Multi-analyte profiling of inflammatory mediators in COPD sputum--the effects of processing.

Prior to using a new multi-analyte platform for the detection of markers in sputum it is advisable to assess whether sputum processing, especially mucus homogenization by dithiothreitol (DTT), affects the analysis. In this study we tested a novel Human Inflammation Multi Analyte Profiling® Kit (v1.0 Luminex platform; xMAP®). Induced sputum samples of 20 patients with stable COPD (mean FEV1, 59.2% pred.) were processed in parallel using standard processing (with DTT) and a more time consuming sputum dispersion method with phosphate buffered saline (PBS) only. A panel of 47 markers was analyzed in these sputum supernatants by the xMAP®. Twenty-five of 47 analytes have been detected in COPD sputum. Interestingly, 7 markers have been detected in sputum processed with DTT only, or significantly higher levels were observed following DTT treatment (VDBP, α-2-Macroglobulin, haptoglobin, α-1-antitrypsin, VCAM-1, and fibrinogen). However, standard DTT-processing resulted in lower detectable concentrations of ferritin, TIMP-1, MCP-1, MIP-1ß, ICAM-1, and complement C3. The correlation between processing methods for the different markers indicates that DTT processing does not introduce a bias by affecting individual sputum samples differently. In conclusion, our data demonstrates that the Luminex-based xMAP® panel can be used for multi-analyte profiling of COPD sputum using the routinely applied method of sputum processing with DTT. However, researchers need to be aware that the absolute concentration of selected inflammatory markers can be affected by DTT.

Methazolamide does not impair respiratory work performance in anesthetized rabbits.

In human medicine, the carbonic anhydrase (CA) inhibitor acetazolamide is used to treat irregular breathing disorders. Previously, we demonstrated in the rabbit that this substance stabilized closed-loop gain properties of the respiratory control system, but concomitantly weakened respiratory muscles. Among others, the highly diffusible CA-inhibitor methazolamide differs from acetazolamide in that it fails to activate Ca(2+)-dependent potassium channels in skeletal muscles. Therefore, we aimed to find out, whether or not methazolamide may exert attenuating adverse effects on respiratory muscle performance as acetazolamide. In anesthetized spontaneously breathing rabbits (n = 7), we measured simultaneously the CO(2) responses of tidal phrenic nerve activity, tidal transpulmonary pressure changes, and tidal volume before and after intravenous application of methazolamide at two mean (+/- SE) cumulative doses of 3.5 +/- 0.1 and 20.8 +/- 0.4 mg/kg. Similar to acetazolamide, low- and high-dose methazolamide enhanced baseline ventilation by 52 +/- 10% and 166 +/- 30%, respectively (P < 0.01) and lowered the base excess in a dose-dependent manner by up to 8.3 +/- 0.9 mmol/l (P < 0.001). The transmission of a CO(2)-induced rise in phrenic nerve activity into volume and/or pressure and, hence, respiratory work performance was 0.27 +/- 0.05 ml x kg(-1) x kPa x unit(-1) under control conditions, but remained unchanged upon low- or high-dose methazolamide, at 0.30 +/- 0.06 and 0.28 +/- 0.07 ml x kg(-1) x kPa x unit(-1), respectively. We conclude that methazolamide does not cause respiratory muscle weakening at elevated levels of ventilatory drive. This substance (so far not used for medication of respiratory diseases) may thus exert stabilizing influences on breathing control without adverse effects on respiratory muscle function.

Food composition and acid-base balance: alimentary alkali depletion and acid load in herbivores.

Alkali-enriched diets are recommended for humans to diminish the net acid load of their usual diet. In contrast, herbivores have to deal with a high dietary alkali impact on acid-base balance. Here we explore the role of nutritional alkali in experimentally induced chronic metabolic acidosis. Data were collected from healthy male adult rabbits kept in metabolism cages to obtain 24-h urine and arterial blood samples. Randomized groups consumed rabbit diets ad libitum, providing sufficient energy but variable alkali load. One subgroup (n = 10) received high-alkali food and approximately 15 mEq/kg ammonium chloride (NH4Cl) with its drinking water for 5 d. Another group (n = 14) was fed low-alkali food for 5 d and given approximately 4 mEq/kg NH4Cl daily for the last 2 d. The wide range of alimentary acid-base load was significantly reflected by renal base excretion, but normal acid-base conditions were maintained in the arterial blood. In rabbits fed a high-alkali diet, the excreted alkaline urine (pH(u) > 8.0) typically contained a large amount of precipitated carbonate, whereas in rabbits fed a low-alkali diet, both pH(u) and precipitate decreased considerably. During high-alkali feeding, application of NH4Cl likewise decreased pH(u), but arterial pH was still maintained with no indication of metabolic acidosis. During low-alkali feeding, a comparably small amount of added NH4Cl further lowered pH(u) and was accompanied by a significant systemic metabolic acidosis. We conclude that exhausted renal base-saving function by dietary alkali depletion is a prerequisite for growing susceptibility to NH4Cl-induced chronic metabolic acidosis in the herbivore rabbit.

NHE3 in the human brainstem: implication for the pathogenesis of the sudden infant death syndrome (SIDS)?

Previous studies have demonstrated an inverse correlation between the degree of respiratory drive and NHE3 mRNA expression in the brainstem of awake rabbits. Here we show that the levels of NHE3 mRNA extractable from kryo-conserved tissue are highly variable also in the human brainstem. As an insufficient drive to breath may be a final event causing sudden infant death, we compared the expression of NHE3 mRNA in a collective of children who died from non-natural causes to an equal number of SIDS victims. Evaluation of signals from NHE3 RT-PCR showed higher values for the SIDS collective than for the control group. We suggest that the level of NHE3 expression in brainstem tissue may contribute to the vulnerability of infants for SIDS.

Role of brainstem sodium/proton exchanger 3 for breathing control during chronic acid base imbalance.

RATIONALE: The sodium/proton exchanger (NHE) 3 is expressed in brainstem areas with prevalence for central chemosensitivity. Selective NHE3 inhibitors can evoke CO(2) mimetic responses both in vitro and in vivo, demonstrating the functional significance of this pH-regulating protein. Moreover, levels of NHE3 expression are inversely correlated to interindividual differences of baseline ventilation in conscious rabbits. OBJECTIVES: We explored the influence of chronic acid-base disturbances on mRNA levels of brainstem NHE3 in relation to breathing control. METHODS: Alveolar ventilation (Va), blood gases, systemic base excess (BE), and metabolic Vco(2) were determined in rabbits shortly after exposure to either CO(2)-enriched air for 3 days (n = 5) or to ammonium chloride with drinking water for 2 days (n = 6). Untreated animals served as controls (n = 24). NHE3 mRNA within the obex region was quantified by real-time reverse transcription-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: After chronic hypercapnia, we found a compensatory rise of BE (mean +/- SEM) to 5.3 +/- 0.5 mmol x L(-1) with slightly elevated Pa(CO(2)). Brainstem NHE3 mRNA as well as Va were not significantly different from control levels. In the NH(4)Cl group, arterial pH was approximately 0.09 units lower than control, and BE decreased to -6.5 +/- 1.6 mmol x L(-1) with slightly decreased Pa(CO(2)), but considerably reduced Va (by approximately 25%; P < 0.05) and Vco(2). Concomitantly, brainstem NHE3 mRNA had increased from control level of 1.45 +/- 0.19 to 3.64 +/- 0.37 fg cDNA/mug RNA; P < 0.01. CONCLUSIONS: Expression of brainstem NHE3 is up-regulated by chronic metabolic acidosis but not by prolonged hypercapnia. It is proposed that elevated brainstem NHE3 expression contributes to limit maladaptive hyperventilation during metabolic acidosis.

Food mineral composition and acid-base balance in preterm infants.

BACKGROUND: Due to a transient age-related low renal capacity for net acid excretion, preterm infants fed formula are at a considerable risk of spontaneously developing incipient late metabolic acidosis, clinically characterized by e.g., disturbed bone mineralization and impaired growth. AIM OF THE STUDY: From acid-base data in blood and urine under different diets of modified human milk or preterm formulas is attempted to explore the impact of food mineral (and protein) composition on renal regulation and systemic acid-base balance in preterm infants. PATIENTS AND METHODS: Data were collected from 48 infants fed their own mother's milk (28 native human milk, 20 enriched with fortifier) and 34 patients on formula (23 on a standard batch, 11 on a modified batch with reduced acid load). Intake of food was measured and acid-base data were determined in blood and timed-urine (8-12 h) samples. RESULTS: Differences in mineral composition of the diets led to considerable differences of daily "alkali-intake", without significant effects on non-respiratory (base excess, BE) and respiratory (PCO(2)) acid-base data in the blood. In contrast, a highly significant proportionality between individual dietary alkali intake and daily renal base (Na(+) + K(+)-Cl(-)) excretion was observed (y = 0.32x-0.70, n = 80, r = 0.77, P < 0.0001), irrespective of the type of the diet. CONCLUSION: Renal base saving mechanisms are normally effective in preterm infants to compensate for differences in dietary acid-base load. Generally, nutritional acid-base challenges can be judged much earlier and more safely by urinary than by blood acid-base analysis. Taking into account the age specific low capacity for renal NAE, the relatively high nutritional acid load of preterm standard formula should be reduced.

Sodium/Proton exchanger 3 in the medulla oblongata and set point of breathing control.

RATIONALE: In vivo inhibition of the sodium/proton exchanger 3 (NHE3) in chemosensitive neurons of the ventrolateral brainstem augments central respiratory drive in anesthetized rabbits. OBJECTIVES: To further explore the possible role of this exchanger for the control of breathing, we examined the individual relationship between brainstem NHE3 abundance and ventilation in rabbits during wakefulness. METHODS: In 32 adult male rabbits on standard nutritional alkali load, alveolar ventilation, metabolic CO2 production, and blood gases were determined, together with arterial and urinary acid-base status and renal base control functions. Expression of NHE3 in brainstem tissue from the obex region was determined by quantitative real-time reverse-transcription polymerase chain reaction analysis. MEASUREMENTS AND MAIN RESULTS: Regarding the distribution above and below the median, we classified high and low brainstem NHE3 animals, expressing a mean (+/- SEM) NHE3 mRNA of 2.08 +/- 0.28 and 0.72 +/- 0.06 fg cDNA/mg RNA, respectively. Alveolar ventilation of high brainstem NHE3 animals was lower than that of low brainstem NHE3 animals (715 +/- 36 vs. 919 +/- 41 ml . minute(-1); p < 0.01), a finding also reflected by a marked difference in Pa(CO2) (5.24 +/- 0.16 vs. 4.44 +/- 0.15 kPa; p < 0.01). Among possible secondary factors, CO2 production, systemic base excess, and fractional renal base reabsorption were not found to be different. CONCLUSIONS: We conclude that the level of brainstem NHE3 expression-most likely via intracellular pH modulation-contributes to the individual control of breathing and Pa(CO2) in conscious rabbits by adjusting the set point and the loop gain of the system.

Food mineral composition and acid-base balance in rabbits.

BACKGROUND: Alkali-rich diets are often recommended in human medicine to prevent the pathological consequences of nutritional acid load in conditions of impaired renal function. AIM OF THE STUDY: This study was undertaken in rabbits as common laboratory animals for basic medical research to explore the impact of high versus low dietary alkali intake on systemic acid-base balance and renal control in a typical herbivore. METHODS: Male rabbits (2.3-4.8 kg) were kept in a metabolism cage. The 24h urine and arterial blood samples were analysed for acid-base data. The metabolic CO2 production was measured to calculate alveolar ventilation. Three randomized groups of animals were fed ad libitum with rabbit chow providing sufficient energy but variable alkali load, assessed by the ashes' cation-anion difference. RESULTS: The average daily nutritional alkali load (+/- SEM) was 67.1 +/- 2.2 mEq x kg(-1) (N = 58) in the group on high, 45.4 +/- 2.5 mEq x kg(-1) (N = 31) in the group on normal and 1.7 +/- 0.5 mEq x kg(-1) (N = 11) in the group on low alkali food. Respective mean arterial base excess values (BE) were 1.4 +/- 0.3 mM, 0.3 +/- 0.4 mM and 0.0 +/- 0.3 mM, being significantly higher on high alkali food (P < 0.05) than in the other groups. Arterial PCO2, alveolar ventilation and metabolic CO2 production were not significantly different between groups. On normal and high-alkali chow, an alkaline urine (pH(u) > 8.0) with 18-20 mmol x kg(-1) bicarbonate/carbonate was excreted daily, typically containing an insoluble precipitate of 35-60% carbonate. On low-alkali diet, the mean pH(u) decreased to 6.26 +/- 0.14, due to a strong reduction of daily excreted soluble bicarbonate and precipitated carbonate to 1.2 +/- 0.6 and 0.7 +/- 0.2 mmol x kg(-1), respectively. Thereby, nearly complete fractional base reabsorption of 97.8 +/- 0.7 % was reached. CONCLUSION: Herbivore nutritional alkali-load elicited large rates of renal base excretion including precipitates, to which the urinary tract of the rabbits appeared to be adapted. Dietary base variations were more accurately reflected in the urine than by the blood acid-base status. A strongly base-deficient diet exerted maximum impact on renal base saving mechanisms, implying a critical precondition for growing susceptibility to metabolic acidosis also in the rabbit.