To Örebro University

BACKGROUND: Procollagen III, aminoterminal peptide (PIIINP) is a degradation product of collagen type III-synthesis. Collagen type III is distributed in many tissues, and an increase in serum PIIINP could reflect an increase in collagen turnover and pro-fibrotic activity. In this study, on a population of younger, healthy adults, we examined whether serum PIIINP correlates with early markers of vascular health, to evaluate its potential as a biomarker for early screening of preclinical cardiovascular risk.

METHODS: PIIINP levels, pulse wave velocity (PWV) and Carotid-intima media thickness (cIMT) was measured in 834 healthy, non-smoking, individuals aged 18-26. In univariable and multivariable linear regression models, we examined the association between PIIINP and vascular measurements, PWV and cIMT with adjustment for serum lipids, liver enzymes and systolic blood pressure.

RESULTS: The average of PIIINP, PWV and cIMT measurements in this population, were low (7.1 and 7.3 µg/L, 5.5 and 5.2 m/s, and 0.50 and 0.49 mm for men and women, respectively). In univariable analyses, PIIINP correlated positively with cIMT (p = 0.0061) and negatively with PWV (p = 0.0069). In multivariable analyses, a statistically significant association remained between PIIINP and cIMT (p < 0.001), but not with PWV.

CONCLUSION: Serum PIIINP correlates with cIMT in a healthy population, indicating its potential as a biomarker of cardiovascular risk at a preclinical stage. PIIINP measurement being easier to perform and less examiner dependent than the more time consuming and cumbersome cIMT, are suggestive of its possible merits as an early screening tool for cardiovascular disease.

To assess the impact of high levels of hemolysis on the laboratory results for free β-hCG, PAPP-A, and TRAb performed on the B·R·A·H·M·S KRYPTOR Compact PLUS. Adapted from the CLSI guidelines EP07-A2, paired difference testing was performed on serum samples from the routine laboratory workflow. Three sample pools for each assessed analyte was prepared and subjected to increased levels of added hemolysate. For β-hCG and PAPP-A, the relative difference in the measured analyte concentration between the sample with 0 g/L added Hb and the samples with increasing free Hb concentrations (up to 6 g/L), was well below the pre-set acceptance criterion of 10% at all levels. The TRAb results showed greater variation than the other analytes, likely a consequence of imprecision rather than hemolysis. Hemolysis has a negligible effect on the analysis results of free beta-hCG, PAPP-A and TRAb measured on the B·R·A·H·M·S KRYPTOR Compact PLUS.

Objectives: To assess the impact of hemolysis on laboratory results under local conditions and to verify the hemolysis index cut-off for potassium using real-world data.

Methods: The statistical bootstrapping method was performed on 54,125 samples collected at the University Hospital of Örebro (USÖ) and the results were compared to a method based on stratification of samples according to hemolysis level, and on paired difference testing.

Results: Setting the acceptable allowable limit of error to 10 %, the three assessed strategies yielded comparable results with respect to the impact of haemolytic interference on test results for potassium. The suggested cut-offs were 111 mg Hb/dL for the bootstrapping method, between 100-125 mg Hb/dL for the method based on stratification, and around 150 mg/dL for the paired difference testing strategy. The impact of hemolysis on potassium measurement is likely different between primary care patients and inpatients.

Conclusions: Using the effect of hemolysis on potassium measurement as a model, a novel approach towards finding clinically acceptable limits for analytical interference is presented, that relies on the bootstrapping method and on actual patient data from routine laboratory operation, hence incorporating local population characteristics, equipment and instrumental settings.

OBJECTIVE: The amino-terminal peptide of type III procollagen (PIIINP) is a byproduct of type III collagen synthesis that exhibits promise as a biomarker of fibrosis, specifically in monitoring hepatic fibrosis in methotrexate treated patients. The Advia Centaur^® PIIINP assay is developed for track-based automated laboratory systems and is suitable for large volume analysis. Reference intervals in children and younger adults have been published previously. Here we measured PIIINP to determine reference ranges, specifically including elderly patients, for whom such are currently lacking.

METHODS: Samples were collected from subjects ranging from 20 to 98 years of age. Blood donors and clinical samples from primary care patients were used for reference interval calculation. Samples were analysed using the Advia Centaur^® PIIINP assay. After exclusion of samples high in alanine transaminase (AST), aspartate transaminase (ALT), and C-reactive protein (CRP) 386 samples were used in the reference interval calculation.

RESULTS AND CONCLUSION: We determined the following reference interval for the Advia Centaur^® PIIINP assay: the lower limit of the reference interval (2.5% percentile with 95% CI) was 4.42 (4.20-4.65) µg/L and the upper limit of the reference interval (97.5% percentile 95% CI) 16.0 (15.04-17.02) µg/L.No significant differences in mean PIIINP concentrations were found between men and women. While differing mean PIIINP concentrations were seen among subjects in different age groups, the differences were small and partitioning of reference range was determined not to be necessary.

Phosphorylation of specific side chains in target proteins by protein kinases is a major means of regulation of cellular processes. In the thylakoid membrane of higher plants, the redox-controlled phosphorylation of photosystem II (PSII) and light-harvesting complex II (LHCII) proteins regulates energy distribution between the two photosystems and PSII protein dynamics. The thylakoid proteins are phosphorylated by several distinct kinases that are subject to different control mechanisms. Regulation of LHCII kinase(s) activation is signalled by the redox-state of the plastoquinone pool and the cytochrome b6/f complex and modulated by the thiol redox-state of the chloroplast. Phosphorylation of PSII core-complex proteins may be achieved by PSII core associated kinase(s) that are regulated by the redox-state of the plastoquinone pool alone without involvement of the cytochrome b6/f complex. The identities of the protein kinases in the thylakoid membrane are still unknown. In this work a kinase enriched fraction obtained from a spinach thylakoid extract by perfusion chromatography is described. This fraction phosphorylates isolated LHCII and the CP43 protein of isolated PSII cores and exhibits a major protein kinase band of 64 kDa.

This work further demonstrates that, in addition to the well-studied redox-regulation of the protein kinases in the thylakoid membrane, phosphorylation can also be modulated at the substrate level. The exposure of the phosphorylation sites of both the LHCII and the CP43 protein to the respective protein kinases is regulated by light-induced conformational changes. This so-called substrate activation increases the phosphorylation level of both proteins. However, illumination for long times and/or in high light in situ, in the intact thylakoid, induces a dramatic decrease in the phosphorylation level of the LHCII under conditions preventing phosphorylation. The implications of these light-induced conformational changes at the substrate level are discussed in relation to energy distribution between the two photosystems and the dynamics of the thylakoid membrane as a whole. Furthermore, the effect of xantophyll pigments on the light-induced conformational changes and thylakoid protein phosphorylation is presented.

Thylakoid membrane proteins are phosphorylated by different enzymes, which are subject to different control mechanisms. Activation of the light harvesting complex (LHCII) kinase is signaled by the redox state of plastoquinone and the cytochrome b/f complex and modulated by the thiol reduction state. Phosphorylation of Photosystem II (PS II) proteins may involve kinase(s) associated with the PS II core complex that do not involve the cytochrome b/f complex. Exposure of the phosphoprotein phosphorylation site(s) to protein kinases is regulated by light-induced conformational changes. Thus, thylakoid protein phosphorylation is regulated at both the enzyme and substrate levels. Thylakoid protein dephosphorylation is also under regulatory control, involving interaction between an immunophilin and a membrane-bound phosphatase. The physiological significance of thylakoid protein phosphorylation is not fully understood. Phosphorylation of LHCII is suggested to have a dual role: i) regulation of the LHCII/PSII/PS I interaction, underlying the mechanism of energy transfer balance and ii) prevention of the light-induced aggregation of LHCII or LHCII-PS II complexes. The formation of such macrodomains may affect the dynamics of the thylakoid membrane, which requires unhindered lateral diffusion of integral protein complexes. Phosphorylation of PS II subunits appear to be essential for the repair of photodamage to its reaction center occurring during light stress conditions.