Cell cycle and apoptosis genes in atherosclerosis

The work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder occurring in the large and medium-sized arteries of the body. Although in the beginning 90s promising lipid lowering therapies predicted a strong reduction in cardiovascular deaths, in westernized societies it is still the underlying cause of about 40% of all deaths, indicating that treatment of atherosclerosis goes beyond lipid lowering solely. In addition to lipids, continuous cell growth (cell cycle) and cell death (i.e. apoptosis and necrosis) processes play a central role in the development of atherosclerosis. To investigate the role of several cell cycle and apoptosis genes (i.e. p53, Rb and Mdm2) in atherosclerosis we generated and characterized several mouse models based on site-specific recombinase (SSR) technology. The studies described in this thesis show that next to therapies aiming at lifestyle interventions, lipid therapies and regulation of inflammation, targeting cell cycle and apoptosis genes on lesional or cellular level might prove the most effective way to reduce the burden of atherosclerosis.TNO-Quality of Life, Gaubius Laboratories (afdeling: Vascular and Metabolic Diseases)UBL - phd migration 201

How low is really low?:Comparison of two C-peptide assays to establish residual C-peptide production in type 1 diabetes

INTRODUCTION: C-peptide is an important marker to assess residual insulin production in individuals with type 1 diabetes (T1D). The accuracy and detection limits of C-peptide assays are important to detect C-peptide microsecretion and to reliably observe changes over time in these people. We compared and verified two commercially available assays able to measure C-peptide in the picomolar range. METHODS: The ultrasensitive Mercodia enzyme-linked immunosorbent C-peptide assay (ELISA) was compared with the Beckman immunoradiometric assay (IRMA) for C-peptide, assessing reproducibility (coefficient of variation [CV]), limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ). RESULTS: For both assays within-run and between-run variation were high at the low (around the detection limit) C-peptide concentration range, with CVs of around 40%. LoB values for the ultrasensitive ELISA and the IRMA were 1.3 and 0.16 pmol/L respectively. LoD values were 2.4 and 0.54 pmol/L respectively. LoQ values were 9.7 and 3.8 pmol/L respectively. Only the IRMA met the specifications claimed by the manufacturer. CONCLUSIONS: The IRMA provided the lowest threshold for quantification of serum C-peptide. LoQ of commercially available assays should be established in-house before applying them in research studies and clinical trials in which low C-peptide levels have clinical or scientific relevance

Residual C-peptide secretion and hypoglycemia awareness in people with type 1 diabetes

INTRODUCTION: This study aimed to assess the association between fasting serum C-peptide levels and the presence of impaired awareness of hypoglycemia (IAH) in people with type 1 diabetes. RESEARCH DESIGN AND METHODS: We performed a cross-sectional study among 509 individuals with type 1 diabetes (diabetes duration 5-65 years). Extensive clinical data and fasting serum C-peptide concentrations were collected and related to the presence or absence of IAH, which was evaluated using the validated Dutch version of the Clarke questionnaire. A multivariable logistic regression model was constructed to investigate the association of C-peptide and other clinical variables with IAH. RESULTS: In 129 (25%) individuals, residual C-peptide secretion was detected, while 75 (15%) individuals reported IAH. The median (IQR) C-peptide concentration among all participants was 0.0 (0.0-3.9) pmol/L. The prevalence of severe hypoglycemia was lower in people with demonstrable C-peptide versus those with absent C-peptide (30% vs 41%, p=0.025). Individuals with IAH were older, had longer diabetes duration, more frequently had macrovascular and microvascular complications, and more often used antihypertensive drugs, antiplatelet agents and cholesterol-lowering medication. There was a strong association between IAH and having a severe hypoglycemia in the preceding year. In multivariable regression analysis, residual C-peptide, either continuously or dichotomous, was associated with lower prevalence of IAH (p=0.040-0.042), while age at diabetes onset (p=0.001), presence of microvascular complications (p=0.003) and body mass index (BMI) (p=0.003) were also independently associated with the presence of IAH. CONCLUSIONS: Higher BMI, the presence of microvascular complications and higher age at diabetes onset were independent risk factors for IAH in people with type 1 diabetes, while residual C-peptide secretion was associated with lower risk of this complication

Klinische chemie voor iedereen: het Wikipedia-project

Public relations (PR) and information to patients are key goals of the Dutch Society of Clinical Chemistry (NVKC). In this paper several initiatives will be discussed that have been undertaken to meet this statement. The Wikipedia project is one of those initiatives in which information concerning clinical chemistry is added or revised to the Dutch version of the online encyclopaedia Wikipedia. Over 100 tests and items of clinical chemistry have been added or revised and are recognized as a separate category within this online encyclopaedia. Statistical analysis showed an increase in the number of visitors to these articles of Wikipedia. Furthermore, by adding and improving the available information, the quality of the information provided online is enhanced. In conclusion: the attribution of health care professionals to this public domain leads to enhanced access of high quality information for the main public to clinical chemistry in general and to specific laboratory blood tests

Urine Concentrating Capacity, Vasopressin and Copeptin in ADPKD and IgA Nephropathy Patients with Renal Impairment

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients have an impaired urine concentrating capacity. Increased circulating vasopressin (AVP) concentrations are supposed to play a role in the progression of ADPKD. We hypothesized that ADPKD patients have a more severely impaired urine concentrating capacity in comparison to other patients with chronic kidney disease at a similar level of kidney function, with consequently an enhanced AVP response to water deprivation with higher circulating AVP concentrations. METHODS: 15 ADPKD (eGFR<60) patients and 15 age-, sex- and eGFR-matched controls with IgA nephropathy (IgAN), underwent a water deprivation test to determine maximal urine concentrating capacity. Plasma and urine osmolality, urine aquaporin-2 (AQP2) and plasma AVP and copeptin (a surrogate marker for AVP) were measured at baseline and after water deprivation (average 16 hours). In ADPKD patients, height adjusted total kidney volume (hTKV) was measured by MRI. RESULTS: Maximal achieved urine concentration was lower in ADPKD compared to IgAN controls (533±138 vs. 642±148 mOsm/kg, p = 0.046), with particularly a lower maximal achieved urine urea concentration (223±74 vs. 299±72 mmol/L, p = 0.008). After water deprivation, plasma osmolality was similar in both groups although change in plasma osmolality was more profound in ADPKD due to a lower baseline plasma osmolality in comparison to IgAN controls. Copeptin and AVP increased significantly in a similar way in both groups. AVP, copeptin and urine AQP2 were inversely associated with maximal urine concentrating in both groups. CONCLUSIONS: ADPKD patients have a more severely impaired maximal urine concentrating capacity with a lower maximal achieved urine urea concentration in comparison to IgAN controls with similar endogenous copeptin and AVP responses

Impact of the Choice of IGF-I Assay and Normative Dataset on the Diagnosis and Treatment of Growth Hormone Deficiency in Children

Background: The analysis of insulin-like growth factor I (IGF-I) is an important tool for pediatricians in the diagnosis and treatment of growth hormone deficiency in children. However, significant differences exist in IGF-I assays and normative datasets, which can have important clinical consequences. Methods: IGF-I analyses were performed using the IDS-iSYS platform on 1,897 samples from pediatric patients (0.5-18 years old). Z-scores were calculated based on normative IGF-I data from Bidlingmaier et al. (SD-BM) [J Clin Endocrinol Metab. 2014 May; 99(5): 1712-21] and normative IGF-I data from the IGF-I harmonization program in the Netherlands (SD-NL). The differences in Z-scores were analyzed at relevant clinical decision points (-2 SD, +2 SD). These normative datasets were also compared to normative data reported by Elmlinger et al. [Clin Chem Lab Med. 2004; 42(6): 654-64]. Results: The difference in Z-score between SD-BM and SD-NL was highest in males between 0 and 3 years old, exceeding 2 SD. Clinically relevant discordance between both Z-scores at-2 and +2 SD was found in 12.7% of all samples. The IGF-I levels at-2 and +2 SD reported in the normative dataset of Elmlinger et al. were up to 100% higher than the IGF-I levels reported by Bidlingmaier et al. or the Dutch harmonization program. Conclusion: Pediatricians and laboratory specialists should be aware of relevant differences that can exist between IGF-I assays and normative data. Well-defined pediatric reference ranges for the IDS-iSYS platform are highly desirable

Misleading FT4 measurement: Assay-dependent antibody interference

Introduction: Commonly used free thyroxine (FT4) immunoassays can be falsely elevated due to interference causing misinterpreted thyroid function. We present two cases with high FT4 concentrations due to antibody interference. This study’s aim was to investigate the source of the FT4 immunoassay interference and possibility of its removal by two different techniques in order to correct the discrepancy between obtained FT4 values and the patient’s clinical status. Materials and methods: Two patients presented at their general practitioners’ with elevated FT4 concentrations in combination with a normal and increased thyroid stimulating hormone (TSH) concentrations. Clinical symptoms differed between patients but did not correspond with the hyperthyroid status suggested by the laboratory results. FT4 concentrations from both patients were measured on four common commercial immunoassays and the dialysis method before and after treatment with heterophilic blocking tubes and protein A/G. Results: Removal of interfering antibodies using protein A/G resulted in normal FT4 concentrations. Conclusion: This report illustrates falsely elevated FT4 concentrations due to assay interference on the Immulite immunoassay analyser caused by heterophilic antibodies, which were eliminated by protein A/G treatment. We point out the importance of a close collaboration between doctors and the laboratory to avoid unnecessary clinical intervention

Comparison of ex vivo stability of copeptin and vasopressin

BACKGROUND: Copeptin, part of the vasopressin precursor, is increasingly used as marker for vasopressin and is claimed to have better ex vivo stability. However, no study has directly compared the ex vivo stability of copeptin and vasopressin. METHODS: Blood of ten healthy volunteers was collected in EDTA tubes. Next, we studied the effect of various pre-analytical conditions on measured vasopressin and copeptin levels: centrifugation speed, short-term storage temperature and differences between whole blood and plasma, long-term storage temperature and repeated freezing and thawing. The acceptable change limit (ACL), indicating the maximal percentage change that can be explained by assay variability, was used as cut-off to determine changes in vasopressin and copeptin. RESULTS: The ACL was 25% for vasopressin and 19% for copeptin. Higher centrifugation speed resulted in lower vasopressin levels, whereas copeptin concentration was unaffected. In whole blood, vasopressin was stable up to 2 h at 25°C and 6 h at 4°C. In plasma, vasopressin was stable up to 6 h at 25°C and 24 h at 4°C. In contrast, copeptin was stable in whole blood and plasma for at least 24h at both temperatures. At -20°C, vasopressin was stable up to 1 month and copeptin for at least 4 months. Both vasopressin and copeptin were stable after 4 months when stored at -80°C and -150°C. Vasopressin concentration decreased after four freeze-thaw cycles, whereas copeptin concentration was unaffected. CONCLUSION: Vasopressin levels were considerably affected by pre-analytical conditions, while copeptin levels were stable. Therefore, a strict sample handling protocol for measurement of vasopressin is recommended