Increased activity of lysosomal enzymes in the peritoneal fluid of patients with gynecologic cancers and pelvic inflammatory disease.

PURPOSE: To investigate whether the activity of lysosomal enzymes is increased in the peritoneal fluid of patients with gynecologic cancers compared to activity in the peritoneal fluid from normal subjects and those with pelvic inflammatory disease, and fluid from benign ovarian cysts. PATIENTS AND METHODS: beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activity was measured in the peritoneal fluid from patients with gynecologic cancer, pelvic inflammatory disease, and normal subjects, and fluid from benign ovarian cysts. RESULTS: The mean+/-SD of beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activity in the gynecologic cancers was 120+/-50 nmol, 203+/-86 nmol, and 240+/-119 nmol 4-methylumbelliferone/ml/h, respectively; in the normal control subjects it was 22+/-9 nmol, 46+/-10 nmol, and 80+/-23 nmol, respectively (P=0.00003, 0.0001, and 0.0001, respectively). The activity was increased even in cases without malignant cells in the peritoneal fluid. In pelvic inflammatory disease it was 148+/-82 nmol, 278+/-112 nmol, and 291+/-140 nmol, respectively. The activity in the fluid of the ovarian cysts was similar to that of the normal peritoneal fluid. There was a significant positive correlation between enzyme activity and stage of cancer, that was stronger for beta-glucuronidase (r=0.889, P=0.003). CONCLUSION: The increased lysosomal enzyme activity in gynecologic cancers, without overlapping between patients and normal subjects or benign ovarian cyst fluid, indicates that such measurements might be applied for diagnostic purposes.

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes.

Plasma 125I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.

Effects of total hip replacement and bed rest on blood rheology and red cell metabolism.

In order to better understand the pathophysiologic changes in the immediate postoperative period after total hip replacement surgery and to distinguish alterations due to the surgical operation from those due to bed rest, we examined rheologic parameters and red cell metabolism of patients before, 1 day after, and 5 to 6 days after total hip replacement and compared the results to those obtained from normal volunteers placed at bed rest for 5 days. Bed rest in the control group led to increases in hematocrit, mean corpuscular hemoglobin concentration, red cell DPG and ATP levels, and plasma concentrations of total proteins, globulins, and fibrinogen, with attendant increases in whole blood viscosity, plasma viscosity, blood viscosity, relative blood viscosity with hematocrit adjusted to 45%, and viscometric aggregation index, and the viscosity of red cell suspensions in Ringer's solution at 45% hematocrit decreased at low shear rate. The patient group, despite the postoperative lowering of their hematocrit, mean corpuscular hemoglobin concentration, and total plasma proteins and a consequent decrease of whole blood viscosity, revealed disproportionate increases in blood viscosity, relative blood viscosity, and viscometric aggregation index. These rheologic changes, which reflect an enhanced red cell aggregability, may contribute to complications of thrombophlebitis. Enforced inactivity, when superimposed on the effects of trauma, blood loss, transfusion with bank blood, and the low-flow state, would exaggerate these rheologic problems. The results suggest that management of total hip replacement patients should include hemorrheologic considerations (e.g., preoperative intentional hemodilution) and early postoperative activity.

Rheology of sickle cells and its role in microcirculatory dynamics.

The alterations in the viscoelastic properties of the hemoglobin S solution following deoxygenation can be correlated, on the one hand, with the gelation of the HbS molecules and, on the other hand, with changes in the rheological behavior of the deoxygenated SS erythrocytes. The abnormal rheology of SS erythrocytes forms the basis of their altered flow behavior through filter pores in vitro and microcirculation in vivo. The results support the notion that the rheological behavior of sickle cells is an important link between the molecular events in hemoglobin S following deoxygenation and the behavior of the cell in the microcirculation. Although recent advances have shed considerable lights on the rheology of sickle cells in a variety of conditions, further studies are needed in order to elucidate the pathophysiological role of cell rheology in this disease, especially in terms of the influence of individual SS cells in the heterogeneous populations and the correlation between in vitro and in vivo behavior of SS cells.

Fibers, crystals, and other forms of HbS polymers in deoxygenated sickle erythrocytes.

We have examined by electron microscopy the formation of fibers and crystals from sickle hemoglobin within sickle erythrocytes following deoxygenation during capillary storage from 1 to 132 days. Intracellular fibers were found on the first day and throughout the period of study. The fibers exhibited a diameter (mean +/- SD) of 17.4 +/- 0.62 nm and were aligned in the cell with a fiber-to-fiber spacing of 18.6 nm (x-axis) by 22.7 nm (y-axis). Between 65 and 132 days, extracellular hemoglobin crystals developed, with a lattice periodicity of 9.63 +/- 0.6 nm. Fibers and crystals coexist as separate structures. These results suggest that crystal formation upon storage of packed deoxygenated sickle erythrocytes may proceed via a phase of fiber dissolution followed by hemoglobin reassembly into extracellular crystals, rather than by a progressive alignment and direct fusion of existing fibers.

Viscoelastic properties of sickle cells and hemoglobin.

The quantitative relationship between deoxygenation and rheological behavior of SS cell suspensions and concentrated HbS solutions has been studied under steady shear (viscosity eta) and oscillatory shear (complex viscosity with viscous component eta' and elastic component eta"). decrease of O2 saturation below 80-85% causes eta, eta' and eta" to increase progressively in SS cell suspensions and HbS solutions. These rheological parameters do not change in AA cell suspensions and HbA solutions following deoxygenation. The deoxygenation-induced increase in eta" of HbS solutions from the unmeasurable level when oxygenated reflects the gelation of HbS. At high O2 saturations, the rheological behavior of the intracellular fluid has relatively insignificant contribution to that of the SS cell suspension. As O2 saturation decreases, the viscoelastic properties of the SS cell suspension become increasingly dominated by that of the intracellular HbS. at a given degree of deoxygenation, eta, eta' and eta" of HbS solutions decrease with increasing shear rate, indicating that HbS aggregates can be dispersed by shear stress.

Variability of intracellular pH within individual populations of SS and AA erythrocytes.

Individual populations of AA and SS erythrocytes were fractionated according to cell density by centrifugation, and the fractions analysed for intracellular pH (PHi), the mole ratio of 2,3-diphosphoglycerate to haemoglobin (DPG:Hb), and cell concentration of haemoglobin (MCHC). The pHi of SS erythrocytes was consistently lower than that of AA erythrocytes throughout the density range, and the lowest pHi of both cell types (AA and SS) was found in cells with the highest density. As the highest density AA and SS erythrocytes are characterized by the lowest DPG:Hb values, their relatively low pHi cannot be ascribed to intracellular organic phosphate. Instead we propose that a redistribution of hydrogen ions across the membrane of both AA and SS erythrocytes is the ultimate result of progressive alterations in these membranes in vivo.