jejunal mucosa, and normal and cancerous liver. While arylesterase markedly reacted only with concanavalin A, arylamidase, alkaline phosphatase, gamma-glutamyltransferase, and cholinesterase were intensely precipitated by lectins from Ricinus communis 120, Canavalia ensiformis, Triticum vulgare and Phaseolus vulgaris S. Agglutinins from Glycine max, Arachis hypogaea and Ulex europaeus proved less effective. The reaction mainly depended on the origin of enzymes not on their species. Desialylation always decreased precipitation, and in extracts of normal liver parenchyma it even totally abolished precipitation, by Triticum vulgare lectin. Sialoenzymes therefore appear to be normal intracellular constituents. Differences between enzymes from normal and cancerous liver were not reflected by variant properties of the corresponding activities in sera. The same held true for multiple forms. The reasons for these differences are discussed.