No evidence for a diminished ovarian reserve among patients with hypertensive disorders of pregnancy: a case control study.

BACKGROUND: Existing evidence suggests a relation between cardiovascular dysfunction and diminished ovarian reserve. While it is known that pre-existent cardiovascular dysfunction is also associated with the development of preeclampsia (PE) during pregnancy, we hypothesize that signs of diminished ovarian reserve may occur more frequently among women with a history of hypertensive disorders of pregnancy (HDP). The aim of our study was therefore to analyse if women with a history of HDP show signs of diminished ovarian reserve, represented by lower anti-Mullarian hormone (AMH) levels, compared to controls. For this retrospective observational case control study, patients included women with a history of HDP, whereas controls constituted of women with a history of an uncomplicated pregnancy. The study was conducted in a tertiary referral centre in which all women underwent a one-time cardiovascular and metabolic assessment. Ovarian reserve and markers of cardiovascular function were evaluated, adjusted for age and body mass index (BMI) using linear regression analyses. RESULTS: 163 patients and 81 controls were included over a time span of 3 years. No signs of diminished ovarian reserve i.e. lower AMH level were observed in the patient group versus controls. A subgroup analysis even showed higher AMH levels in late onset HDP as compared to controls (2.8 vs. 2.0 µg/L, p = 0.025). As expected, cardiovascular function markers were significantly less favourable in the patient group compared to controls; higher levels of systolic blood pressure (BP) (5%), diastolic BP (4%), triglycerides (29%), glucose (4%) and insulin levels (81%) (all p < 0.05), whereas high density lipid (HDL) cholesterol was 12% lower (NS). CONCLUSIONS: Despite unfavourable cardiovascular risk profile, the present study does not substantiate the hypothesis that women with HDP show accelerated ovarian ageing as compared to healthy parous controls. Although HDP patients should be warned about their cardiovascular health, they shouldn't be concerned about unfavourable ovarian reserve status.

Mid-regional proadrenomedullin, C-terminal proendothelin-1 values, and disease course are not different in critically ill SARS-CoV-2 pneumonia patients with obesity.

BACKGROUND/OBJECTIVES: Patients affected by obesity and Coronavirus disease 2019, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), appear to have a higher risk for intensive care (ICU) admission. A state of low-grade chronic inflammation in obesity has been suggested as one of the underlying mechanisms. We investigated whether obesity is associated with differences in new inflammatory biomarkers mid-regional proadrenomedullin (MR-proADM), C-terminal proendothelin-1 (CT-proET-1), and clinical outcomes in critically ill patients with SARS-CoV-2 pneumonia. SUBJECTS/METHODS: A total of 105 critically ill patients with SARS-CoV-2 pneumonia were divided in patients with obesity (body mass index (BMI) ≥ 30 kg/m2, n = 42) and patients without obesity (BMI < 30 kg/m2, n = 63) and studied in a retrospective observational cohort study. MR-proADM, CT-proET-1 concentrations, and conventional markers of white blood count (WBC), C-reactive protein (CRP), and procalcitonin (PCT) were collected during the first 7 days. RESULTS: BMI was 33.5 (32-36.1) and 26.2 (24.7-27.8) kg/m2 in the group with and without obesity. There were no significant differences in concentrations MR-proADM, CT-proET-1, WBC, CRP, and PCT at baseline and the next 6 days between patients with and without obesity. Only MR-proADM changed significantly over time (p = 0.039). Also, BMI did not correlate with inflammatory biomarkers (MR-proADM rho = 0.150, p = 0.125, CT-proET-1 rho = 0.179, p = 0.067, WBC rho = -0.044, p = 0.654, CRP rho = 0.057, p = 0.564, PCT rho = 0.022, p = 0.842). Finally, no significant differences in time on a ventilator, ICU length of stay, and 28-day mortality between patients with or without obesity were observed. CONCLUSIONS: In critically ill patients with confirmed SARS-CoV-2 pneumonia, obesity was not associated with differences in MR-proADM, and CT-proET-1, or impaired outcome. TRIAL REGISTRATION: Netherlands Trial Register, NL8460.

Low-Iodine Diet of 4 Days Is Sufficient Preparation for 131I Therapy in Differentiated Thyroid Cancer Patients.

CONTEXT: No consensus exists about the optimal duration of the low-iodine diet (LID) in the preparation of 131I therapy in differentiated thyroid cancer (DTC) patients. OBJECTIVE: This work aimed to investigate if a LID of 4 days is enough to achieve adequate iodine depletion in preparation for 131I therapy. In addition, the nutritional status of the LID was evaluated. METHODS: In this prospective study, 65 DTC patients treated at 2 university medical centers were included between 2018 and 2021. The patients collected 24-hour urine on days 4 and 7 of the LID and kept a food diary before and during the LID. The primary outcome was the difference between the 24-hour urinary iodine excretion (UIE) on both days. RESULTS: The median 24-hour UIE on days 4 and 7 of the LID were not significantly different (36.1 mcg [interquartile range, 25.4-51.2 mcg] and 36.5 mcg [interquartile range, 23.9-47.7 mcg], respectively, P = .43). On day 4 of the LID, 72.1% of the DTC patients were adequately prepared (24-hour UIE < 50 mcg), and 82.0% of the DTC patients on day 7 (P = .18). Compared to the self-reported regular diet, DTC patients showed a significantly (P < .01) lower percentage of nutrient intake (calories, protein, calcium, iodine, and water) during the LID. CONCLUSION: The 24-hour UIE on day 4 of the LID did not differ from day 7, and therefore shortening the LID from 7 to 4 days seems justified to prepare DTC patients for 131I therapy in areas with sufficient iodine intake and may be beneficial to maintain a sufficient nutritional intake during DTC treatment.

Endothelium-associated biomarkers mid-regional proadrenomedullin and C-terminal proendothelin-1 have good ability to predict 28-day mortality in critically ill patients with SARS-CoV-2 pneumonia: A prospective cohort study.

PURPOSE: We assessed the ability of mid-regional proadrenomedullin (MR-proADM) and C-terminal proendothelin-1 (CT-proET-1) to predict 28-day mortality in critically ill patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia. METHODS: Biomarkers were collected during the first seven days in this prospective observational cohort study. We investigated the relationship between biomarkers and mortality in a multivariable Cox regression model adjusted for age and SOFA score. RESULTS: In 105 critically ill patients with confirmed SARS-CoV-2 pneumonia 28-day mortality was 28.6%. MR-proADM and CT-proET-1 were significantly higher in 28-day non-survivors at baseline and over time. ROC curves revealed high accuracy to identify non-survivors for baseline MR-proADM and CT-proET-1, AUC 0.84, (95% CI 0.76-0.92), p < 0.001 and 0.79, (95% CI 0.69-0.89), p < 0.001, respectively. The AUC for prediction of 28-day mortality for MR-proADM and CT-proET-1 remained high over time. MR-proADM ≥1.57 nmol/L and CT-proET-1 ≥ 111 pmol/L at baseline were significant predictors for 28-day mortality (HR 6.80, 95% CI 3.12-14.84, p < 0.001 and HR 3.72, 95% CI 1.71-8.08, p 0.01). CONCLUSION: Baseline and serial MR-proADM and CT-proET-1 had good ability to predict 28-day mortality in critically ill patients with SARS-CoV-2 pneumonia. TRIAL REGISTRATION: NEDERLANDS TRIAL REGISTER, NL8460.

The relationships of sex hormone-binding globulin, total testosterone, androstenedione and free testosterone with metabolic and reproductive features of polycystic ovary syndrome.

OBJECTIVE: A recent Mendelian randomization study has suggested a causal role for sex hormone-binding globulin (SHBG), total testosterone and free testosterone in the pathogenesis of polycystic ovary syndrome (PCOS). The aim of this study was to assess the relationships of SHBG, androstenedione, total and free testosterone with the individual metabolic and reproductive features of PCOS. DESIGN: Cross-sectional data in PCOS patients (n=96) prospectively collected in a secondary/tertiary clinic for menstrual cycle disorders. METHODS: Multivariable regression analyses were conducted to study the associations between SHBG, androstenedione, total and free testosterone with metabolic (BMI, waist circumference, systolic and diastolic blood pressure, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides and homeostatic model assessment for insulin resistance [HOMA2-IR]) and reproductive features (menstrual cycle length, antral follicle count, anti-Müllerian hormone, luteinizing hormone, follicle-stimulating hormone and Ferriman-Gallwey score) of PCOS. RESULTS: Serum SHBG and free testosterone, but not total testosterone or androstenedione, were significantly associated with BMI, waist circumference, serum triglycerides, HDL cholesterol, LDL cholesterol and HOMA2-IR. The strength of the associations with serum lipids was reduced after adjustment for BMI, but not for HOMA2-IR. Total testosterone was significantly associated with antral follicle count. SHBG, total testosterone and androstenedione were significantly associated with serum AMH. Only the strength of the association for SHBG was reduced after adjustment for BMI. CONCLUSIONS: Serum SHBG is associated with primarily metabolic features, whereas total testosterone and androstenedione are associated with reproductive features of PCOS. These results suggest a differential underlying pathophysiology for the metabolic and reproductive features of PCOS.

The Effect of Bolus Vitamin D3 Supplementation on Distal Radius Fracture Healing: A Randomized Controlled Trial Using HR-pQCT.

Vitamin D is an important factor in bone metabolism. Animal studies have shown a positive effect of vitamin D3 supplementation on fracture healing, but evidence from clinical trials is inconclusive. A randomized controlled trial was performed to assess the effects of vitamin D3 supplementation on fracture healing using HR-pQCT-based outcome parameters. Thirty-two postmenopausal women with a conservatively treated distal radius fracture were included within 2 weeks postfracture and randomized to a low-dose (N = 10) and a high-dose (N = 11) vitamin D intervention group receiving a 6-week bolus dose, equivalent to 700 and 1800 IU vitamin D3 supplementation per day, respectively, in addition to a control group (N = 11) receiving no supplementation. After the baseline visit 1-2 weeks postfracture, follow-up visits were scheduled at 3-4, 6-8, and 12 weeks postfracture. At each visit, HR-pQCT scans of the fractured radius were performed. Cortical and trabecular bone density and microarchitectural parameters and microfinite element analysis-derived torsion, compression, and bending stiffness were assessed. Additionally, serum markers of bone resorption (CTX) and bone formation (PINP) were measured. Baseline serum levels of 25OHD3 were <50 nmol/L in 33% of all participants and <75 nmol/L in 70%. Compared with the control group, high-dose vitamin D3 supplementation resulted in a decreased trabecular number (regression coefficient ß: -0.22; p < 0.01) and lower compression stiffness (B: -3.63; p < 0.05, together with an increase in the bone resorption marker CTX (B: 0.062; p < 0.05). No statistically significant differences were observed between the control and low-dose intervention group. In conclusion, the bolus equivalent of 700 U/day vitamin D3 supplementation in a Western postmenopausal population does not improve distal radius fracture healing and an equivalent dose of 1800 IU/day may be detrimental in restoring bone stiffness during the first 12 weeks of fracture healing. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Relationship between de novo lipogenesis and serum sex hormone binding globulin in humans.

OBJECTIVE: Obesity and liver fat are associated with decreased levels of serum sex hormone binding globulin (SHBG). Laboratory studies suggest that hepatic de novo lipogenesis (DNL) is involved in the downregulation of SHBG synthesis. The aim of the present study was to address the role of DNL on serum SHBG in humans. DESIGN: A cross-sectional study examining the association between DNL, measured by stable isotopes, and serum SHBG, stratified by sex. PARTICIPANTS: Healthy men (n = 34) and women (n = 21) were combined from two cross-sectional studies. Forty-two per cent of participants had hepatic steatosis, and the majority were overweight (62%) or obese (27%). RESULTS: DNL was inversely associated with SHBG in women (ß: -0.015, 95% CI: -0.030; 0.000), but not in men (ß: 0.007, 95% CI: -0.005; 0.019) (p for interaction = .068). Adjustment for study population, age and body mass index did not materially change these results, although statistical significance was lost after adjustment for serum insulin. CONCLUSIONS: An inverse association between DNL and SHBG may explain the decreased SHBG levels that are observed in obesity, at least in women.

Kidney and vascular function in adult patients with hereditary fructose intolerance.

Objective: Previous studies have shown that patients with hereditary fructose intolerance (HFI) are characterized by a greater intrahepatic triglyceride content, despite a fructose-restricted diet. The present study aimed to examine the long-term consequences of HFI on other aldolase-B-expressing organs, i.e. the kidney and vascular endothelium. Methods: Fifteen adult HFI patients were compared to healthy control individuals matched for age, sex and body mass index. Aortic stiffness was assessed by carotid-femoral pulse wave velocity (cf-PWV) and endothelial function by peripheral arterial tonometry, skin laser doppler flowmetry and the endothelial function biomarkers soluble E-selectin [sE-selectin] and von Willebrand factor. Serum creatinine and cystatin C were measured to estimate the glomerular filtration rate (eGFR). Urinary glucose and amino acid excretion and the ratio of tubular maximum reabsorption of phosphate to GFR (TmP/GFR) were determined as measures of proximal tubular function. Results: Median systolic blood pressure was significantly higher in HFI patients (127 versus 122 mmHg, p = .045). Pulse pressure and cf-PWV did not differ between the groups (p = .37 and p = .49, respectively). Of all endothelial function markers, only sE-selectin was significantly higher in HFI patients (p = .004). eGFR was significantly higher in HFI patients than healthy controls (119 versus 104 ml/min/1.73m2, p = .001, respectively). All measurements of proximal tubular function did not differ significantly between the groups. Conclusions: Adult HFI patients treated with a fructose-restricted diet are characterized by a higher sE-selectin level and slightly higher systolic blood pressure, which in time could contribute to a greater cardiovascular risk. The exact cause and, hence, clinical consequences of the higher eGFR in HFI patients, deserves further study.

Longitudinal changes in placental biomarkers in women with early versus late placental dysfunction.

Objective: To evaluate longitudinal changes of angiogenic biomarkers in early- (EO-PD) versus late-onset (LO-PD) placental dysfunction. Methods: Serum PlGF and sFlt-1 measured at different intervals in EO-PD (n= 43), LO-PD (n= 31) and controls (n = 133). Results: sFlt-1/PlGF ratio was higher at 16 weeks (30.6 vs 17.5), 20 weeks (29.3 vs 8.9) and 30 weeks (16.6 vs 6.7) in EO-PD vs controls (all p< 0.05), but not in LO-PD. Longitudinal changes for all intervals had higher AUC than single measurements. Conclusion: Longitudinal biomarker change between 12 and 30 weeks could improve prediction of EO-PD compared to single measurements.

Can Fetal Growth Velocity and First Trimester Maternal Biomarkers Improve the Prediction of Small-for-Gestational Age and Adverse Neonatal Outcome?

BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the value of adding fetal growth velocity and first trimester maternal biomarkers to baseline screening, for the prediction of small-for-gestational age (SGA) and adverse neonatal outcomes. METHOD: A retrospective cohort study was conducted of singleton pregnancies in the Maastricht University Medical Centre between 2012 and 2016. The biomarkers PAPP-A, ß-hCG, PlGF, and sFlt-1 were measured at 11-13 weeks of gestational age (GA) and two fetal growth scans were performed (18-22 and 30-34 weeks of GA). Differences in biomarkers and growth velocities were compared between appropriate-for-gestational age (AGA; birth weight percentile 10-90) and SGA (birth weight percentile <10). Combinations of the biomarkers and fetal growth velocity were added to baseline screening for the prediction of SGA and adverse neonatal outcome. RESULTS: We included 296 singleton pregnancies. Compared to AGA (n = 251), SGA neonates (n = 45) had significantly lower growth velocities in the abdominal circumference (mm/week): 10.1 ± 0.98 versus 10.8 ± 0.98, p = 0.001. Compared with AGA, the SGA neonates had higher sFlt-1 multiples of the median (MoM): 0.89 (0.55) versus 0.76 (0.44), p = 0.023, and a higher sFlt-1/PlGF MoM ratio: 1.09 (1.03) versus 0.90 (0.64), p = 0.027. For a 15% false-positive rate, the prediction of SGA neonates increased from 44.8% for the baseline screening model to 56.5% after the addition of fetal growth velocities, and to 73.9% after the further addition of maternal biomarkers (PPV 9.6%, NPV 82.4%). The corresponding AUC for the three models were 0.722, 0.804, and 0.839, respectively. In addition, AGA neonates with reduced fetal growth velocity had more adverse neonatal outcomes compared to the AGA reference group (12.4 vs. 3.9%, p = 0.013). CONCLUSIONS: Combining fetal growth velocity with first trimester biomarkers resulted in a better prediction of SGA compared to baseline screening parameters alone. This approach could possibly result in reduced adverse neonatal outcomes in neonates, who are at a potential risk due to late mild placental dysfunction.

Patients With Aldolase B Deficiency Are Characterized by Increased Intrahepatic Triglyceride Content.

CONTEXT: There is an ongoing debate about whether and how fructose is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). A recent experimental study showed an increased intrahepatic triglyceride (IHTG) content in mice deficient for aldolase B (aldo B-/-), the enzyme that converts fructose-1-phosphate to triose phosphates. OBJECTIVE: To translate these experimental findings to the human situation. DESIGN: Case-control study. SETTING: Outpatient clinic for inborn errors of metabolism. PATIENTS OR OTHER PARTICIPANTS: Patients with hereditary fructose intolerance, a rare inborn error of metabolism caused by a defect in aldolase B (n = 15), and healthy persons matched for age, sex, and body mass index (BMI) (n =15). MAIN OUTCOME MEASURE: IHTG content, assessed by proton magnetic resonance spectroscopy. RESULTS: IHTG content was higher in aldo B-/- patients than controls (2.5% vs 0.6%; P = 0.001) on a background of lean body mass (median BMI, 20.4 and 21.8 kg/m2, respectively). Glucose excursions during an oral glucose load were higher in aldo B-/- patients (P = 0.043). Hypoglycosylated transferrin, a surrogate marker for hepatic fructose-1-phosphate concentrations, was more abundant in aldo B-/- patients than in controls (P < 0.001). Finally, plasma ß-hydroxybutyrate, a biomarker of hepatic ß-oxidation, was lower in aldo B-/- patients than controls (P = 0.009). CONCLUSIONS: This study extends previous experimental findings by demonstrating that aldolase B deficiency also results in IHTG accumulation in humans. It suggests that the accumulation of fructose-1-phosphate and impairment of ß-oxidation are involved in the pathogenesis.

Contribution of Liver Fat to Weight Loss-Induced Changes in Serum Hepatokines: A Randomized Controlled Trial.

CONTEXT: Hepatokines have emerged as potential mediators of obesity-associated comorbidities, such as type 2 diabetes, cardiovascular disease, fractures, and central hypogonadism. OBJECTIVE: To assess whether weight loss-induced changes in hepatokines are mediated by intrahepatic triglyceride (IHTG) content. DESIGN: Cross-sectional study and randomized controlled trial. SETTING: General community. PARTICIPANTS: Metabolically healthy, lean men (waist <94 cm; n = 25) and men with abdominal obesity (waist 102 to 110 cm; n = 52). INTERVENTION: Men with abdominal obesity were randomized to 8-week dietary weight loss or no weight loss. MAIN OUTCOME MEASURES: IHTG and serum hepatokines, that is, serum IGF1, IGF binding protein 1 (IGFBP1), SHBG, fibroblast growth factor 21 (FGF21), fetuin A, and plasma fetuin B. RESULTS: All hepatokines, except for fetuin B, were significantly different between lean men and men with obesity. After the weight-loss intervention (-10.3 kg; 95% CI, -11.4 to-9.2), serum IGF1, IGFBP1, SHBG, and fetuin A approached the values observed in lean men. Cross-sectional associations were observed between IHTG and IGF1 (ß = -0.51; 95% CI, -0.82 to -0.20), IGFBP1 (ß = -4.2; 95% CI, -7.7 to -0.7), and FGF21 (ß = 2.1; 95% CI, 1.3 to 2.9) in lean men and men with abdominal obesity combined. Weight loss resulted in a reduction of IHTG (treatment effect, -2.2%; 95% CI, -3.4% to -1.2%) that was associated with a change in IGF1 (ß = -0.9; 95% CI, -1.3 to -0.4), IGFBP1 (ß = -0.17; 95% CI, -0.31 to -0.03), and SHBG levels (ß = -0.18; 95% CI, -0.29 to -0.07). Mediation analyses showed that only the weight loss-induced change in serum IGF1 was mediated by IHTG (mediated effect, 32.7%; 95% CI, 4.6% to 79.2%). CONCLUSIONS: Dietary weight loss has differential effects on hepatokines. This study shows that the change in serum IGF1 levels after dietary weight loss is mediated by the change in IHTG content.

Diurnal Rhythm of Cardiac Troponin: Consequences for the Diagnosis of Acute Myocardial Infarction.

BACKGROUND: Interpretation of serial high-sensitivity cardiac troponin (hs-cTn) measurements for the diagnosis of acute myocardial infarction (AMI) assumes random fluctuation of hs-cTn around an individual's homeostatic set point. The aim of this study was to challenge this diagnostic concept. METHODS: Study 1 examined the presence of a diurnal hs-cTn rhythm by hourly blood sampling, day and night, in 24 individuals without a recent history of AMI. Study 2 assessed morning vs evening diagnostic accuracy of hs-cTnT and hs-cTnI in a prospective multicenter diagnostic study of 2782 unselected patients, presenting to the emergency department with acute chest pain. RESULTS: In study 1, hs-cTnT, but not hs-cTnI, exhibited a diurnal rhythm, characterized by gradually decreasing concentrations throughout daytime, rising concentrations during nighttime, to peak concentrations in the morning (mean 16.2 ng/L at 8:30 AM and 12.1 ng/L at 7:30 PM). In study 2, the hs-cTnT rhythm was confirmed by higher hs-cTnT concentrations in early-morning presenters compared to evening presenters with an adjudicated diagnosis of noncardiac disease. The diagnostic accuracy [area under the receiver-operation characteristics curve (AUC)] of hs-cTnT at presentation, 1 h, and for the combination of absolute changes with presenting concentration, were very high and comparable among patients presenting early morning as compared to evening (all AUC >0.93). hs-cTnI exhibited no diurnal rhythm with no differences in AUC among early-morning and evening presenters. CONCLUSIONS: Rhythmic diurnal variation of hs-cTnT is a general phenomenon that is not seen with hs-cTnI. While the diurnal hs-cTnT rhythm does not seem to affect the diagnostic accuracy of hs-cTnT for AMI, it should be considered when using hs-cTnT for screening purposes. CLINICAL TRIAL REGISTRATION: 1. Circadian Variation of Cardiac Troponin, NCT02091427, www.clinicaltrials.gov/ct2/show/NCT02091427. 2. Advantageous Predictors of Acute Coronary Syndrome Evaluation (APACE) Study, NCT00470587, www.clinicaltrials.gov/ct2/show/NCT00470587.

A multicenter evaluation of dysthyroxinemia in a defined patient cohort.

BACKGROUND: In the region Limburg (The Netherlands) almost all of the five participating laboratories use a different immunoassay platform to determine thyroid stimulating hormone (TSH) and free thryoxine (FT4). With the frequent transfer of patients within the region, harmonization of test result interpretation is necessary. In this study, we investigated dysthyroxinemia classification between participating laboratories and developed procedures for improvement. METHODS: Two ring surveys with an interval of 2 years were performed. Four patient groups (n=100) with different dysthyroxinemia classification were based on biochemical results of the Autodelphia analyzer. Samples were tested in five participating laboratories. In each group the percentage of patients classified with dysthyroxinemia was calculated and differences were analyzed by the Fisher's exact test. RESULTS: After the first survey, the percentage of patients with hyperthyroxinemia was more than 20% lower in three laboratories compared to the other two. Bhattacharya analysis revealed that the upper reference limit of FT4 was 20%-30% too high in two laboratories. Adjustments of reference ranges appeared to be effective in the second survey. The third laboratory reported significantly lower percentages of patients with hyperthyroxinemia in the second survey. New FT4 reference ranges were determined for this laboratory, resulting in adequate classification of hyperthyroxinemia. CONCLUSIONS: This study illustrates the potential of a multicenter evaluation of dysthyroxinemia in a biochemical-defined patient cohort. In particular, classification of hyperthyroxinemia differed between laboratories. Adjustments of reference ranges resulted in better agreement of dysthyroxinemia classification. Even using internal and external quality assurance programs, application of multicenter ring surveys is advised to prevent inadequate reference ranges.

A SELDI-TOF-MS study in Lacunar Stroke with Subsequent Haptoglobin Phenotyping.

Using Surface-Enhanced Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS), we aimed to detect differences in protein profile in serum samples of two lacunar stroke subtypes. SELDI-TOF-MS, followed by protein identification, was performed in samples of 8 first-ever lacunar stroke patients with MR imaging showing a single symptomatic lacunar lesion (type 1), and 8 with multiple additional "silent" lacunar lesions and extensive white matter lesions (type 2). A 16 kDa protein, identified as alpha-2-chain of haptoglobin (Hp), was found to be overrepresented in type 1 compared to type 2 (peak intensity 12.5 vs. 5.0; p=0.02). As a polymorphism with two alleles, Hp-1 and Hp 2, determines the presence of alpha-1 and/or alpha-2-chains in the Hp-molecule, Hp phenotypic analysis was performed. Hp 1 : Hp-2 allele frequency was 0.562 : 0.438 in type 1 and 0.812 : 0.188 in type 2 (population reference approximately 0.4 : 0.6). We conclude that the overrepresentation of the alpha-2-chain in lacunar stroke type 1 compared to type 2 relates to a higher Hp-2 allele frequency in the former. Yet, compared to population reference, the phenotype distribution in both patient groups deviates towards a high Hp-1 allele frequency. Our findings suggest a role for the Hp gene in the etiology of cerebral small vessel disease. Larger studies are now needed to explore this new candidate gene.

A specific immunoprecipitation method for isolating isoforms of insulin-like growth factor binding protein-3 from serum.

BACKGROUND: This report describes an in-house developed immunoprecipitation method to isolate insulin-like growth factor binding protein-3 (IGFBP-3) and its isoforms from serum. The method was compared to other existing immunoprecipitation methods. The study of IGFBP-3 isoforms is relevant for further studies on congenital defects in glycosylation (CDG), galactosemia, and alcoholic liver cirrhosis. METHODS: Monoclonal and/or polyclonal anti-human IGFBP-3 antibodies were covalently immobilised on protein-A Sepharose beads using dimethyl pimelimidate as cross-linker. By incubation with these immobilised antibodies, intact IGFBP-3 and fragments of IGFBP-3 were isolated from serum. Enzyme-linked immunosorbent assay (ELISA) and one-dimensional gel electrophoresis (1-DE) experiments were performed to define the optimal immunoprecipitation method. Isolated proteins were separated by 1-DE and two-dimensional gel electrophoresis (2-DE) and visualised by Western blotting. RESULTS: ELISA and 1-DE results illustrated that an optimal isolation was performed using PBS for the incubation with serum. Laemmli sample buffer, containing 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride and sodium dodecyl sulfate, or urea/CHAPS was optimal for the elution. Clinical validation was performed using CDG-Ia serum samples. The 2-DE experiments showed characteristic isoform patterns for CDG-Ia. CONCLUSIONS: The optimized in-house developed immunoprecipitation method resulted in specific detection of IGFBP-3 isoforms and is suitable for further studies on glycosylation defects.