Characterization of cholesterol homeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts reveals a Niemann-Pick disease type C-like phenotype with enhanced lysosomal Ca2+ storage.

Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1-/--MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1-/--MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1-/--MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1-/--MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1-/--MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis.

Intradialytic hypertension and its association with endothelial cell dysfunction.

BACKGROUND AND OBJECTIVES: Intradialytic hypertension is associated with adverse outcomes, yet the mechanism is uncertain. Patients with intradialytic hypertension exhibit imbalances in endothelial-derived vasoregulators nitric oxide and endothelin-1, indirectly suggesting endothelial cell dysfunction. We hypothesized that intradialytic hypertension is associated in vivo with endothelial cell dysfunction, a novel predictor of adverse cardiovascular outcomes. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS: We performed a case-control cohort study including 25 hemodialysis (HD) subjects without (controls) and 25 with intradialytic hypertension (an increase in systolic BP pre- to postdialysis ≥10 mmHg ≥4/6 consecutive HD sessions). The primary outcome was peripheral blood endothelial progenitor cells (EPCs) assessed by aldehyde dehydrogenase activity (ALDH(br)) and cell surface marker expression (CD34(+)CD133(+)). We also assessed endothelial function by ultrasonographic measurement of brachial artery flow-mediated vasodilation (FMD) normalized for shear stress. Parametric and nonparametric t tests were used to compare EPCs, FMD, and BP. RESULTS: Baseline characteristics and comorbidities were similar between groups. Compared with controls, 2-week average predialysis systolic BP was lower among subjects with intradialytic hypertension (144.0 versus 155.5 mmHg), but postdialysis systolic BP was significantly higher (159.0 versus 128.1 mmHg). Endothelial cell function was impaired among subjects with intradialytic hypertension as measured by decreased median ALDH(br) cells and decreased CD34(+)CD133(+) cells (ALDH(br), 0.034% versus 0.053%; CD34(+)CD133(+), 0.033% versus 0.059%). FMD was lower among subjects with intradialytic hypertension (1.03% versus 1.67%). CONCLUSIONS: Intradialytic hypertension is associated with endothelial cell dysfunction. We propose that endothelial cell dysfunction may partially explain the higher event rates observed in these patients.

Biochemical markers of muscular damage.

Muscle tissue may be damaged following intense prolonged training as a consequence of both metabolic and mechanical factors. Serum levels of skeletal muscle enzymes or proteins are markers of the functional status of muscle tissue, and vary widely in both pathological and physiological conditions. Creatine kinase, lactate dehydrogenase, aldolase, myoglobin, troponin, aspartate aminotransferase, and carbonic anhydrase CAIII are the most useful serum markers of muscle injury, but apoptosis in muscle tissues subsequent to strenuous exercise may be also triggered by increased oxidative stress. Therefore, total antioxidant status can be used to evaluate the level of stress in muscle by other markers, such as thiobarbituric acid-reactive substances, malondialdehyde, sulfhydril groups, reduced glutathione, oxidized glutathione, superoxide dismutase, catalase and others. As the various markers provide a composite picture of muscle status, we recommend using more than one to provide a better estimation of muscle stress.

Clinical importance of adherence to treatment with eicosapentaenoic acid by patients with hypercholesterolemia.

BACKGROUND: Despite the risk of critical heart disease, poor adherence to treatment is common in patients with lifestyle-related diseases such as hypercholesterolemia. The association between adherence to treatment and clinical outcome was examined in JELIS (Japan EPA Lipid Intervention Study) and strategies for avoiding poor adherence were explored. METHODS AND RESULTS: Patients taking 80% or more of the study medications were considered to exhibit good adherence. The primary endpoint was either sudden cardiac death or myocardial infarction. Adherence was lower in the eicosapentaenoic acid (EPA) + statin group (66.5%) than in the statin alone group (72.5%). In good adherers with previous coronary artery disease, EPA substantially reduced the risk compared with statin alone (hazard ratio 0.55, 95% confidence intervals 0.34-0.88, P<0.014). Furthermore, the clinical benefit of EPA + statin was significantly larger in patients with good adherence than in those with poor adherence (P=0.041). Finally, a 5-year risk prediction model constructed from the data indicated that complete adherence would lead to 51% reduction of risk compared with non-adherence. CONCLUSIONS: Good adherence to medication was associated with a lower cardiovascular risk than with poor adherence, and the assistance of a pharmacist is of great importance in achieving persistent adherence during treatment.

Meta-analysis: accuracy of rapid tests for malaria in travelers returning from endemic areas.

BACKGROUND: Microscopic diagnosis of malaria is unreliable outside specialized centers. Rapid tests have become available in recent years, but their accuracy has not been assessed systematically. PURPOSE: To determine the accuracy of rapid diagnostic tests for ruling out malaria in nonimmune travelers returning from malaria-endemic areas. DATA SOURCES: The authors searched MEDLINE, EMBASE, CAB Health, and CINAHL (1988 to September 2004); hand-searched conference proceedings; checked reference lists; and contacted experts and manufacturers. STUDY SELECTION: Diagnostic accuracy studies in nonimmune individuals with suspected malaria were included if they compared rapid tests with expert microscopic examination or polymerase chain reaction tests. DATA EXTRACTION: Data on study and patient characteristics and results were extracted in duplicate. The main outcome was the likelihood ratio for a negative test result (negative likelihood ratio) for Plasmodium falciparum malaria. Likelihood ratios were combined by using random-effects meta-analysis, stratified by the antigen targeted (histidine-rich protein-2 [HRP-2] or parasite lactate dehydrogenase [LDH]) and by test generation. Nomograms of post-test probabilities were constructed. DATA SYNTHESIS: The authors included 21 studies and 5747 individuals. For P. falciparum, HRP-2-based tests were more accurate than parasite LDH-based tests: Negative likelihood ratios were 0.08 and 0.13, respectively (P = 0.019 for difference). Three-band HRP-2 tests had similar negative likelihood ratios but higher positive likelihood ratios compared with 2-band tests (34.7 vs. 98.5; P = 0.003). For P. vivax, negative likelihood ratios tended to be closer to 1.0 for HRP-2-based tests than for parasite LDH-based tests (0.24 vs. 0.13; P = 0.22), but analyses were based on a few heterogeneous studies. Negative likelihood ratios for the diagnosis of P. malariae or P. ovale were close to 1.0 for both types of tests. In febrile travelers returning from sub-Saharan Africa, the typical probability of P. falciparum malaria is estimated at 1.1% (95% CI, 0.6% to 1.9%) after a negative 3-band HRP-2 test result and 97% (CI, 92% to 99%) after a positive test result. LIMITATIONS: Few studies evaluated 3-band HRP-2 tests. The evidence is also limited for species other than P. falciparum because of the few available studies and their more heterogeneous results. Further studies are needed to determine whether the use of rapid diagnostic tests improves outcomes in returning travelers with suspected malaria. CONCLUSIONS: Rapid malaria tests may be a useful diagnostic adjunct to microscopy in centers without major expertise in tropical medicine. Initial decisions on treatment initiation and choice of antimalarial drugs can be based on travel history and post-test probabilities after rapid testing. Expert microscopy is still required for species identification and confirmation.

Deletion of a phenylalanine in the N-terminal region of human cytochrome P-450(17 alpha) results in partial combined 17 alpha-hydroxylase/17,20-lyase deficiency.

Steroid 17 alpha-hydroxylase and 17,20-lyase activities reside within the same polypeptide chain (cytochrome P-450(17 alpha)), and consequently human 17 alpha-hydroxylase deficiencies are characterized by defects in either or both of these activities. Human mutants having these deficiencies represent an excellent source of material for investigation of P-450(17 alpha) structure-function relationships. The CYP17 gene from an individual having partial combined 17 alpha-hydroxylase/17,20-lyase deficiency has been characterized structurally and the homozygous mutation found to be the deletion of the phenylalanine codon (TTC) at either amino acid position 53 or 54 in exon 1. Reconstruction of this mutation into a human P-450(17 alpha) cDNA followed by expression in COS 1 cells led to production of the same amount of immunodetectable P-450(17 alpha) protein as found with expression of the normal human P-450(17 alpha) cDNA. However, 17 alpha-hydroxylase activity of this mutant protein measured in intact cells was less than 37% of that observed upon expression of the wild-type enzyme, whereas 17,20-lyase activity of the mutant was less than 8% of that observed with the normal enzyme. When estimated in intact cells, the Km for 17 alpha-hydroxylation of progesterone was increased by a factor of 2 in the mutant enzyme, whereas the Vmax was reduced by a factor of 3. In order to estimate the kinetic parameters for the 17,20-lyase reaction, microsomes were isolated from transfected COS 1 cells to enrich for this activity. Surprisingly, the specific activity of the mutant 17 alpha-hydroxylase in microsomes was 3-fold less than that observed in intact cells, indicating that the structure of mutant P-450(17 alpha) was dramatically altered upon disruption of COS 1 cells. Apparently the deletion of a single phenylalanine in the N-terminal region of P-450(17 alpha) alters its folding in such a way that both enzymatic activities are dramatically decreased, leading to the partial combined deficiency observed in this individual.

Induction of labour in sheep after fetal hypophysectomy: an investigation of the possible involvement of a fetal pituitary secretion in the activation of placental enzymes by fetal cortisol.

Activities of 17 alpha-hydroxylase, C-17, 20 lyase and aromatase were measured in placentae of intact and hypophysectomized fetuses after premature induction of parturition by fetal administration of Synacthen. Activities of these enzymes were compared with concentrations of steroids produced by the placenta, which were measured in maternal plasma before and during parturition. Hypophysectomy was assessed by determining fetal plasma concentrations of LH before and after administering LH-RH, and histologically post partum. Concentrations of progesterone, which decreased before parturition, and of 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one and oestradiol-17 beta, which increased, were not affected by fetal hypophysectomy. However, fetal hypophysectomy reduced the normal rise in oestrone sulphate which precedes parturition and abolished the normal rise in androstenedione. Although placental activities of C-17, 20 lyase and aromatase (but not 17 alpha-hydroxylase) tended to be reduced by fetal hypophysectomy, these effects were not statistically significant. The data support an earlier suggestion that an unidentified product of the fetal pituitary may be involved in the activation of C-17, 20 lyase by fetal cortisol before parturition. However, the results of the present study show that the action of such a factor is more likely to be permissive than obligatory.

[Diagnostic procedures in hereditary fructose intolerance (author's transl)]. / Möglichkeiten zur Diagnostik einer hereditären Fructose-Intoleranz.

In order to diagnose hereditary fructose intolerance up to now, there were only the dangerous fructose-load and the biochemical evidence of this metabolic defect from biopsies of liver, intestine or kidney. Since there are no screening tests nor tests for heterocygote carriers or prenatal diagnostic procedures, we tested a simple method to determine serum activities of the two enzymes concerned in this defect (fructose-1-phosphate aldolase, fructose-1,6-diphosphate aldolase). Even in completely healthy children we could measure both activities in a good range. Children with known liver lesion caused other than HFI had significantly increased activities of both enzymes. In 4 cases with HFI we could not measure any activity of fructose-1-phosphate aldolase and a decreased activity of fructose-1,6-di-phosphate aldolase in serum, despite an apparently damaged liver. We propose to define those two serum activities in any case of an obscure liver lesion, frequent vomiting and postprandial hypoglycemia in early childhood, in order to exclude HFI or to demonstrate its possible presence.

Intraspecific red cell enzyme variation in the pigtailed macaque (Macaca nemestrina).

The erythrocytes of 350 pigtailed macaques (Macaca nemestrina) were examined for electrophoretic variation of hemoglobin and 26 enzymes. Seven enzymes showed variation in more than 1% of individuals: phosphoglucose isomerase, phosphoglucomutase-1, soluble NADP-dependent isocitric dehydrogenase, peptidase A, peptidase C, 2,3-diphosphoglycerate mutase, and acid phosphatase. Variation with lesser frequency was found in soluble glutamic-oxalacetic transaminase, phosphoglycerate kinase, lactic dehydrogenase, and hemoglobin. Only eight samples were tested for esterase D, and one of these had a variant phenotype. Enzymes with no clear variation were adenylate kinase, adenosine deaminase, phosphofructokinase, hexokinase, pyruvate kinase, glyceraldehyde 3-phosphate dehydrogenase, aldolase, phosphoglycerate mutase, phosphopyruvate hydratase (enolase), phosphoglucomutase-3, and superoxide dismutase. There was father-to-son transmission of PGI, PGM-1, peptidase C, 6PGD, 2,3-DPGAM, NADP-ICD, and acid phosphatase variants, suggesting that these loci are autosomal as in man.

[Liver biopsy and blood serum enzyme content in patients with rheumatic heart disease with circulatory insufficiency]. / Gepatobiopsiia i soderzhanie fermentov v syvorotke krovi u bol'nykh revmaticheskimi porokami serdtsa s nedostatochnost'iu krovoobrashcheniia

Puncture biopsy of the liver and a comparison of the blood serum enzymes-lactate-dehydrogenase and fructose-1-phosphate-aldolase-with the nature of histomorphological alterations were effected in 18 patients with rheumatic heart diseases, circulatory insufficiency of the II-III degree and rheumatism in the I-II stage of activity. Morphological changes in the liver were not specific, being characterized largely by congestive manifestations and deranged intrahepatic circulation. With a declining level of glycogen and nucleoproteins in the hepatocytes the blood serum of such patients showed an increased content of fructose-1-phosphate-aldolase and lactate-dehydrogenase. Histomorphological analysis of the puncture material facilitate the evaluation of the nature of intravital changes in the liver of patients with different degrees of circulatory insufficiency.