Standardization of two immunological HbA1c routine assays according to the new IFCC reference method.

The measurement of HbA1c is meanwhile well established as the most important parameter in clinical chemistry for monitoring the long term metabolic control of diabetic patients. However, the comparability of HbA1c values obtained by different methods in different laboratories and different countries is limited since there was no internationally agreed reference method available. Additionally the % HbA1c values, based on the most common DCCT values are too high due to well known non specificity of the DCCT standardization protocol. Recently a new reference method for the determination of HbA1c was developed and has been approved by the IFCC as the future basis for the worldwide standardization of HbA1c routine assays. The two routine methods from Roche Diagnostics (both based on the immunoturbidimetric determination of the stable glucose adduct to the N-terminal group of the hemoglobin beta chain, Roche-Hitachi/Tina-quant [a] and COBAS INTEGRA) are directly standardized against this new reference method. Both routine methods are based on a two step approach: in a first step the total Hb is quantified by a colorimetric method and in a second step the stable glucose adduct to the N-terminal group of the hemoglobin beta chain is quantified by immunoturbidity (HbA1c mass). Consequently, first the standardization of the total Hb assay was reconfirmed by method comparisons against the cyanomethemoglobin reference method. Second, the standardization of the HbA1c mass was done by running method comparisons against the new IFCC reference method. The resulting new calibrator values for HbA1c [mass] and total Hb [mass] allow a direct estimation of % HbA1c according to IFCC just by calculating the HbA1c [mass]/total Hb [mass] ratio. Good linear correlations were obtained when comparing the routine methods against the new IFCC reference, indicating the high specificity of these immunological approaches. The following correlations are obtained: Hitachi / Tina-quant [a] (Y) versus IFCC reference method (X): Y = -0.031 + 1.009 x X; r = 0.995. COBAS INTEGRA (Y) versus IFCC reference method (X): Y = -0.156 + 1.006 x X; r = 0.997. Hitachi / Tina-quant [a] (Y) versus Integra (X); (both standardized according to IFCC): Y = 0.057 + 1.003 x X; r = 0.997. The HbA1c values obtained with this new IFCC standardization are significantly lower than the well known DCCT values. Due to the specificity of the immunological approach which is very close to the analyte as defined by the IFCC reference method, no further corrections of the obtained % HbA1c values are necessary. A slope/intercept correction formula is derived which allows the transformation of IFCC values into the DCCT numbers if requested.

Subunit composition of the zinc proteins alpha- and beta-lipovitellin from chicken.

Chicken alpha- and beta-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 +/- 0.2 mol of zinc/275 kDa per alpha-lipovitellin and 1.4 +/- 0.2 mol of zinc/155 kDa per beta-lipovitellin, respectively. The subunits of beta-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3-16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of alpha- and beta-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from alpha- and beta-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of alpha- and beta-Lv, respectively, and two peptides of the 30-kDa subunit of alpha- and beta-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of alpha- and beta-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of alpha-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor-product relationship of Vtg I.

Automated Cobas Core PSA assays: sensitive, precise, and specific measurement of PSA-total and PSA-total/PSA-free ratio.

We describe the analytical performance of the newly developed Roche Cobas Core PSA Total EIA and the Cobas Core PSA Free EIA. The assays are designed for the random access immunochemistry analyzer Cobas Core. Both assays are highly precise (CV's < 4%) and highly sensitive (PSA Total: analytical sensitivity < 0.02 ng/ml; PSA Free: < 0.01 ng/ml). The PSA Total EIA measures PSA-ACT and PSA free equimolarly. The assays are standardized against the reference material of the Stanford group and correlate very well with the designated golden standard Hybritech Tandem-R assays. The high-dose-hook is beyond 20,000 ng/ml and 4500 ng/ml for the PSA Total and PSA Free assay, respectively. In summary, the data indicate clearly the reliability of the measuring system and the achievement of the demands of PSA diagnostics.

Iron center, substrate recognition and mechanism of peptide deformylase.

Eubacterial proteins are synthesized with a formyl group at the N-terminus which is hydrolytically removed from the nascent chain by the mononuclear iron enzyme peptide deformylase. Catalytic efficiency strongly depends on the identity of the bound metal. We have determined by X-ray crystallography the Fe2+, Ni2+ and Zn2+ forms of the Escherichia coli enzyme and a structure in complex with the reaction product Met-Ala-Ser. The structure of the complex, with the tripeptide bound at the active site, suggests detailed models for the mechanism of substrate recognition and catalysis. Differences of the protein structures due to the identity of the bound metal are extremely small and account only for the observation that Zn2+ binds more tightly than Fe2+ or Ni2+. The striking loss of catalytic activity of the Zn2+ form could be caused by its reluctance to change between tetrahedral and five-fold metal coordination believed to occur during catalysis. N-terminal formylation and subsequent deformylation

Isolation and crystallization of functionally competent Escherichia coli peptide deformylase forms containing either iron or nickel in the active site.

Three metallo forms of peptide deformylase (PDF, EC 3.5.1.31) of Escherichia coli were prepared and crystallized (space group C2, diffraction limit 1.9 A) for initiating the X-ray structure determination of the metal center in correlation with the catalytic functionality of this enzyme. The native Fe2+ containing enzyme species was directly isolated from overproducing bacteria by using catalase as a buffer additive, which stabilizes the catalytic activity against oxidative destruction. The Ni2+ containing form, which is oxygen-insensitive, was obtained by metal exchange with free Ni2+ and found to be catalytically equally effective (kcat/KM = 10(5) M-1 s-1 for N-formyl-Met-Ala). The Zn2+ form, prepared from the apoenzyme or by displacement of bound Ni2+ by free Zn2+, proved virtually inactive.

Constituents of wing gland and abdominal hair pencil secretions of male African sugarcane borer,Eldana saccharina walker (Lepidoptera: Pyralidae).

In addition totrans-3,7-dimethyl-6-octen-4-olide (eldanolide), vanillin, and 4-hydroxybenzaldehyde, identified by French workers in the wing gland and abdominal hair pencil secretions of the male African sugarcane borer,Eldana saccharina, we have, in an earlier note, reported the presence of several other terpenoid, aromatic, and unbranched-chain compounds such as, (Z)-3,7-dimethylocta-2,6-dienoic acid, 6,10,14-trimethyl-2-pentadecanol, 4-hydroxy-3-methoxybenzyl alcohol, 1-octadecane thiol, 16-hexadecanolide, and 18-octadecanolide in these secretions. In the present paper experimental details and spectral evidence supporting the identification of these compounds, as well as the identification of (Z)-9-hexadecenal and cw-3,7-di-methyl-6-octen-4-olide (cis-eldanolide), are reported. Using electroantennography it was found that male and female antennae reacted approximately equally strongly to both secretions. This result was confirmed in analyses of the secretions using coupled gas chromatography-electroantennography and it was found that male as well as female antennae responded to eldanolide. Vanillin, substituted phenols related to vanillin, and some oxygenated monoterpenes elicited weak responses in male and female antennae. In some analyses 6,10,14-trimethyl-2-pentadecanol, present in the secretions of the insect, gave a strong antennal response. The results obtained in dynamic and static headspace determinations showed that several of the organic compounds present in the glandular secretions are released in detectable quantities and are present in widely varying quantitative ratios in the effluvia of individual calling male moths.