Urinary excretion of 2,3-dinor-thromboxane B2 in man under normal conditions, following drugs and during some pathological conditions

Quantitation of 2,3-dinor-thromboxane B2 (2,3-dinor-TxB2) was performed by gas chromatography-mass spectrometry. Under normal conditions the urinary excretion of 2,3-dinor-TxB2 was relatively constant in the same individual from day to day but during a 24-hour period a somewhat higher excretion rate was found during the first few hours after awakening. A pronounced reduction of the urinary excretion of 2,3-dinor-TxB2 was found after oral administration of 500 mg of aspirin or 50 mg of indomethacin, while 500 mg of paracetamol did not affect the urinary excretion. Increased excretion of 2,3-dinor-TxB2 was found in normal pregnancies and in diseases such as diabetes mellitus and homocysteinuria in comparison to the urinary excretion in normal healthy subjects. We also report one case, where the urinary excretion of 2,3-dinor-TxB2 was increased for a short period following the first symptoms of a myocardial infarction and those data indicate that thromboxane A2 (TxA2) may be of pathophysiological importance in human myocardial infarction. The results strongly indicate that measurements of the urinary excretion of 2,3-dinor-TxB2 should be meaningful as a tool for investigation of the involvement of thromboxane in various pathophysiological processes in vivo in man.     

Measurement of urinary 2,3-dinor-thromboxane B2 and thromboxane B2 using bonded-phase phenylboronic acid columns and capillary gas chromatography-negative-ion chemical ionization mass spectrometry

The use of bonded-phase phenylboronic acid columns to selectively extract 2,3-dinor-thromboxane B2 and thromboxane B2 from urine is reported. The compounds were first derivatized as the methoxime and then applied to the phenylboronic acid columns. Subsequent purification by thin-layer chromatography and derivatization to the pentafluorobenzyl ester, trimethylsilyl ether followed by capillary gas chromatography-negative-ion chemical ionization mass spectrometry, monitoring specific ions, allows quantitation in the low-picogram/milliliter range. In healthy male volunteers, the median excretions of 2,3-dinor-thromboxane B2 and thromboxane B2 were 10.3 ng/h (range, 4.5-24 ng/h) and 2.8 ng/h (range, 0.5-7.3 ng/h), respectively. The method offers a noninvasive, specific approach to the study of thromboxane synthesis and platelet function in man. It is much less labor intensive than currently available methods employing electron-impact chromatography-mass spectrometry.     

Urinary prostaglandin E2 excretion in renal allotransplantation in man

The urinary prostaglandin E₂ excretion was measured daily for 28 days in 15 patients (10 men and 5 women) after renal allotransplantation. Patients with acute oliguric renal failure immediately after the transplantation showed high urinary PGE₂ concentrations, but no or minimal increase in the total excretion rates. The median PGE₂ excretion was 211 μg/24 h after establishment of stable renal function, but with great individual variations. Rejection crises were characterized by a two-fold increase in PGE₂ excretion, with a subsequent fall induced by the steroid treatment. The PGE₂ excretion correlated better with urinary sodium excretion than diuresis.The pathophysiological role of the renal prostaglandin ssynthesis remains incompletely defined. The prostaglandin E₂ (PGE₂) appears to act as a modulator of the renal salt and water excretion (1,2) and prostaglandins are important mediators of the immunresponses (3,4). The eraly renal allograft rejection is an event characterized by salt and water retention together with decreasing renal function (5). Antibodies against renal tissue as well as cytotoxic leukocytes (“killer cells”) are active in the process (6,7) and many hormonal systems are involved, among them renin and vasopressin (8). Both hormones are known to stimulate the synthesis of prostaglandin in the kidneys and interact with its effect (9,10,11). The present material was therefore designed to study the urinary excretion of PGE₂ in the kidney allografts before and during rejection crises.     

Effects of antitussive drugs under normal and pathological conditions

The antitussive effect codeine and 1-propoxyphene have been studied in non-anaesthetized healthy cats and cats with respiratory tract inflammation elicited by undiluted croton oil. The drugs were administered intraperitoneally in doses of 10 and 20 mg/kg body weight. The antitussive effect was studied on the 4th day, after inflammation had set in. Cough induced in nonanaesthetized cats by mechanical irritation of the laryngopharyngeal and tracheobronchial areas was evaluated by changes of the lateral tracheal pressure. Experimentally induced inflammatory changes of the respiratory tract due to the antitussive activity of 1-porpoxyphene were significantly reduced, but that of codeine had not changed at all.     

Urinary excretion of electrolytes during prolonged physical activity in normal man

Nine normal young male students were studied during 2 days of relative rest, during 2 days of physical training and again during the succeeding 2 days of relative rest. Twenty-four hour urine collections showed that sodium and potassium excretion were lower during the exercise days, while urinary aldosterone excretion was increased. No differences in the 24-h urinary excretion of creatinine, calcium, and magnesium were found between the resting and exercise days. Hemoglobin concentration, hematocrit and red cell counts were decreased at 14 h and 42 h after exercise; these findings together with the increased serum bilirubin concentration could result from hemolysis. Plasma renin activity, angiotensin II and aldosterone concentration were increased 14 h after exercise but returned to baseline 42 h after exercise. Our data shows that one should take into account previous exercise when interpreting results of certain of these tests.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2α on human umbilical cord vessels

The effect of six naturally occurring prostaglandins on isolated umbilical arteries and veins has been studied. All six prostaglandins had a constricting effect on the umbilical vessels. On the umbilical artery preparations the potencies in decreasing order were A₂>B₂>F_2α>B₁>E₂>A₁. Prostaglandin B₂ was more potent than PGA₂ on the umbilical vein. Polyphloretin phosphate (PPP) antagonised the constricting effect of all six prostaglandins without altering responses to 5-hydroxytryptamine.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Urinary excretion of prostaglandin E2 and prostaglandin F2α in potassium-deficient rats

Potassium-deficiency was induced in rats by dietary deprivation of potassium. The animals became polyuric and urine osmolality decreased more then three-fold compared to controls. Urinary excretion of prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) did not increase during 2 weeks of potassium depletion. Partial inhibition of renal prostaglandin synthesis by meclofenamate did not increase the urine osmolality after water deprivation. These results make unlikely the hypothesis that the polyuria of potassium-deficiency, is the result of enhanced renal synthesis of prostaglandins with subsequent antagonism of the hydro-osmotic effect of vasopressin. Male animals consistently excreted less PGE₂ than female animals.     

Holotranscobalamins in B12 and non B12 requiring prokaryotes and eukaryotes

Transcobalamins, vitamin B₁₂ binding proteins, deliver B₁₂ to cell surface receptors which then permit B₁₂ to cross cell membranes for metabolic use. There is little documentation concerning B₁₂ binding proteins in bacteria and protists. We found that prokaryotes and eukaryotes requiring B₁₂, as well as those protists synthesizing B₁₂, also produce several transcobalamins for functionally transporting B₁₂ similar to humans.     

Synthesis of thromboxane B2 metabolites

This paper reports the synthesis of 11-dehydrothromboxane B₂ methyl ester (II), 15-dehydrothromboxane B₂ methyl ester (III), 15-dehydro-13,14-dihydrothromboxane B₂ (XII) and 2,3-dinorthromboxane B₂ methyl ester (XV). These compounds, as their free acids, have been reported to be thromboxane metabolites.     

Urinary Thromboxane B2 in Cardiac Transplant Patients as a Screening Method of Rejection

Noninvasive methods for regular monitoring of cardiac transplant patients for acute rejection are preferable to the only currently accepted method involving frequent endomyocardial biopsies. Thromboxane A₂ (TXA₂) is synthesized in large amounts by monocytes/macrophages during organ graft rejection. It enhances T-lymphocyte clonal expansion and cytotoxic function as well as upregulating the major histocompatibility class II expression on antigen presenting cells. Experimentally increased urinary excretion of TXA₂ metabolites is associated with cardiac transplant rejection. We therefore compared urinary immunoreactive thromboxane B₂ (i-TXB₂) levels to the rejection score of the endomyocardial biopsies. In addition we graded the degree of activated lymphocytes in peripheral blood. Urinary i-TXB₂ was significantly higher in patients exhibiting medium to severe rejection than in patients without rejection (1236 ± 372 vs. 526 ± 57 pg/mL). The urine i-TXB₂ (704 ± 48 pg/mL) of all patients who participated in this study, whose endomyocardial biopsy indicated rejection, was also significantly higher than in the non-rejecting group. Increased levels of urine i-TXB₂ were associated with increased biopsy scores. Circulating activated lymphocytes was also significantly increased in patients with moderate/severe rejection compared to patients with no rejection (66 ± 11 vs. 39 ± 4 per mm (3)) (p < 0.01). Further, this study shows that urine i-TXB₂ is associated with increased endomyocardial biopsy scores (acute rejection scores) and blood lymphocyte activation. Thus we conclude that urine i-TXB₂ may be of potential value as a diagnostic screening test for helping identify cardiac transplant patients undergoing acute rejection.     

Development of a GC-MS method for quantitation of 2,3-dinor-6-keto-PGF1∝ and determination of the urinary excretion rates in healthy humans under normal conditions and following drugs

Tetradeuterated 2,3-dinor-6-keto-PGF₁∝ was used as internal standard in the development of a method for quantitation of 2,3-dinor-6-keto-PGF₁∝ in human urine based on gas chromatography - mass spectrometry. The urinary excretion rates of 2,3-dinor-6-keto-PGF₁∝ in twenty normal healthy males and females were 9.7 ± 4.6 and 8.8 ± 8.5 (mean ± SD) ng/h respectively. A considerable inter- and intra-individual variation was found under normal conditions. It was also found that the urinary excretion of 2,3-dinor-6-keto-PGF₁∝ was increased about fivefold during and shortly after 30 min of strenuous jogging. Any data about the effect of nonsteroidal antiinflammatory drugs on the excretion rate of 2,3-dinor-6-keto-PGF₁∝ are difficult to interpret when considering the above findings. However, oral administration of 500 mg of aspirin did not seem to reduce the excretion rate of 2,3-dinor-6-keto-PGF₁∝.     

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production

The metabolism of thromboxane B₂ was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5, 6, 7, 8, 9, 11, 12, 14, 15-³H₈]-Thromboxane B₂ was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods.In urine the major metabolites were identified as 11-dehydro-TXB₂ and 2, 3, 4, 5-tetranor-TXB₁, and other prominent products were 11-dehydro-2, 3, 4, 5-tetranor-TXB₁, 2, 3-dinor-TXB₁ and 2, 3-dinor-TXB₂. In the circulation, TXB₂ was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB₂, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB₂ was not converted into 11-dehydro-TXB₂ by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver.The results of the present study suggest that 11-dehydro-TXB₂ maybe a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB₂ and 11-dehydro-TXB₂.     

Rb2[B12(OH)12]·2H2O and Rb2[B12(OH)12]·2H2O2: Hydrate and perhydrolate of rubidium dodecahydroxo-closo-dodecaborate

The orthorhombically crystallizing salts Rb₂[B₁₂(OH)₁₂]·2H₂O (a = 1576.81(9), b = 813.08(5), c = 1245.32(7) pm) and Rb₂[B₁₂(OH)₁₂]·2H₂O₂ (a = 1616.54(9), b = 814.29(5), c = 1260.12(7) pm) could be prepared from Rb₂[B₁₂H₁₂] and hydrogen peroxide. Both crystal structures were determined by X-ray single crystal diffraction and refined in the space group Cmce. They are not isostructural to the other compounds containing icosahedral dodecahydroxo-closo-dodecaborate dianions [B₁₂(OH)₁₂]²⁻ and potassium, rubidium or cesium cations already known to literature, but both title compounds crystallize quasi-isotypically exhibiting Rb⁺ cations in 10-fold oxygen coordination. The hydrogen peroxide adduct (Rb₂[B₁₂(OH)₁₂]·2H₂O₂) is explosive on shock and heat, while the hydrate (Rb₂[B₁₂(OH)₁₂]·2H₂O) is not.     

Regulation of urinary thromboxane B2 in man: Influence of urinary flow rate and tubular transport

Thromboxane B₂ (TxB) is excreted in human urine, but the mechanism of renal excretion and the quantitative relationship of urinary TxB to the active parent compound, thromboxane A₂, of renal or extrarenal origin is not established. To determine the effects of vasoactive hormones, uricosuric agents and urinary flow rate on TxB excretion, urinary TxB was measured by radioimmunoassay and mass spectrometry, and renal metabolism of blood TxB was determined by radiochromatography of urine after i.v. [³H]-TxB infusions. Basal TxB was 6.7 ± 1.1 ng/h during an oral water load, and TxB fell with s.q. antidiuretic hormone (to 3.4 ± 0.4 ng/h, P<0.01) and with fluid restriction (to 2.6 ± 0.5 ng/hr, P=0.001) in parallel with urinary volume. Urinary excretion of unmetabolized [³H]-TxB also fell (by 56%) with fluid restriction, implicating altered metabolism rather than synthesis as the mechanism of the urinary flow effect. Angiotensin II infusions slightly reduced both TxB and urine volume, consistent with a flow effect. In contrast, probenecid did not alter urine volume, but increased urinary uric acid (by 244%), TxB (from 5.6 ± 0.9 to 11.1 ± 2.9 ng/h) and urinary excretion of blood [³H]-TxB (by 243%) by similar amounts (all P<0.05), suggesting that TxB is actively reabsorbed in the proximal tubule, similarly to uric acid. Thus, urinary excretion of TxB of renal and extrarenal origin is regulated by proximal and distal tubule factors.     

Flow cytometry of isolated mitochondria during development and under some pathological conditions

Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .