Sitosterolemia and xanthomatosis together are a disease characterized by premature cardiovascular disease, and by elevated plasma concentrations of total sterols and of plant sterols, especially sitosterol which is hyperabsorbed. In order to determine whether this abnormal metabolism also involved other sterols, a patient with sitosterolemia was fed a diet high in shellfish that contain significant quantities of noncholesterol sterols, some of which are less well absorbed than cholesterol in humans. Compared with control subjects (n = 8), the sitosterolemic subject had an increased absorption of 22-dehydrocholesterol (71.5% vs. 43.8 +/- 11.4%, mean +/- SD), C-26 sterol (80.6% vs. 49.3 +/- 11.4%), brassicasterol (51.8% vs. 4.8 +/- 4.2%), and 24-methylene cholesterol (60.5% vs. 16.0 +/- 8.3%). This enhanced absorption was associated with an increased plasma total shellfish sterol level (13.1 mg/dl vs. 1.9 +/- 0.7 mg/dl in normals). In the sitosterolemic subject, as in normals, the shellfish sterols were not preferentially concentrated in any lipoprotein class, and 50-65% of these sterols were in the esterified form in plasma. Bile acids and neutral sterols were quantitated in bile obtained by duodenal aspiration. The bile acid composition did not differ significantly in the sitosterolemic subject compared with the normal controls. The sitosterolemic subject, though, was unable to concentrate normally the neutral shellfish sterols in bile. The normal controls concentrated the shellfish sterols in bile 6.3 +/- 1.7-fold relative to the plasma shellfish sterol concentration whereas the study subject was only able to concentrate them 2.1-fold. We propose that sitosterolemia and xanthomatosis occur from a generalized abnormality in the usual ability of the gut mucosa and other tissues of the body to discriminate among many different sterols. This has important implications for the understanding of the pathophysiology of this disease and for therapeutic recommendations.
R E Gregg, W E Connor, D S Lin, H B Brewer Jr
We produced an antiserum by immunizing rabbits with purified human megakaryocyte colony stimulating factor (Meg-CSF). With the use of an anti-Meg-CSF IgG fraction (AM-IgG), we detected immunoreactive Meg-CSF both in human aplastic anemia serum (AAS) and normal serum. Based on our immunological and biological analyses, Meg-CSF appeared to be antigenically as well as functionally distinct from human urinary erythropoietin (EPO) and thrombopoietic stimulating factor. The AM-IgG fraction was able to suppress the ability of both aplastic anemia serum and purified Meg-CSF to promote megakaryocyte colony formation. In addition, the supernatant formed after immune precipitation of the AAS with AM-IgG no longer possessed Meg-CSF-like activity. The AM-IgG did not suppress the ability of EPO, phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM), or PHA-LCM + EPO to promote erythroid, granulocyte-macrophage, or mixed colony formation, respectively. The use of this antibody has further defined the dependency of human megakaryocytopoiesis on Meg-CSF.
H H Yang, E Bruno, R Hoffman
Two daughters of a propositus with documented McArdle's disease were shown by enzyme assay, gel electrophoresis, and immunoblotting to be partially deficient in skeletal muscle phosphorylase and, presumably, heterozygous for the trait. Both exhibited only the adult form of the skeletal muscle isozyme. By 31P-nuclear magnetic resonance, both heterozygotes showed a greater production of acid during fully aerobic exercise than when blood flow was occluded in ischemic exercise. This pattern is in contrast to that of control subjects, where there is significantly greater acid production in ischemic versus aerobic exercise, and distinct from that of phosphorylase-negative patients in which no acid is produced in either circumstance. We suggest that these heterozygotes may have adapted to their diminished phosphorylase by enhancing utilization of plasma glucose. If so, this mechanism could account for the observation that most of the symptoms of McArdle's disease are often manifest only in adulthood. These studies also show that although there are very high concentrations of phosphorylase in skeletal muscle (approximately 2% of the soluble protein), such a high level is essential for normal muscle glycogenolysis.
R T Bogusky, R G Taylor, L J Anderson, K L Angelos, J S Lieberman, D A Walsh
Serum oxalate rises in uremia because of decreased renal clearance, and crystals of calcium oxalate occur in the tissues of uremic patients. Crystal formation suggests that either uremic serum is supersaturated with calcium oxalate, or local oxalate production or accumulation causes regional supersaturation. To test the first alternative, we ultrafiltered uremic serum and measured supersaturation with two different methods previously used to study supersaturation in urine. First, the relative saturation ratio (RSR), the ratio of the dissolved calcium oxalate complex to the thermodynamic calcium oxalate solubility product, was estimated for 11 uremic (before and after dialysis) and 4 normal serum samples using a computer program. Mean ultrafiltrate oxalate predialysis was 89 +/- 8 microM/liter (+/- SEM), 31 +/- 4 postdialysis, and 10 +/- 3 in normals. Mean RSR was 1.7 +/- 0.1 (predialysis), 0.7 +/- 0.1 (postdialysis), and 0.2 +/- 0.1 (normal), where values greater than 1 denote supersaturation, less than 1, undersaturation. Second, the concentration product ratio (CPR), the ratio of the measured calcium oxalate concentration product before to that after incubation of the sample with calcium oxalate monohydrate crystal, was measured in seven uremic and seven normal serum ultrafiltrates. Mean oxalate was 91 +/- 11 (uremic) and 8 +/- 3 (normal). Mean CPR was 1.4 +/- 0.2 (uremic) and 0.2 +/- 0.1 (normal). Predialysis, 17 of 18 uremic ultrafiltrates were supersaturated with respect to calcium oxalate. The degree of supersaturation was correlated with ultrafiltrate oxalate (RSR, r = 0.99, r = 29, P less than 0.001; CPR, r = 0.75, n = 11, P less than 0.001). A value of ultrafiltrate oxalate of 50 microM/liter separated undersaturated from supersaturated samples and occurred at a creatinine of approximately 9.0 mg/dl.
E M Worcester, Y Nakagawa, D A Bushinsky, F L Coe
We examined two inhibitors of DNA synthesis, hydroxyurea (HU) and aphidicholin (APC), and two inhibitors of prostaglandin cyclooxygenase, indomethacin and flufenamic acid, for their effects on the resorptive responses of fetal rat long-bone cultures to epidermal growth factor (EGF) and parathyroid hormone (PTH). As we have previously found, HU decreased unstimulated 45Ca release but had little effect on the resorptive response to PTH. HU also did not block resorption stimulated by EGF. Addition of the cyclooxygenase inhibitor, indomethacin, did not alter the resorptive responses of unstimulated or PTH-treated cultures in either the presence or absence of HU or the resorptive response of bones cultured with EGF alone. However, indomethacin completely blocked the resorptive response to EGF of bones that were cultured with HU. The effects of indomethacin on EGF-mediated resorption in HU-treated cultures appeared to be related to an inhibition of prostaglandin synthesis since flufenamic acid had similar effects. However, the effects of HU on the resorptive response to EGF may not have resulted solely from its inhibitory action on DNA synthesis since APC, in the absence of cyclooxygenase inhibitors, completely blocked EGF-mediated resorption without significantly affecting the response to PTH. These results demonstrate that the mechanisms regulating PTH- and EGF-mediated resorption in fetal rat long-bone cultures differ, and imply that a component of EGF-mediated resorption in these cultures is dependent on sustained DNA synthesis.
J A Lorenzo, J Quinton, S Sousa, L G Raisz
Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life.
U Vetter, J Zapf, W Heit, G Helbing, E Heinze, E R Froesch, W M Teller
Vascular endothelium possesses multiple procoagulant properties, including synthesis and expression of Factor V. We studied the effects of homocysteine on the regulation of endothelial cell Factor V activity. Elevated levels of homocysteine are associated with the congenital thrombotic disorder homocystinuria. Treatment of cultured endothelial cells with 0.5-10 mM homocysteine had no effect on cell morphology, but did increase Factor V activity and prothrombin activation by Factor Xa. A radioimmunoassay for endothelial cell Factor V demonstrated that homocysteine treatment did not increase Factor V antigen levels. 125I-prothrombin was activated by treated endothelial cells and Factor Xa in the presence of thrombin inhibitors. Exogenous 125I-Factor V was cleaved by homocysteine-treated but not control endothelial cells. 125I-Factor V cleavage products distinct from those generated by thrombin and Factor Xa were identified. These data provide evidence for regulation of endothelial cell Factor V activity, and indicate that increased Factor V activity associated with homocysteine-treated vascular endothelium results primarily from induction of an activator of Factor V.
G M Rodgers, W H Kane
Pulmonary alveolar macrophages (PAM), obtained by bronchoalveolar lavage from 47 individuals, reduced hexavalent chromium [Cr(VI)] and decreased its mutagenicity. Their specific activity--mostly mediated by cytosolic, enzyme-catalyzed mechanisms--was significantly higher than in corresponding preparations of mixed-cell populations from human peripheral lung parenchyma or bronchial tree, or from rat lung or liver. At equivalent number of PAM, Cr(VI) reduction, total protein, and some oxidoreductase activities were significantly increased in smokers. No appreciable variation could be detected between lung cancer and noncancer patients. In rats, the Cr(VI)-reducing activity of PAM preparations was induced by Aroclor 1254. Thus, alveolar macrophages provide crucial defense mechanisms not only by phagocytizing metals, but also by metabolically reducing Cr(VI). The epithelial-lining fluid (ELF) also displayed some Cr(VI) reduction. Together with already investigated metabolic processes occurring inside lung cells, these mechanisms are expected to determine thresholds in the pulmonary carcinogenicity of chromium.
F L Petrilli, G A Rossi, A Camoirano, M Romano, D Serra, C Bennicelli, A De Flora, S De Flora
Two groups of adult male Munich-Wistar rats and a third group of nondiabetic age-matched and weight-matched normal control rats underwent micropuncture study 1 mo, and morphologic studies 14 mo, after induction of streptozotocin diabetes or sham treatment. All animals were fed standard rat chow. Diabetic rats received daily ultralente insulin to maintain stable moderate hyperglycemia (approximately 350 mg/dl). In addition, one group of diabetic rats was treated with the angiotensin I converting enzyme inhibitor, enalapril, 15 mg/liter of drinking water. Average kidney weight, whole kidney and single-nephron glomerular filtration rate, and glomerular plasma flow rate were elevated to similar values in both groups of diabetic rats, relative to normal control rats. Non-enalapril-treated diabetic rats exhibited significant elevations in mean glomerular capillary hydraulic pressure and transcapillary hydraulic pressure gradient, compared with the other groups studied, and only this group eventually developed marked and progressive albuminuria. Likewise, histological examination of the kidneys at 14 mo disclosed a high incidence of glomerular structural abnormalities only in non-enalapril-treated diabetic rats. These findings indicate that prevention of glomerular capillary hypertension in rats with diabetes mellitus effectively protects against the subsequent development of glomerular structural injury and proteinuria. This protection is afforded despite pronounced hyperglycemia and elevated levels of glucosylated hemoglobin, further supporting our view that hemodynamic rather than metabolic factors predominate in the pathogenesis of diabetic glomerulopathy.
R Zatz, B R Dunn, T W Meyer, S Anderson, H G Rennke, B M Brenner
Circulating immune complexes (CIC) containing IgA and C3 were elevated in 48% of IgA nephropathy patients; IgA1 was the predominant subclass. IgA1-IgG CIC were detected in 44%, IgA2-IgG CIC in 7%, and IgM-IgA1 CIC in 16% of the patients. No IgM-IgA2 CIC were detectable. Sucrose gradient ultracentrifugation indicated that IgG-IgA1 CIC were predominantly of intermediate (13-19S) size whereas IgA1-C3 CIC sedimented from 11S to 19S. At acid pH, isolated CIC revealed the presence of substantial amounts of 7S IgA. One third of the patients had elevated serum IgA rheumatoid factor (RF) of both polymeric and monomeric forms despite normal levels of IgM-RF; 87% of patients with elevated IgA-RF had IgA1-IgG CIC. These results indicate that the IgA1 component of CIC in patients with IgA nephropathy is not necessarily of mucosal origin and suggest that a portion of these CIC consists of IgA RF immunologically complexed with autologous IgG.
C Czerkinsky, W J Koopman, S Jackson, J E Collins, S S Crago, R E Schrohenloher, B A Julian, J H Galla, J Mestecky