Yoshifumi Horikawa

Clinical significance of measurement of plasma annexin V concentration of patients in the emergency room

Annexin V, a calcium-binding protein, is widely present in various organs and tissues. In the present study, plasma annexin V concentration was measured in 158 patients who were brought to the emergency room, including 25 patients suffering from acute myocardial infarction (AMI), 14 with cerebrovascular disease, 11 with trauma of the extremities, 11 with severe trauma associated with visceral damage, and 35 with witnessed cardiac arrest. Annexin V concentration in normal healthy individuals (n=110) was 1.9+/-0.7 ng/ml. Annexin V concentration in AMI and cardiac arrest patients was 11.0+/-4.9 and 15.3+/-7.9 ng/ml, respectively, being significantly higher than that in patients with cerebrovascular disease (5.4+/-2.7 ng/ml). The value in severe trauma patients was 15.9+/-9.4 ng/ml, being significantly higher than that in patients with trauma of the extremities (5.6+/-1.2 ng/ml). Annexin V concentrations in the cardiac arrest and AMI patients who survived more than 24 h after admission were lower than those in patients who died within 24 h after the onset of symptoms. Annexin V content in the lungs and myocardium in normal rats was extremely high in comparison to that in brain and skeletal muscle. These results suggest that the high levels of plasma annexin V in patients with AMI, cardiac arrest and severe trauma reflect the severity of damage of the myocardium and/or other visceral organs, and measurement of plasma annexin V concentration may help to assess the prognosis of patients brought to the emergency room.

Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease.

To confirm the significance of excretion of annexin V into the urine and the change of urinary annexin V concentration in kidney disease, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two monoclonal antibodies. Urinary annexin V concentration was measured in healthy individuals and patients with kidney and other diseases. Urinary annexin V did not change over a range of pH between 5.0 and 8.0, and was stable during the course of the study for 24 h at room temperature and for 8 days at 4 degrees C. The mean urinary annexin V concentration in 105 normal healthy individuals was 1.5+/-1.5 ng/ml, while that in patients with nephrotic syndrome and systemic lupus erythematosis (SLE) nephritis was 9.3+/-9.1 and 6.6+/-6.7 ng/ml, respectively, and that in IgA nephropathy and chronic renal failure was 2.6+/-2.1 and 1.3+/-0.7 ng/ml, respectively. Annexin level correlated with urinary protein concentration (r=0. 717), but not the serum creatinine concentration, blood urea nitrogen (BUN) and 24-h creatinine clearance. Mean urinary annexin V concentration in patients with ischemic heart disease, hypertension, and diabetes mellitus was 1.4+/-1.0, 1.4+/-1.1, and 1.7+/-1.3 ng/ml, respectively. In one case of relapsing nephrotic syndrome, the urinary annexin V concentration was markedly increased in the early phase after admission and then decreased. This patient later required hemodialysis. These results suggest that a high urinary annexin V concentration may be an indicator of acute renal injury related to the urinary protein level.

Localization of annexin V in rat normal kidney and experimental glomerulonephritis.

The localization of annexin V, a calcium binding protein, was immunochemically and immunohistologically studied in experimental rat glomerulonephritis using annexin V polyclonal antibody. Plasma and urinary annexin V levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Urinary annexin V level, which was correlated with urinary l-lactate dehydrogenase activity, N-acetyl-β-d-glucosaminidase activity and protein level, increased time-dependently after the injection of nephritogenic antigen (bovine glomerular basement membrane), progressively increasing to attain a peak level at 4 weeks of 51.5±11.3 ng/h. However, plasma annexin V level showed no increase during the study period. Normal kidneys showed strong staining for annexin V in distal tubules, being particularly strong in tubules of the inner stripe of the outer medulla, but could not be detected in proximal tubules. Annexin V was seen in visceral epithelial cells, Bowman’s capsule of the glomerulus, the vascular endothelium of arterioles and interlobular arteries, and vascular smooth muscle. In nephritis, the lumen of distal tubules and the luminal cell membrane were deeply stained, with leakage of annexin V being observed from tubular cells. In the present study, renal annexin V was markedly excreted into urine, and its urinary level reflected the severity of damage of renal tissue and the progression of nephritis. These changes of annexin V in the distal tubule and visceral epithelial cells may be of significance in cell injury of the kidney.

The role of cardiac mitochondria in the regulation of intracellular calcium during ischemia and reperfusion: X-ray microanalysis using freeze-dried sections.

SummaryThe calcium concentration in papillary muscles was measured by X-ray microanalysis in order to clarify the role played by mitochondria in intracellular calcium regulation during ischemia and reperfusion. Rat hearts perfused by the Langendorff method were rapidly frozen prior to and during ischemia, as well as following reperfusion. Sections prepared by cryoultramicrotomy were freeze-dried, carbon-coated, and analyzed in an electron microscope. A new freeze-drying procedure was developed, in which the ultrastructure was well-preserved, with sarcomeres, triads, and mitochondria easily recognized. Calcium accumulation into the mitochondria occurred during 30-min ischemia (29.7 ± 17.0 mmol/kg dry weight) and increased further after 15-min reperfusion (157.1 ± 104.5), the calcium concentration decreased after 60-min reperfusion (58.1 ± 29.0). However, the calcium concentration in the cytosol did not change significantly. It is thought that mitochondrial calcium accumulation is reversible, to a certain degree, and that the mitochondria play a part in intracellular calcium regulation in pathological states.

A new technique to prevent rehydration of freeze-dried sections for X-ray microanalysis: Papillary muscles and myocyte specimens

There is considerable interest of the changes in intracellular electrolytes in various pathophysiological states in cardiology. X-ray microanalysis is a useful method to measure the concentration of intracellular elements, and freeze-drying is a required technique for X-ray microanalysis to preserve elements. In this study, we utilized a new technique by which ultrathin sections were evaporated by carbon in an external freeze-dryer after freeze-drying and we were able to obtain clear electron micrographs of papillary muscles and myocytes. Calcium concentrations of mitochondria and cytosol were measured during 120 min ischemia. We showed that mitochondria played a role in intracellular calcium regulation during 30 min ischemia.