The identification of urinary bile alcohols by gas chromatography–mass spectrometry in patients with liver disease and in healthy individuals

Abstract. The neutral steroid fractions in the urine of eleven patients suffering from various forms of liver disease with cholestasis and of ten healthy individuals were studied by glass capillary gas chromatography-mass spectrometry. The steroid conjugates in urine were enzymatically solvolysed, the liberated steroids extracted and transformed into the trimethylsilylether for measurements.
The excretion rates of androstane and pregnane metabolites of patients with liver disease were far lower than those of healthy persons. The main compounds in the urine of the former were the bile alcohols 27 - nor -3α, 7α, 12α, 24, 25 - pentahydroxy - 5β - cholestane and 3α, 7α, 12α, 25, 26 - pentahydroxy - 5β - cholestane. Our data suggest a correlation between the excretion rates of these bile alcohols and the serum levels of bilirubin. While the excretion rate of the two bile alcohols in the urine of healthy individuals was approximately 0.24 mg/24 h (0.6 μmol/24 h) a patient with a serum bilirubin of 841 μmol/1 excreted 4 mg/24 h (9 μmol/24 h). The accumulation of bile alcohols described in this study possibly indicates alternative pathways of cholic acid formation in liver disease.     

Effects of cyclooxygenase inhibitors, prostaglandins F2α and I2, on isolated coeliac and basilar arteries of alloxan-diabetic dogs

Abstract. The effects of prostaglandin synthetase inhibitors PGF_2α and PGI₂ on the tone of isolated basilar and coeliac arteries were studied in healthy and alloxan-diabetic dogs. PGF_2α (1 μmol l^-1) produced a significantly higher tone in diabetic basilar arteries (1·15 ± 0·16 mN) than in normal cerebral vessels (0·7 + 0·10 mN). By contrast, the contractile responses of normal and diabetic coeliac arteries to PGF_2α did not differ. The cyclooxygenase inhibitors indomethacin (3 μmol l^-1) and suprofen (0·58 μmol l^-1) potentiated the PGF_2α-evoked contractions in all of the vessels studied. The percent potentiation was greater (50–60%) in the basilar arteries from alloxan-treated dogs than in normal basilar vessels (22–30%). There was not such a difference between diabetic and normal coeliac arteries. Prostacyclin produced a concentration-related relaxation in the presence of indomethacin or indomethacin + PGF_2α. The relaxant potencies of PGI₂ were similar in the vessels from metabolically healthy and diabetic dogs. The IC₅₀ values for PGI₂ were 11·6 ± 1·3 and 11·8 ± 1·8 nmol l^-1 in normal and diabetic basilar arteries, respectively; they were 25·4 ± 3·2 and 26·2 ± 3·9 nmol l^-1 in control and alloxan-treated coeliac vessels. These results indicate that normal and diabetic vessels may have differential reactivity to cyclooxygenase inhibitors, this difference being dependent on the vascular region.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

α1-adrenoceptor subtype(s) mediating noradrenaline-induced contractions of the guinea-pig aorta

Summary— The effect of α₁-adrenoceptor subtype selective antagonists, WB 4101, SZL-49 and chloroethylclonidine on noradrenaline-induced contractions of the guinea-pig aorta has been studied in an attempt to identify the α₁-adrenoceptor subtype(s) involved in the response. Noradrenaline and SDZ NVI 085 induced contractions of the aorta. Noradrenaline-induced contractions were competitively antagonised by WB 4101 (pA₂ = 8.92, slope = 1.05). The contractions were significantly reduced by SZL-49 but not by chloroethylclonidine, indicating an action on α_1A-adrenoceptor subtype. Noradrenaline-induced contractions of the aorta were not inhibited by nifedipine (10⁻⁶ M). The results are interpreted to suggest that α_1A-adrenoceptor subtype mediates noradrenaline-induced contractions of the guinea-pig aorta and that activation of α_1A-adrenoceptor subtype in the guinea-pig aorta is probably linked to intracellular Ca⁺⁺ release.     

Differential response of hypertrophied rat hearts to various α1-adrenoceptor agonists

Summary— With respect to the heart, the prolonged existence of hypertension, both in man and in experimental animals is predominantly characterized by an increase in left ventricular myocardial mass. In this process, the autonomic nervous system plays an important role. Although endogenous catecholamine stimulation of the heart is mainly exerted via the β-adrenoceptors, in several mammalian species, the stimulation of cardiac α-adrenoceptors also mediates positive inotropic actions. We investigated the functional responses of isolated hypertrophied hearts taken from spontaneously hypertensive rats (SHR) and rats with an induced aortic stenosis (ASR) to various α₁-adrenoceptor agonists and compared them with those from age matched Wistar Kyoto (WKY) and "sham" operated controls. Accordingly, we studied the functional response to: methoxamine (α₁), cirazoline (α₁) and phenylephrine (α₁ > β₁). The inotropic response to the α₁-adrenoceptor agonists cirazoline and methoxamine proved to be significantly weaker in hypertrophied hearts from SHR and ASR than in non-hypertrophied hearts from WHY and "sham" operated controls ( p < 0.05). The inotropic response to phenylephrine remained intact in hypertrophied myocardial tissue. However, it was significantly reduced when the hearts were pre-treated with the intracellular Ca²⁺-antagonists ryanodine and TMB-8. These findings show that the mechanism of sarcolemmal Ca²⁺ release, activated by phenylephrine, is still intact in the hypertrophied myocardial cell. In conclusion, these data show that cardiac hypertrophy, be it of genetical or mechanical origin, leads to a reduced response of the isolated heart to α₁-adrenoceptor stimulation.     

Multiple ways to switch platelet integrins on and off

Summary.  In the classical concept of platelet integrin activation, it is considered that unidirectional conformational changes of α_IIbβ₃ and α₂β₁ regulate the adhesiveness of platelets for fibrin(ogen) and collagen, respectively. Here, we summarize recent evidence that these conformational changes: (i) can also occur in the reverse direction; and (ii) are not independent events. Platelet stimulation through the P2Y₁₂ receptors provokes only transient α_IIbβ₃ activation via signaling routes involving phosphoinositide 3-kinases and Rap1b. Furthermore, α_IIbβ₃ can be secondarily inactivated in platelets with prolonged high Ca²⁺ rises, which expose phosphatidylserine and bind coagulation factors. Thus, platelet stimulation with strong agonists (collagen and thrombin) also results in transient integrin activation. Integrin α₂β₁ is found to be activated by a mechanism that is directly linked to α_IIbβ₃ activation. Integrin α₂β₁ can adopt different activation states, depending on the trigger. Conclusively, reversibility and synchrony of platelet integrin activation are newly identified mechanisms to restrict thrombus growth and to allow optimal coagulation factor binding. Back-shifting of activated integrins towards their resting state may be a novel goal of antithrombotic medication.     

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β₁ (TGF β₁) on the angiotensin II (Ang II)-induced increase in cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). [Ca²⁺]_i in VSMC was measured using the fluorescent dye fura-2. When TGF β₁ was applied 30 s prior to Ang II, the Ang II-induced [Ca²⁺]_i increase was significantly enhanced in VSMC from SHR ( P < 0.05 compared to control), whereas after the preincubation with TGF β₁ for 30 min, the Ang II-induced [Ca²⁺]_i increase was significantly reduced in VSMC from both strains. Using the manganese-quenching technique, it was confirmed that short-term exposure to TGF β₁ enhanced the Ang II-induced trans-plasma-membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β₁ on the Ang II-induced [Ca²⁺]_i increase. It is concluded that TGF β₁ modulates the Ang II-induced calcium handling in VSMC.     

Serum β2-microglobulin at remission and relapse in patients with multiple myeloma

Abstract. Serum β₂-microglobulin (S-β₂m) was determined before treatment in fifty patients with multiple myeloma (MM), twenty-two of which had pure Bence Jones (BJ) myeloma. S-β₂m was related to clinical stage before, but not after, a correction of S-β₂m values for co-existent raised S-creatinine values (> 106 μmol 1^-1)- However, S-β₂m as well as corrected β₂m was a parameter of prognostic value. The median expected survival time was 14 months at S-β₂m values > 8·0 mg 1^-1 and 56 months at values<3·5 mg 1^-1. A response to treatment was associated with a decrease of S-β₂m and a stationary course with unchanged values, whereas a relapse or progressive disease was connected with an increase. In β₂m producers', i.e. patients with a clear initial high S-β₂m, serial determinations are of value for monitoring patients with MM. In particular, this is the case in BJ myelomas, as they lack a quantifiable serum M-component. With respect to β₂m no difference was found between BJ and other patients with MM.     

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

Summary.  Platelet adhesion to surface-bound fibrinogen depends on integrin α_IIbβ₃. In the present study, we investigated the role of the regions ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T of the β₃ cytoplasmic tail in the regulation of platelet adhesion under flow conditions, by introducing peptide mimetics in platelets. Introduction of peptide EATSTFTN (E–N) increased surface coverage by 35%, an effect caused by 25% more adhesion. In contrast, peptide TNITYRGT (T–T) decreased surface coverage by 16%, as a result of 25% less adhesion. An S→P substitution in the E–N peptide, thereby mimicking a mutation in Glanzmann's thrombasthenia, abolished the effect of E–N. A suboptimal concentration of cytochalasin D is known to enhance ligand binding to α_IIbβ₃ in platelet suspensions. Under flow, cytochalasin D (1 µmol L⁻¹) induced 50% more platelet adhesion, with a strong reduction in platelet spreading. Both peptides opposed the increase in adhesion by cytochalasin D and partly (E–N) and completely (T–T) restored platelet spreading. Thus, the ⁷⁴⁹EATSTFT⁷⁵⁶N and ⁷⁵⁵TNITYRG⁷⁶²T regions of β₃ contribute to the regulation of αIIbβ3 anchorage to the cytoskeleton and platelet spreading to an adhesive surface.     

Effects of high-altitude chronic hypoxia on platelet α2-receptors in man

During chronic high-altitude (HA) exposure, basal and exercise-induced noradrenaline (NA) increases do not parallel blood pressure (BP) changes observed; unlike β-adrenergic receptors, to our knowledge no data are available on α-receptors. We studied platelet α₂- and leucocyte β-receptors and basal catecholamine levels in 11 trained climbers before and after they had spent a 15-day period at a height of over 4400 m. In six of the climbers we also evaluated catecholamines after maximal bicycle ergometer exercise. After chronic high-altitude exposure, a significant decrease was found in platelet α₂-receptor density and affinity [ B _max from 92.6 ± 6.7 to 54.6 ± 4.2 fmol mg⁻¹ protein ( P  < 0.001) and K _D from 1.271 ± 0.034 to 1.724 ± 0.077 nmol L⁻¹ ( P  < 0.05)], although no changes to β-receptors were observed. No changes were found in basal pre- and post-expedition NA and adrenaline (A), and there was only a slight decrease in post-expedition NA after maximal exercise. Our results suggest that prolonged exposure to hypoxia induces a down-regulation of α₂-receptors, which may be a contributory factor in the regulation of the physiological vascular response to acclimatization.     

Association of the antagonism of von Willebrand factor but not fibrinogen by platelet αIIbβ3 antagonists with prolongation of bleeding time

Summary.  Background:  The α _IIb β ₃ antagonists inhibit platelet aggregation and are used as antithrombotic agents for cardiothrombotic disease. The present study investigates the correlation of inhibition of fibrinogen and von Willebrand factor (VWF) binding by α _IIb β ₃ antagonists with the inhibition of platelet aggregation and prolongation of bleeding time (BT). Methods:  Inhibition of fibrinogen and VWF binding were assessed in a purified α _IIb β ₃-binding assay. As an in vitro cell-based assay, platelet aggregation and VWF-mediated adhesion studies were performed using human platelets. In vivo effects on BT were measured using a template device in dogs at the same time as an ex vivo aggregation study was performed. Results:   In vitro studies demonstrated that the antiaggregatory effects of α _IIb β ₃ antagonists correlate with their inhibition of fibrinogen binding, but not VWF. Interestingly, the effects of α _IIb β ₃ antagonists on BT could be differentiated from the inhibition of platelet aggregation. Furthermore, this differentiation was strongly correlated with the different inhibitory potencies between fibrinogen and VWF binding, as well as that between VWF-mediated adhesion and aggregation. Conclusions:  Our study provides novel evidence showing that the inhibitory effect of α _IIb β ₃ antagonists on VWF, but not fibrinogen binding, correlates with their ability to prolong BT.     

Protective role of glutathione on α1 proteinase inhibitor inactivation by the myeloperoxidase system. Hypothetic study for therapeutic strategy in the management of smokers' emphysema

Summary— In smoking subjects with obvious emphysema, the interaction between neutrophil-derived MPO and H₂O₂ produced by alveolar inflammatory cells (alveolar macrophages (AM) and polymorphonuclear neutrophils (PMN)) has the ability to spontaneously inactivate, in vitro , the α₁ proteinase inhibitor (α₁PI). This inactivation can induce a desequilibrium of the protease-antiprotease balance in the lungs. In this study, we investigated the ability of glutathione to protect α₁PI. In a cellular model of α₁PI inactivation mimicking the effects of alveolar inflammatory cells present in the lower respiratory tract of smoking patients with emphysema, we demonstrated that glutathione can protect α₁PI against the oxidative inactivation by these activated cells. This protection has been computed in a cellular experimentation (AM and MPO-system) with a 50% inhibitory concentration of 62 μM. Moreover, glutathione has an important inhibitory effect directly on H₂O₂ released by PMA-stimulated AM (IC₅₀ = 30 μM) or PMA stimulated PMN (IC₅₀ = 70 μM). The mechanism, which governs glutathione may be a result of a scavenging effect on H₂O₂ as demonstrated in a free cellular experiment. With this in vitro demonstrated effectiveness, glutathione as a therapeutic antioxidant, via the aerosol, has been proposed, in order to prevent tissue damage, inflicted by an excess of activated phagocytic cells, in some lung diseases such as smoking patients with emphysema.     

Relationship between fractional calcium absorption and gastric emptying

Abstract. The relationship between calcium absorption and gastric emptying and the precision of measurement of fractional calcium absorption using a single isotope technique were evaluated in 14 normal postmenopausal women (age range 61–72 years). On two occasions separated by between 5 and 15 days, each subject was given 250 mL water containing 0.2 MBq of ⁴⁵Ca in 20 mg of calcium carrier as the chloride, 20 mg kg^-1 paracetamol and 9 MBq of ^99mTc sulphur colloid. Venous blood samples were taken at -2, 15, 30, 45, 60, 90, 120, 150 and 180 min after consumption of the drink, and gastric emptying (GE) was monitored with a gamma camera. Fractional calcium absorption in the first hour (α₆) was calculated from the blood samples obtained at 15, 30, 45, 60, 90 and 120 min. An absorption rate was also derived from the 60 min sample using only a calibration curve (α₁). There were close correlations between radiocalcium absorption on the two study days ( r = 0.89, P < 0.001 for both α₁ and α₆) and between α₁ and α₆ ( r = 0.93, P < 0.001). Plasma paracetamol concentrations at 15 min were directly related to the early phase of GE ( r = 0.42, P < 0.05). In contrast, calcium absorption was inversely related to GE ( r — 0.45, P < 0.05). We conclude that radiocalcium absorption is not greatly influenced by gastric emptying rate and that the single blood sample procedure has similar precision to the six-blood sample test.     

Effects on renal tubular protein reabsorption of the infusion of free haemoglobin

Abstract. Hydroxy-ethyl-starch cryopreserved blood with a mean free haemoglobin content of 260 μmol/l (SD 57 μmol/l) was infused into five normal volunteers. The renal clearance of six proteins of differing molecular weight was measured together with an analysis of the qualitative changes in protein excretion during the infusion and over the following 48 h. Other aspects of renal tubular function were assessed by the measurement of urinary N -acetyl-β- D -glucosaminidase, phosphate and amino acids. Increased clearance of low molecular weight proteins and N -acetyl-β- D -glucosaminidase occurred during the 12 h following infusion. β₂ microglobulin clearance × 10³ creatinine clearance rose from a mean of 0·4 (SD 0·1) to a mean of 74·5 (SD 32·3) and N -acetyl-β- D -glucosaminidase from 54·2 units/mmol creatinine (SD 18·0) to 1525 units/mmol creatinine (SD 2318). Increased amounts of low molecular weight proteins were detected in the urine by crossed immunoelectrophoresis. Creatinine clearance remained unaltered. It is argued that these changes may be caused by the competitive inhibition of low molecular weight protein reabsorption in the renal tubule by filtered haemoglobin.     

Study of the pharmacokinetics of cefpirome sulphate in the elderly

Objective: To determine the appropriate method of administration of the cephem antibiotic cefpirome sulphate in elderly patients. Method: We studied cefpirome's pharmacokinetics in patients with urinary tract infections. Patients received cefpirome sulphate 0·5 g by intravenous drip infusion over 30 mins. Results: Patients with a creatinine clearance rate (Ccr) of 80 ml/min had an AUC of 96·7 μg·h/ml and a T _1/2 of 2·36 h, whereas those with Ccr of 40–80 ml/min had an AUC of 172·0 μg·h/ml and a T _1/2 of 3·45 h and those with Ccr of < 40 ml/min had an AUC of 152 μg·h/ml and a T _1/2 of 4·86 h. Conclusion: These results indicate that decreased kidney function can cause increases in the AUC and T _1/2 of cefpirome. Thus in elderly patients and perhaps also in other patients with decreased kidney function, cefpirome should be administered at an initial dose of 0·5 g.     

Gamma-interferon inhibits Pneumocystis carinii attachment to lung cells by decreasing expression of lung cell-surface integrins

Pneumocystis carinii (PC) is a leading cause of pneumonia in immunocompromised patients. Previous work has shown that fibronectin (Fn) and Fn-binding integrins mediate PC attachment to lung cells in vitro . Gamma-interferon (γ-IFN) is a major factor in host defence against PC infection. To determine the effect of γ-IFN on PC attachment to lung cells, the alveolar epithelial cell line A549 was incubated with γ-IFN (0–10⁴ U mL⁻¹) and attachment of ⁵¹Cr-labelled PC to the A549 cells was quantified. PC attachment was significantly decreased ( P  < 0.01) by addition of γ-IFN with no evidence of injury to either the PC or A549 cells. Effects of γ-IFN on PC attachment were observed after 24 h and reached a maximum after 48 h of incubation. To investigate the mechanism of this decrease, we examined integrin expression on γ-IFN-treated A549 cells. A549 cell expression of the α₅ and β₁ integrin subunits was decreased, whereas expression of the α_v subunit was unchanged. Northern blot analysis showed a similar decrease in mRNA for the α₅ and β₁ integrins. Therefore, γ-IFN-mediated inhibition of PC infection may, in part, result from inhibition of PC attachment to alveolar epithelial cells caused by γ-IFN-induced decreases in alveolar integrin expression.     

Granulocyte Collagenase, Elastase and Plasma Protease Inhibitors in Purulent Sputum

Abstract. Sputum was collected from patients with purulent chronic bronchitis. Immuno-chemical techniques using rabbit antiserum against human granulocyte collagenase and elastase showed the presence of both enzymes. Also the serum protease inhibitors α₁-antitrypsin and α₂-macroglobulin were demonstrated. Their protease inhibiting capacity was saturated. Granulocyte elastase and collagenase occurred not only in complexes with the inhibitors, but also as free enzymes. All sputa showed free proteolytiC., elastolytic and collagenolytic activity. The concentration of collagenase was equal in the sol phase and in the gel phase of the sputa, but most of the elastase was bound to the gel phase.     

Alzheimer amyloid β-peptides exhibit ionophore-like properties in human erythrocytes

Abstract. There is growing evidence that the amyloid β-peptide (β₁_₄₀) is involved in the aetiology of Alzheimer's disease also implicating an altered calcium homeostasis of affected cells. Beta₁_₄₀ has been proposed to form calcium channels in synthetic bilayer membranes [1]. We wanted to investigate in the present study whether β₁-₄₀ (or fragments thereof) could act as ionophores in a biological membrane like the one in human erythrocytes. Incubation of the cells for 2h and 4h at 37°C together with 6μmolL^-1 of β₁-₄₀ or of fragments β₁_₂₈and β₂₅-₃₅, resulted in a significantly decreased energy charge qualitatively similar to the one obtained by a known calcium ionophore (A 23187, 0.05μmolL^-1). Moreover, β₁_₄₀ and its two fragments induced a significant alteration of ⁴⁵Ca permeability in human red blood cells of the same type as the one achieved by the calcium ionophore. The ionophoric action of β₁_₄₀ and its two fragments may lead to an increase of the intracellular calcium ion concentration, in turn resulting in enhanced Ca²⁺-ATPase activity and a decrease in energy charge. This may be valid also for neuronal plasma membranes and could, therefore, be a possible aetiological mechanism in Alzheimer's disease.