Harriet S. Gilbert

Characterization of hypocholesterolemia in myeloproliferative disease: Relation to disease manifestations and activity

Characterization of the hypocholesterolemia observed in polycythemia vera and agnogenic myeloid metaplasia revealed significant reductions in plasma total cholesterol, low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol in an age- and sex-matched comparison with the Framingham population. Men with myeloproliferative disease also had significantly lower total and LDL cholesterol levels than did those with relative or secondary polycythemia. LDL and HDL cholesterol were significantly correlated, suggesting a generalized disturbance of cholesterol metabolism, unexplained by nutritional status. Evaluation of the relationship among hematic cell proliferation, degree of myeloid metaplasia and hypocholesterolemia by multiple regression analysis revealed that spleen size was the variable of most significance in explaining the variation in plasma total, LDL and HDL cholesterol levels. Uncontrolled disease activity was accompanied by a decline in LDL cholesterol levels. Splenectomy or control of proliferation with chemotherapy or splenic irradiation reversed this abnormality. Levels of plasma total and lipoprotein cholesterol provide information that may be of value in diagnosis and assessment of myeloproliferative disease activity.

A method to correct for errors caused by generation of interfering compounds during erythrocyte lipid peroxidation

A commonly used method for quantification of lipid peroxidation depends upon measurement of a malonaldehyde-thiobarbituric acid derivative with absorbance at 532 nm. Investigation of this assay demonstrated that erythrocyte peroxidation produces compounds that react with thiobarbituric acid to interfere with the malonaldehyde assay. Interference results from carryover absorbance at 532 nm, equivalent to 20% of the intensity of the maximum absorption peak at 453 nm. These compounds are not products of lipid peroxidation but are derived from erythrocyte hemolysate and reduced glutathione. A specific HPLC assay for malonaldehyde corroborated the improved accuracy of measuring absorbance at 453 nm and correcting for the absorbance of the interfering compounds at 532 nm when assaying erythrocyte malonaldehyde production.

The pyrogallol assay for superoxide dismutase: Absence of a glutathione artifact

Reduced glutathione was found to affect the assay for superoxide dismutase when autooxidation of epinephrine, but not pyrogallol, was used as the indicator. Glutathione concentrations in the micromolar range, which correspond to levels in erythrocyte extracts, were capable of perturbing the epinephrine assay method and causing overestimation of enzyme content. The pyrogallol method was not significantly affected by large excesses of glutathione and appears to be a superior method for tissue extracts likely to be rich in glutathione.

Reduced plasma concentrations of total, low density lipoprotein and high density lipoprotein cholesterol in patients with Gaucher type I disease.

Plasma lipid and serum apoprotein concentrations were determined in twenty‐nine individuals with Gaucher type I disease. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol were all significantly reduced in the patients with Gaucher disease compared to a group of matched control subjects. Total, LDL and HDL cholesterol were lower in males than in females with Gaucher disease. These sex differences appeared to be inversely correlated with the severity of disease manifestations which were greater in the males. Serum levels of apoprotein‐B and apoprotein‐AI, the major structural apoproteins of LDL and HDL, respectively, were decreased in the subjects with Gaucher disease. Thus, the reductions in LDL and HDL cholesterol were associated with reduced numbers of lipoprotein particles in plasma. In contrast, apoprotein‐E, a protein which is secreted by several tissues, including activated macrophages and which may mediate hepatic catabolism of lipoproteins, was elevated in the patients. Since macrophages may also catabolize lipoproteins, Gaucher disease may serve as a model for the effect of activated macrophages upon human lipoprotein metabolism.

Comparison of erythroid progenitor cell growth in vitro in polycythemia vera and chronic myelogenous leukemia: Only polycythemia vera has endogenous colonies☆

The ability of erythroid cultures to distinguish among myeloproliferative disorders was examined. We studied 14 patients with polycythemia vera (PV), 11 with chronic myelogenous leukemia (CML), four with non-PV erythrocytosis, two with agnogenic myeloid metaplasia, as well as three normal fetuses and greater than 25 normal adults. Endogenous, i.e. grew without added erythropoietin, bone marrow CFU-E-derived colonies were observed in all but one PV patient. However, endogenous blood BFU-E-derived bursts were observed in only eight of 14 PV patients. Endogenous erythroid colonies were not seen in cultures from any normal adults or fetuses, or patients with CML, erythrocytosis, or myeloid metaplasia. In PV, relative HbF synthesis was always greater in cultures without erythropoietin, while in cultures from all other patients relative HbF synthesis was similar to that observed in cultures from normal individuals. We conclude that PV and CML are distinguishable in culture since CML patients do not have endogenous growth. Most important, endogenous bone marrow CFU-E-derived colonies are the only consistently unique observation in patients with PV, and endogenous CFU-E- and BFU-E-derived colonies and bursts are not uniformly observed in PV blood cultures. In-vitro studies of erythropoiesis to confirm the diagnosis of PV, therefore, require marrow when endogenous colonies and bursts are absent from blood cultures.

Increased Low-Density-Lipoprotein Catabolism in Myeloproliferative Disorders

Hypocholesterolemia reported in patients with myeloproliferative disorders prompted our investigation of lipoprotein metabolism in these patients. The production and fractional catabolic rates of very-low-density lipoprotein (VLDL) apoprotein-B were measured using 131I-VLDL; those of VLDL triglyceride, using 3H-glycerol; and those of low-density lipoprotein (LDL) apoprotein-B, using 125I-LDL. Plasma total and LDL cholesterol levels (mean +/- SD) were significantly reduced in seven patients with myeloproliferative diseases, compared to five normal subjects (93.1 +/- 20.3 mg/dL versus 166.8 +/- 24.6 mg/dL and 50.3 +/- 14.8 mg/dL versus 107 +/- 20.8 mg/dL, respectively). The production rates of VLDL apoprotein-B and VLDL triglyceride were normal in the patients. The fractional catabolic rate of LDL apoprotein-B was increased in the patients with myeloproliferative diseases (0.89 +/- 0.32/d versus 0.52 +/- 0.10/d; p less than 0.05); this increased rate was associated with reduced plasma LDL apoprotein-B levels (41.7 +/- 7.1 mg/dL versus 57.0 +/- 11.3 mg/dL; p less than 0.05) despite normal or elevated LDL apoprotein-B production (16.7 +/- 5.3 mg/kg body weight . d versus 12.9 +/- 1.2 mg/kg body weight . d). The site (or sites) of increased LDL catabolism in these hypocholesterolemic patients with myeloproliferative disorders is under investigation.

A Tumor-Related Vitamin B12 Binding Protein in Adolescent Hepatoma

Abstract Extraordinary elevations of serum vitamin B12 content and serum levels of vitamin B12 binding protein were found in three young patients with hepatoma. All had normal white-cell counts and no evidence of megaloblastosis. Despite a superficial resemblance to the alterations of vitamin B12 and vitamin B12 binders observed in chronic myeloid leukemia, the vitamin B12 binding protein in hepatoma serum exhibited distinct and unique properties that suggest a novel, tumor-related protein. (N Engl J Med 289:1053–1056, 1973)

Isolation and characterization of a novel vitamin B12-binding protein associated with hepatocellular carcinoma.

High levels of a novel vitamin B12-binding protein (hepatoma B12 BP) have been observed recently in plasma obtained from three adolescent patients with hepatocellular carcinoma. This protein has now been isolated in homogeneous form from the plasma and pleural fluid of two of these patients by the use of affinity chromatography with vitamin B12-Sepharose. The hepatoma B12 BP belongs to the R-type group of B12-binding proteins and is essentially indistinguishable from the recently isolated human milk and saliva R-type proteins in terms of: (a) immunologic properties based on immunodiffusion and immunoprecipitation assays; (b) amino acid composition; (c) molecular weight based on amino acid and carbohydrate content; and (d) absorption spectra. Both hepatoma B12 BPs contain more sialic acid and less fucose than the milk and saliva B12 BPs. All four proteins contain similar amounts of galactose, mannose, galactosamine, and glucosamine. Differences in sialic acid content appear to account for the differences in electrophoretic mobility that were observed among the four proteins. Differences in total carbohydrate content appear to account for the differences in apparent molecular weight that were observed with both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor tissue from one of the patients contained 10 times as much R-type protein as did normal liver tissue from the same patient. This suggests, although it does not prove, that synthesis by the tumor is the cause of the high levels of R-type protein found in the plasma of certain patients with hepatocellular carcinoma. Plasma survival studies performed with rabbits indicate that the hepatoma B12 BP has a prolonged plasma survival and suggests that his parameter is also of importance.

Increased circulating levels of transcobalamin ii in gaucher's disease.

The presence of several serum protein abnormalities in Gauchers disease prompted a study of vitamin B12 binding proteins, in which 14 of 15 consecutive patients displayed increased circulating transcobalamin II unassociated with elevations of serum vitamin B12 or other vitamin B12 binders. Transcobalamin II levels were most significantly increased in nine patients with disease severe enough to require splenectomy (P less than 0.01), but were not correlated with liver size or levels of any other laboratory feature of Gauchers disease studied. Splenectomy, per se, did not alter circulating transcobalamin II. Chracterization of the binder in Gauchers disease revealed identity with normal serum transcobalamin II in acid inhibition of vitamin B12 binding, chromatographic behavior, immunologic specificity and functional integrity in vitamin B12 delivery. This observation suggests a relation between reticuloendothelial-cell activity and transcobalamin II metabolism. Elevated transcobalamin II levels may provide an additional means for diagnosis and assessment of Gauchers disease.

Hypocholesterolemia as a manifestation of disease activity in chronic myelocytic leukemia

A patient with chronic myelocytic leukemia and hypocholesterolemia displayed marked fluctuations in plasma cholesterol in response to several therapeutic maneuvers. During chemotherapy there was a reciprocal relation between low density lipoprotein (LDL) cholesterol levels and the degree of leukocytosis and splenomegaly. LDL cholesterol increased after splenectomy, but continued to cycle inversely with the leukocyte count. Receptor‐mediated degradation of 125I‐labeled LDL (125‐LDL) by mononuclear cells in vitro also showed cyclical changes which were unrelated to the number of immature myeloid cells in the population or the level of plasma cholesterol. 125I‐LDL degradation rate was normal or slightly increased during relapse and after remission was achieved, but was greatly increased when tested during periods of remission induction. This patient illustrates that significant changes in plasma total and LDL cholesterol occur in chronic myelocytic leukemia in association with alterations in proliferative state. Tumor load, the presence of an enlarged spleen, and changes in lipid metabolism of circulating cells all appear to contribute to the reduction in LDL cholesterol levels.