Journal of Laboratory and Clinical Medicine最新文献

Lycopene is a natural carotenoid antioxidant that is present in tomatoes and tomato products. The pharmacologic function of lycopene in platelets is not yet understood. Therefore, in this study we sought to systematically examine the effects of lycopene in the prevention of platelet aggregation and thrombus formation. We found that lycopene concentration-dependently (2–12 μmol/L) inhibited platelet aggregation in human platelets stimulated by agonists. Lycopene (6 and 12 μmol/L) inhibited phosphoinositide breakdown in platelets labeled with tritiated inositol, intracellular Ca⁺² mobilization in Fura-2 AM–loaded platelets, and thromboxane B₂ formation stimulated by collagen. In addition, lycopene (6 and 12 μmol/L) significantly increased the formations of cyclic GMP and nitrate but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of 47,000 Da (P47), a marker of protein kinase C activation, was triggered by PDBu (60 nmol/L). This phosphorylation was markedly inhibited by lycopene (12 μmol/L) in phosphorus-32–labeled platelets. In an in vivo study, thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Lycopene (5, 10, and 20 mg/kg) significantly prolonged the latency period for the induction of platelet-plug formation in mesenteric venules. These results indicate that the antiplatelet activity of lycopene may involve the following pathways: (1) Lycopene may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane B₂ formation, thereby leading to inhibition of intracellular Ca⁺² mobilization. (2) Lycopene also activated the formations of cyclic GMP/nitrate in human platelets, resulting in the inhibition of platelet aggregation. The results may imply that tomato-based foods are especially beneficial in the prevention of platelet aggregation and thrombosis.

Results from animal experiments have suggested that reactive oxygen species (ROS) play an important role in tissue damage associated with diabetes. To determine whether ROS are involved in patients with diabetic nephropathy, we measured the plasma and urinary levels of malondialdehyde (MDA), an important marker of lipid peroxidation, and assessed the immunoreactivity of MDA and superoxide dismutase (SOD) in glomeruli of patients and experimental rats with diabetic nephropathy. Both plasma and urinary MDA levels were significantly higher in patients with diabetic glomerulosclerosis (DGS) than those of diabetic patients without proteinuria, proteinuric patients without diabetes, and normal controls. In DGS patients, the plasma MDA was significantly correlated with urinary MDA (p<0.05). The urinary MDA, but not plasma MDA, was significantly correlated with the degree of glomerulosclerosis and the index of mesangial expansion (both p<0.01) in DGS patients. The immunostaining score of glomerular MDA and SOD were also significantly higher in DGS patients than in control kidneys. In rats with diabetes for more than one month, the glomerular immunostaining for both MDA and SOD were also significantly higher than in controls rats, and both were increased with the progression of diabetes. Our results suggest that oxidative stress is involved in the pathogenesis and the progression of DGS.

D-dimer and factor VIII levels raise in advanced cirrhosis. We investigated the behavior and the diagnostic usefulness of D-dimer and factor VIII in cirrhotic patients with asymptomatic portal venous thrombosis. Factor VIII coagulant and D-dimer values were measured in 136 consecutive outpatients with stable cirrhosis divided according to Child-Pugh (CP) classification, who underwent color/power ultrasonography to detect portal thrombosis. Portal thrombosis was found in 33 patients (24.2%). In patients without thrombosis, factor VIII was significantly higher in CP class C compared with class A and B. Conversely, class C patients with portal thrombosis had lower factor VIII levels than those without thrombosis. In both groups, D-dimer was significantly increased in class C compared with class A and B. In class C, thrombotic patients showed higher D-dimer values than did patients without thrombosis. In class C, a D-dimer value ≥ 0.55 μg/mL provided a sensitivity and a negative predictive value for portal thrombosis of 100%, and a factor VIII coagulant level ≤ 80% showed a specificity and a negative predictive value of 76% and 84%, respectively. In class B, a D-dimer value ≥ 0.225 μg/mL had a sensitivity of 89% and a negative predictive value of 82%. In conclusion, our study shows that factor VIII values increase in severe cirrhosis but significantly decrease in the presence of concomitant portal thrombosis, likely because of consumption during thrombosis; D-dimer is enhanced by both liver failure and portal thrombosis; in severe cirrhosis, normal D-dimer and factor VIII values may safely exclude the presence of asymptomatic portal thrombosis.

In surgical pathology, correct immunohistochemical identification of AL amyloidosis poses a particular problem. Immunostaining for λ- or κ-light chains is commonly encountered even in non-immunoglobulin-derived amyloidoses, which leads to a false-positive classification as AL amyloidosis. In this respect, microextraction of amyloid proteins from surgical pathology specimens and their subsequent biochemical characterization may prove useful in reaching the correct diagnosis. In this study, we investigated systematically the influence of fixation on the extraction of amyloid proteins from amyloid-containing tissue samples. Tissue samples were obtained from a patient with generalized AA amyloidosis and from a second patient with generalized AL amyloidosis. The samples were stored either unfixed or fixed in phosphate buffered 4% p-formaldehyde, methacarn, or Bouin for 3 days, 1 week, or 1 month. Thereafter, proteins were extracted according to the procedure of Layfield et al, separated by SDS-PAGE and subjected to Western blotting, using antibodies directed against AA amyloid and immunoglobulin-derived λ-light chain. Following this procedure, a variety of differently sized AA amyloid or λ-light chain immunoreactive protein bands were found in both patients, which is typical for amyloid proteins. Fixation time did not per se prohibit the extraction of these amyloid proteins from tissue samples, which remained detectable irrespective of fixation time. Although all three fixatives impaired the resolution of some, but not all, individual amyloid proteins, this procedure may help to confirm or reject a diagnosis of AL amyloidosis, because detection of several λ- or κ-light chain immunoreactive protein bands in the low-molecular-weight range (<20 kDa) is a common characteristic of their amyloid nature.

Gallbladder Na⁺ absorption and biliary Ca²⁺ are both increased during gallstone formation and may promote cholesterol nucleation. Na⁺/H⁺exchange (NHE) is a major pathway for gallbladder Na⁺ transport. Ca²⁺-dependent second messengers, including protein kinase C (PKC), inhibit basal gallbladder Na⁺ transport. Multiple PKC isoforms with species- and tissue-specific expression have been reported. In this study we sought to characterize Ca²⁺-dependent PKC isoforms in gallbladder and to examine their roles in Na⁺ transport during gallstone formation. Gallbladders were harvested from prairie dogs fed either nonlithogenic chow or 1.2% cholesterol-enriched diet for varying periods to induce various stages of gallstone formation. PKC was activated with the use of phorboldibutyrate, and we assessed gallbladder NHE regulation by measuring unidirectional Na⁺ flux and dimethylamiloride-inhibitable ²²Na⁺ uptake. We measured gallbladder PKC activity with the use of histone III-S phosphorylation and used Gö 6976 to determine PKC-α contributions. Gallbladder PKC isoform messenger RNA and protein expression were examined with the use of Northern- and Western-blot analysis, respectively. Prairie dog and human gallbladder expresses PKC-α, βII, and δ isoforms. The PKC activation significantly decreased gallbladder J^Na_ms and reduced baseline ²²Na⁺ uptake by inhibiting NHE. PKC-α mediated roughly 42% of total PKC activity under basal conditions. PKC-α regulates basal gallbladder Na⁺ transport by way of stimulation of NHE isoform NHE-2 and inhibition of isoform NHE-3. PKC-α blockade reversed PKC-induced inhibition of J^Na_ms and ²²Na⁺ uptake by about 45% in controls but was progressively less effective during gallstone formation. PKC-α contribution to total PKC activity is progressively reduced, whereas expression of PKC-α mRNA, and protein increases significantly during gallstone formation. We conclude that PKC-α regulation of gallbladder NHE becomes progressively more dysfunctional and may in part account for the increased Na⁺ absorption observed during gallstone formation.