Kinetics of Physiological Responses as a Measure of Intensity and Hydration Status During Experimental Physical Stress in Human Volunteers.

INTRODUCTION: Strenuous physical stress induces a range of physiological responses, the extent depending, among others, on the nature and severity of the exercise, a person's training level and overall physical resilience. This principle can also be used in an experimental set-up by measuring time-dependent changes in biomarkers for physiological processes. In a previous report, we described the effects of workload delivered on a bicycle ergometer on intestinal functionality. As a follow-up, we here describe an analysis of the kinetics of various other biomarkers. AIM: To analyse the time-dependent changes of 34 markers for different metabolic and immunological processes, comparing four different exercise protocols and a rest protocol. METHODS: After determining individual maximum workloads, 15 healthy male participants (20-35 years) started with a rest protocol and subsequently performed (in a cross-over design with 1-week wash-out) four exercise protocols of 1-h duration at different intensities: 70% W max in a hydrated and a mildly dehydrated state, 50% W max and intermittent 85/55% W max in blocks of 2 min. Perceived exertion was monitored using the Borg' Rating of Perceived Exertion scale. Blood samples were collected both before and during exercise, and at various timepoints up to 24 h afterward. Data was analyzed using a multilevel mixed linear model with multiple test correction. RESULTS: Kinetic changes of various biomarkers were exercise-intensity-dependent. Biomarkers included parameters indicative of metabolic activity (e.g., creatinine, bicarbonate), immunological and hematological functionality (e.g., leukocytes, hemoglobin) and intestinal physiology (citrulline, intestinal fatty acid-binding protein, and zonulin). In general, responses to high intensity exercise of 70% W max and intermittent exercise i.e., 55/85% W max were more pronounced compared to exercise at 50% W max . CONCLUSION: High (70 and 55/85% W max ) and moderate (50% W max ) intensity exercise in a bicycle ergometer test produce different time-dependent changes in a broad range of parameters indicative of metabolic activity, immunological and hematological functionality and intestinal physiology. These parameters may be considered biomarkers of homeostatic resilience. Mild dehydration intensifies these time-related changes. Moderate intensity exercise of 50% W max shows sufficient physiological and immunological responses and can be employed to test the health condition of less fit individuals.

Plasma citrulline concentration, a marker for intestinal functionality, reflects exercise intensity in healthy young men.

BACKGROUND & AIMS: Plasma citrulline concentration is considered to be a marker for enterocyte metabolic mass and to reflect its reduction as may occur during intestinal dysfunction. Strenuous exercise can act as a stressor to induce small intestinal injury. Our previous studies suggest that this comprises the intestinal ability to produce citrulline from a glutamine-rich protein bolus. In this study we investigated the effects of different exercise intensities and hydration state on citrulline and iFABP levels following a post-exercise glutamine bolus in healthy young men. METHODS: Fifteen healthy young men (20-35 yrs, VO2 max 56.9 ± 3.9 ml kg-1 min-1) performed in a randomly assigned cross-over design, a rest (protocol 1) and four cycle ergometer protocols. The volunteers cycled submaximal at different percentages of their individual pre-assessed maximum workload (Wmax): 70% Wmax in hydrated (protocol 2) and dehydrated state (protocol 3), 50% Wmax (protocol 4) and intermittent 85/55% Wmax in blocks of 2 min (protocol 5). Immediately after 1 h exercise or rest, subjects were given a glutamine bolus with added alanine as an iso-caloric internal standard (7.5 g of each amino acid). Blood samples were collected before, during and after rest or exercise, up to 24 h post onset of the experiment. Amino acids and urea were analysed as metabolic markers, creatine phosphokinase and iFABP as markers of muscle and intestinal damage, respectively. Data were analysed using a multilevel mixed linear statistical model. p values were corrected for multiple testing. RESULTS: Citrulline levels already increased before glutamine supplementation during normal hydrated exercise, while this was not observed in the dehydrated and rest protocols. The low intensity exercise protocol (50% Wmax) showed the highest increase in citrulline levels both during exercise (43.83 µmol/L ± 2.63 (p < 0.001)) and after glutamine consumption (50.54 µmol/L ± 2.62) compared to the rest protocol (28.97 µmol/L ± 1.503 and 41.65 µmol/L ± 1.96, respectively, p < 0.05). However, following strenuous exercise at 70% Wmax in the dehydrated state, citrulline levels did not increase during exercise and less after the glutamine consumption when compared to the resting condition and hydrated protocols. In line with this, serum iFABP levels were the highest with the strenuous dehydrated protocol (1443.72 µmol/L ± 249.9, p < 0.001), followed by the high intensity exercise at 70% Wmax in the hydrated condition. CONCLUSIONS: Exercise induces an increase in plasma citrulline, irrespective of a glutamine bolus. The extent to which this occurs is dependent on exercise intensity and the hydration state of the subjects. The same holds true for both the post-exercise increase in citrulline levels following glutamine supplementation and serum iFABP levels. These data indicate that citrulline release during exercise and after an oral glutamine bolus might be dependent on the intestinal health state and therefore on intestinal functionality. Glutamine is known to play a major role in intestinal physiology and the maintenance of gut health and barrier function. Together, this suggests that in clinical practice, a glutamine bolus to increase citrulline levels after exercise might be preferable compared to supplementing citrulline itself. To our knowledge this is the first time that exercise workload-related effects on plasma citrulline are reported in relation to intestinal damage.

Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette (ABC) multidrug transporter that confers resistance to various anticancer drugs like topotecan and mitoxantrone. To obtain more insight in its cellular functioning, we investigated phosphorylation and N-linked glycosylation of BCRP. In the epithelial Madin-Darby canine kidney (MDCK) cell line, we did not detect phosphorylation of BCRP, in contrast to MRP2, which was phosphorylated. In the ovarian carcinoma cell line T8 also no phosphorylated BCRP was detected. As BCRP in both lines effectively transports drugs, it appears that phosphorylation of BCRP (if it occurs at all) is not needed for drug transport. We further mutated the asparagine residues 418, 557 and 596 in three putative N-linked glycosylation motifs of BCRP to alanines. Mutant proteins were expressed in CHO9 and MDCKII cells by transient transfection and characterized by Western blot and immunofluorescence analysis. We found that only BCRP-N596A and a mutant with all three asparagines mutated (triple mutant) were not glycosylated anymore, indicating that only asparagine 596 is normally glycosylated. The mutation of asparagine 596 (or 418) had little effect on the subcellular localization of BCRP, indicating that N-linked glycosylation is not essential for routing to the plasma membrane. However, BCRP-N557A and the triple mutant were mainly localized intracellularly, probably in the endoplasmic reticulum, suggesting that this mutation disrupted proper routing of BCRP.

[High prevalence of vitamin D deficiency in the south-west Netherlands]. / Hoge prevalentie van vitamine D-deficiëntie in Zuidwest-Nederland.

OBJECTIVE: To determine how often vitamin D deficiency occurs in the populations of a city, the countryside and urbanised areas of the countryside, and also the influence of the seasons. DESIGN: Cross-sectional study carried out in the winter and summer of 2013. Trend analysis from requests for determination of vitamin D levels by general practitioners between January 2010 and August 2012. METHOD: In a random sample survey of all blood samples received by SHL-Groep (diagnostic center for primary care) between 21-01-2013 - 10-03-2013 (winter period) and 01-08-2013 - 30-08-2013 (summer period), Vitamin D levels of the residual material were measured. We reported the results by age group, gender and postal code area in the following areas: The Hague (city), Province of Zeeland (countryside) and West Brabant (urbanised countryside). In addition, the average vitamin D concentration obtained from all the requests for determination of vitamin D levels between January 2010 and August 2012 was measured against the time of year. RESULTS: During the winter period 58.8% of the 2503 participants had a vitamin D serum concentration of < 50 nmol/l (The Hague: 65.6%; Zeeland: 50.9%). A total of 29.9% had vitamin D levels of < 30 nmol/l. In men under the age of 50 years, this was 38.2%. Of the 1910 people tested during the summer, 35.4% had a vitamin D level of < 50 nmol/l (The Hague: 43.7%; Zeeland: 33.5%). 11.6% had a vitamin D level < 30 nmol/l. General practitioners requested the vitamin D levels of 50,441 patients. The average vitamin D level varies considerably with the seasons. CONCLUSION: Vitamin D deficiency occurs frequently, even in relatively young people and more so in cities than in the countryside. The average vitamin D concentration varies with the seasons.

Implementing neonatal screening for haemoglobinopathies in the Netherlands.

BACKGROUND: The birth prevalence of severe haemoglobinopathies such as sickle cell disease (SCD) in the Netherlands has been estimated to be at least 50 newborns per year. Neonatal screening for SCD was added to the Dutch screening programme in January 2007. We here evaluated three high performance liquid chromatography (HPLC) systems for application in neonatal screening for haemoglobinopathies, and present the results of a subsequent pilot screening programme. METHODS: The Variant NewBorn Screening (Vnbs) HPLC system (Bio-Rad) was validated by analysing 131 blood samples and blood mixtures. Subsequently, the performance of the G7 (Tosoh BioScience) and Ultra (Primus Corporation) was compared with the Vnbs. The three HPLC analysers were tested in a pilot screening programme on 21,969 dried blood spot samples from the routine Dutch neonatal screening programme. RESULTS: The pilot screening resulted in 188 abnormal patterns. The three HPLC devices presented comparable within- and between-run precision and detected the abnormal samples similarly. The high throughput, sampling systems, presentation of results, and integration of the chromatograms, however, were different. CONCLUSION: All three analysers detected the same abnormal haemoglobins satisfactorily, but integrated the chromatograms with variable imprecision. Comparison of the results suggested that the Bio-Rad Vnbs was the preferred system. However, software adjustments were required to improve the diagnostic potential of this device for screening for beta- and alpha-thalassaemia.

rab4 regulates transport to the apical plasma membrane in Madin-Darby canine kidney cells.

The small GTPase rab4 is associated with early endosomes and regulates membrane recycling in fibroblasts. rab4 is present in epithelial cells; however, neither its localization nor function has been established in this cell type. We transfected Madin-Darby canine kidney cells with rab4, the GTPase-deficient mutant rab4Q67L, and the dominant negative mutant rab4S22N that poorly binds guanine nucleotides. Confocal immunofluorescence microscopy showed that rab4 was concentrated on internal structures at the lateral side of the cell around the nucleus. Quantitative immunoelectron microscopy revealed that the majority of rab4 was localized in the upper third of the cytoplasm. In cell surface binding experiments with (125)I-transferrin, we found a redistribution of transferrin receptor from the basolateral to the apical plasma membrane in cells expressing rab4 and rab4Q67L. After accumulation of transferrin at 16 degrees C in basolateral early endosomes, rab4 and rab4Q67L increased the amount of apically targeted transferrin receptor. A qualitatively similar effect was obtained in control cells treated with brefeldin A. The effects of brefeldin A and rab4 on apical targeting of transferrin receptor were not additive, suggesting that brefeldin A and rab4 may act in the same transport pathway from common endosomes.

Rab4 function in membrane recycling from early endosomes depends on a membrane to cytoplasm cycle.

The monomeric GTPase rab4 is associated with early endosomes and regulates recycling vesicle formation. Because the function of rab proteins in the biosynthetic pathway does not appear to depend on cycling between membranes and cytosol, we were interested to investigate whether or not this holds true for rab function in the endocytic pathway. We created a chimeric rab4 protein (NHrab4cbvn) in which the carboxyl-terminal prenylation motif was replaced by the transmembrane domain of cellubrevin. The chimeric protein was permanently attached to membranes, properly targeted to early endosomes, and bound guanine nucleotide to the same extent as wild type rab4. However, in transport assays we found that basolaterally endocytosed transferrin was less efficiently transported to the apical cell surface in Madin-Darby canine kidney cells transfected with NHrab4cbvn than in cells expressing wild type rab4. Hence, rab4 function requires ongoing cycles of association and dissociation from early endosomes. This cycle is altered during mitosis when rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. We show here, using a rab4 construct that is permanently hooked onto membranes, that the membrane-bound pool of rab4 is targeted by a mitotic kinase.