Recent Developments in Clinical Plasma Proteomics—Applied to Cardiovascular Research

Funding Information: Funding: Odense University Hospital Research Fund (grant no. A3130 and A3329) and The Danish Heart Foundation.The human plasma proteome mirrors the physiological state of the cardiovascular system, a fact that has been used to analyze plasma biomarkers in routine analysis for the diagnosis and monitoring of cardiovascular diseases for decades. These biomarkers address, however, only a very limited subset of cardiovascular diseases, such as acute myocardial infarct or acute deep vein thrombosis, and clinical plasma biomarkers for the diagnosis and stratification cardiovascular diseases that are growing in incidence, such as heart failure and abdominal aortic aneurysm, do not exist and are urgently needed. The discovery of novel biomarkers in plasma has been hindered by the complexity of the human plasma proteome that again transforms into an extreme analytical complexity when it comes to the discovery of novel plasma biomarkers. This complexity is, however, addressed by recent achievements in technologies for analyzing the human plasma proteome, thereby facilitating the possibility for novel biomarker discoveries. The aims of this article is to provide an overview of the recent achievements in technologies for proteomic analysis of the human plasma proteome and their applications in cardiovascular medicine.publishersversionpublishe

Causes of vitamin K deficiency in patients on haemodialysis

Background: A low vitamin K status is common in patients on haemodialysis, and this is considered one of the reasons for the accelerated atherosclerosis in these patients. The vitamin is essential in activation of the protein Matrix Gla Protein (MGP), and the inactive form, dp-ucMGP, is used to measure vitamin K status. The purpose of this study was to investigate possible underlying causes of low vitamin K status, which could potentially be low intake, washout during dialysis or inhibited absorption capacity. Moreover, the aim was to investigate whether the biomarker dp-ucMGP is affected in these patients. Method: Vitamin K intake was assessed by a Food Frequency Questionnaire (FFQ) and absorption capacity by means of D-xylose testing. dp-ucMGP was measured in plasma before and after dialysis, and phylloquinine (vitamin K1) and dp-ucMGP were measured in the dialysate. Changes in dp-ucMGP were measured after 14 days of protein supplementation. Results: All patients had plasma dp-ucMGP above 750 pmol/L, and a low intake of vitamin K. The absorption capacity was normal. The difference in dp-ucMGP before and after dialysis was −1022 pmol/L (p < 0.001). Vitamin K1 was not present in the dialysate but dp-ucMGP was at a high concentration. The change in dp-ucMGP before and after protein supplementation was −165 pmol/L (p = 0.06). Conclusion: All patients had vitamin K deficiency. The reason for the low vitamin K status is not due to removal of vitamin K during dialysis or decreased absorption but is plausibly due to a low intake of vitamin K in food. dp-ucMGP is washed out during dialysis, but not affected by protein intake to a clinically relevant degree

Oral Magnesium Supplementation in Chronic Kidney Disease Stages 3 and 4:Efficacy, Safety, and Effect on Serum Calcification Propensity-A Prospective Randomized Double-Blinded Placebo-Controlled Clinical Trial

Chronic kidney disease (CKD) is associated with high cardiovascular morbidity and mortality. Recent evidence suggests that increases in both serum and intracellular magnesium (Mg) can slow or even prevent the development of vascular calcification seen in CKD. Serum calcification propensity (T50) is a novel functional test, which is associated with all-cause mortality in CKD and measures the ability of serum to delay the formation of crystalline nanoparticles. Theoretically, increasing serum Mg should improve T50 and thereby reduce the propensity towards ectopic calcification. Methods: We conducted a randomized placebo-controlled double-blinded clinical trial to investigate the safety of 2 different doses of oral Mg supplementation in subjects with CKD stages 3 and 4 as well as their effects on intracellular Mg and T50. Thirty-six subjects with CKD stages 3 and 4 were randomized to one of 3 groups (placebo, elemental Mg 15 mmol/d or elemental Mg 30 mmol/d) given as slow-release Mg hydroxide and followed for 8 weeks. Results: Thirty-four subjects completed the trial. Intracellular Mg remained stable throughout the trial despite significant increases in both serum and urine Mg. T50 increased significantly by 40 min from 256 ± 60 (mean ± SD) to 296 ± 64 minutes (95% confidence interval, 11–70, P < 0.05) in the Mg 30 mmol/d group after 8 weeks. No serious adverse events related to the study medication were reported during the study. Discussion: Oral Mg supplementation was safe and well tolerated in CKD stages 3 and 4 and improved T50, but did not increase intracellular Mg. Further studies are needed to investigate the long-term effects of Mg supplementation in CKD stage 3 and 4 and whether improvement in calcification propensity is related to clinical endpoints

Classification of amyloidosis by model‐assisted mass spectrometry‐based proteomics

Funding Information: Funding: This research was partly funded by a “Center of Clinical Excellence” research grant from the Health Region of Southern Denmark to Odense Amyloidosis Center (AmyC). Publisher Copyright: © 2021 by the authors. Li-censee MDPI, Basel, Switzerland.Amyloidosis is a rare disease caused by the misfolding and extracellular aggregation of proteins as insoluble fibrillary deposits localized either in specific organs or systemically through-out the body. The organ targeted and the disease progression and outcome is highly dependent on the specific fibril‐forming protein, and its accurate identification is essential to the choice of treat-ment. Mass spectrometry‐based proteomics has become the method of choice for the identification of the amyloidogenic protein. Regrettably, this identification relies on manual and subjective inter-pretation of mass spectrometry data by an expert, which is undesirable and may bias diagnosis. To circumvent this, we developed a statistical model‐assisted method for the unbiased identification of amyloid‐containing biopsies and amyloidosis subtyping. Based on data from mass spectrometric analysis of amyloid‐containing biopsies and corresponding controls. A Boruta method applied on a random forest classifier was applied to proteomics data obtained from the mass spectrometric analysis of 75 laser dissected Congo Red positive amyloid‐containing biopsies and 78 Congo Red negative biopsies to identify novel “amyloid signature” proteins that included clusterin, fibulin‐1, vitronectin complement component C9 and also three collagen proteins, as well as the well‐known amyloid signature proteins apolipoprotein E, apolipoprotein A4, and serum amyloid P. A SVM learning algorithm were trained on the mass spectrometry data from the analysis of the 75 amyloid-containing biopsies and 78 amyloid‐negative control biopsies. The trained algorithm performed su-perior in the discrimination of amyloid‐containing biopsies from controls, with an accuracy of 1.0 when applied to a blinded mass spectrometry validation data set of 103 prospectively collected am-yloid‐containing biopsies. Moreover, our method successfully classified amyloidosis patients ac-cording to the subtype in 102 out of 103 blinded cases. Collectively, our model‐assisted approach identified novel amyloid‐associated proteins and demonstrated the use of mass spectrometry‐based data in clinical diagnostics of disease by the unbiased and reliable model‐assisted classification of amyloid deposits and of the specific amyloid subtype.publishersversionpublishe

Galectin-3 and fibulin-1 in systolic heart failure:relation to glucose metabolism and left ventricular contractile reserve

Abstract Background Heart failure (HF) patients with diabetes (DM) have an adverse prognosis and reduced functional capacity, which could be associated with cardiac fibrosis, increased chamber stiffness and reduced left ventricular (LV) contractile reserve. Galectin-3 (Gal-3) and fibulin-1 are circulating biomarkers potentially reflecting cardiac fibrosis. We hypothesize that plasma levels of Gal-3 and fibulin-1 are elevated in HF patients with DM and are associated with reduced LV contractile reserve in these patients. Methods A total of 155 patients with HF with reduced ejection fraction underwent a low-dose dobutamine echocardiography and blood sampling for biomarker measurements. Patients were classified according to history of DM and an oral glucose tolerance test (OGTT) as: normal glucose tolerance (NGT) ( n \u2009=\u200970), impaired glucose tolerance (IGT) ( n \u2009=\u200925) and DM ( n \u2009=\u200960). Results Galectin-3 levels were elevated in DM patients as compared to non-diabetic patients ( P \u2009=\u20090.02), while higher fibulin-1 levels were observed in HF patients with IGF and DM ( P \u2009=\u20090.07). Reduced LV contractile reserve was associated with increasing Gal-3 levels (\u3b2\u2009=\u2009\u22120.19, P \u2009=\u20090.03) although, this association was attenuated after adjustment for estimated glomerular filtration rate ( P \u2009=\u20090.66). Fibulin-1 was not associated with LV contractile reserve ( P \u2009=\u20090.71). Conclusions Galectin-3 and fibulin-1 levels were elevated in HF patients with impaired glucose metabolism. However, reduced LV contractile reserve among HF patients with DM does not to have an independent impact on plasma Gal-3 and fibulin-1 levels

AFM Characterization of the Internal Mammary Artery as a Novel Target for Arterial Stiffening

Using the atomic force microscopy- (AFM-) PeakForce quantitative nanomechanical mapping (QNM) technique, we have previously shown that the adventitia of the human internal mammary artery (IMA), tested under dehydrated conditions, is altered in patients with a high degree of arterial stiffening. In this study, we explored the nanoscale elastic modulus of the tunica media of the IMA in hydrated and dehydrated conditions from the patients with low and high arterial stiffening, as assessed in vivo by carotid-femoral pulse wave velocity (PWV). In both hydrated and dehydrated conditions, the medial layer was significantly stiffer in the high PWV group. The elastic modulus of the hydrated and dehydrated tunica media was significantly correlated with PWV. In the hydrated condition, the expression activity of certain small leucine-rich repeat proteoglycans (SLRPs), which are associated with arterial stiffening, were found to be negatively correlated to the medial elastic modulus. We also compared the data with our previous work on the IMA adventitia. We found that the hydrated media and dehydrated adventitia are both suitable for reflecting the development of arterial stiffening and SLRP expression. This comprehensive study of the nanomechanical properties integrated with the proteomic analysis in the IMAs demonstrates the possibility of linking structural properties and function in small biological samples with novel AFM methods. The IMA is a suitable target for predicting arterial stiffening