The IFCC Working Group for the Standardisation of Cystatin C (WG-SCC), in collaboration with the Institute for Reference Materials and Measurements (IRMM), announces the availability of the new certified reference material ERM-DA471/IFCC. The material was characterised using a pure protein primary reference preparation (PRP) as calibrant. The PRP was prepared from recombinant cystatin C, and its concentration measured using dry mass determination. The characterisation of ERM-DA471/IFCC was performed by particle enhanced immuno-nephelometry, particle enhanced immuno-turbidimetry, and enzyme amplified single radial immuno-diffusion. The certified cystatin C mass concentration in ERM-DA471/IFCC, if reconstituted according to the specified procedure, is 5.48 mg/L, the expanded uncertainty (k=2) being 0.15 mg/L.

We describe a fully automated particle-enhanced turbidimetric assay for cystatin C in undiluted serum and EDTA-plasma. The throughput is 90 samples per hour and urgent samples can be analyzed in 7 min. The assay range (0.4-14.1 mg/L) covers the concentration range in health and disease. The within- and between-run imprecision is 0.9% and 2.2%, respectively. Analytical recovery of additions of recombinant cystatin C averaged 98%. Rheumatoid factors (< or = 323,000 IU/L), bilirubin (< or = 150 mumol/L), hemoglobin (< or = 1.2 g/L), and triglycerides (< or = 8.5 mmol/L) do not interfere in the assay. In view of the superior (by ROC analysis) diagnostic accuracy of serum concentrations of cystatin C for reduced glomerular filtration rate (GFR) in comparison with creatinine, cystatin C seems an attractive alternative to creatinine for estimation of GFR.

The albumin levels decreased in the second and third trimesters, whereas the levels of chi2-macroglobulin were unchanged, which is compatible with a virtually unaltered transfer of chi2-macroglobulin between the intra- and extravascular space during pregnancy and a significantly increased extravascular fraction of albumin.

In this study, the maternal plasma levels of beta2-microglobulin, beta-trace protein and cystatin C were all significantly elevated in pre-eclampsia compared to those of healthy pregnant women, and displayed similar diagnostic performance for diagnosing pre-eclampsia. The results indicate that low molecular mass proteins are useful as markers of renal impairment in pre-eclampsia.

A Primary Reference Preparation has been produced using pure, recombinant, Cystatin C in a solvent of 0.1 mol/L KCl. Dry mass determination of the Primary Reference Preparation resulted in a Cystatin C concentration of 5.20 g/L. Agarose-electrophoresis and SDS-electrophoresis, as well as N-terminal sequencing, verified the purity, homogeneity and identity of Cystatin C in the Primary Reference Preparation. For the Secondary Reference Preparation, a serum pool was collected and stabilized. A pilot batch was made to verify the selected procedure and spiking with the pure, recombinant Cystatin C. The final Secondary Reference Preparation is now produced (4468 vials) and ready for value assignment and further characterization.

The processes of activation and deactivation of ribulose-l,5-bisphosphate carboxylase purified from wheat have been investigated. Two forms of the enzyme are indistinguishable in terms of ribulose-l,5-bisphosphate carboxylation and oxidation but exhibit different rates of activation. One form is slowly activated in saturating COz and Mg2+ at moderate temperatures (t0.5 zz 120 min at 25 "C), the other form rapidly activated z 8 s). In the presence of the effectors 6-phosphogluconate or NADPH, significantly lower concentrations of the activating co-factors can achieve full activation of both enzyme species. However, with another effector, fructose 1,6-bisphosphate, for the slowly activating species the mode of action is the same as with 6-phosphogluconate or NADPH, whereas the activation of the rapidly activating species is significantly inhibited. The substrate, ribulose 1,5-bisphosphate, also inhibits this rapid activation process.A mechanism is proposed for the reactions involving activation that accounts for the differential rates of activation and the response to effectors Ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) catalyses two competing reactions, the carboxylation of ribulosePz to two molecules of 3-phosphoglycerate and, in the presence of molecular oxygen, the oxidation of ribulose-P2 to 2-phosphoglycolate and 3-phosphoglycerate. The two catalytic reactions are considered to involve the same site on the enzyme, because they exhibit a similar response to activating co-factors, and certain effectors and inhibitors. The enzyme requires activation with COz and Mg2+ ion both for full carboxylase and oxygenase activities.Activation of the enzyme is an ordered and reversible process involving, firstly, the binding of a COZ molecule to an 8-amino group of a lysine residue on the enzyme and, then, addition of the Mg"+ to the enzyme-CO2 complex [I]. The activating COz molecule is thus quite distinct from the substrate COZ consumed during the carboxylase reaction [2]. Details of the involvement of the activating COz and Mg2+ ion in the catalytic reactions are lacking. However, most recent proposals suggest that the co-factors bind within the active-site region of the enzyme [3].The activation of the carboxylase from a number of sources has been studied [4-61 particularly the spinach enzyme with, most recently, details of the effects of sugar phosphates on the activation process [7,8]. In this report we give details of the activation of the carboxylase purified from wheat leaves. Previous work [9] has shown that two forms of the wheat enzyme can exist that exhibit different activation kinetics. One is a slowly activating species requiring prolonged incubation in COz and ME'', the other is a rapidly activating form similar to the carboxylases from other sources. In the present work, we have investigated bothAbbreviations. Ribulose-P2, ribulose 1,5-bisphosphate; fructose-Pz, fructose 3,6-bisphosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine.Enzyme. Rib...

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The reactions catalysed by ribulosebisphosphate carboxylase/oxygenase have been studied by high resolution proton NMR spectroscopy. The protons of the substrate ribulose-Pz and the products of the carboxylase and oxygenase reactions are readily assigned to the various carbon centres. Half of the 3-phosphoglycerate molecules generated during carboxylase turnover of ribulose-P2 in saturating CO2 concentrations acquire deuterium from the solution at the Cz position, whereas the other 3-phosphoglycerate molecules and also the 3-phosphoglycerate produced as a result of the oxygenase activity contain no deuterium of this type. However, the addition of activated enzyme (i.e., in the presence of an effector) to a solution of ribulose-Pz in the near absence of either CO2 or 02 catalyses the exchange of the CS proton with the deuterium of the solution. Ribu~osebisphosphate carbo~fase/oxygen~e NMR Enediol Ribulose b~phosphateProto~/deaterium exchange