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Biomedical subjects

K Winkler

Publications and source records attributed to K Winkler.

At least 91 records · Page 5Linked to original sources
PubMed1995

Oxygraphic evaluation of mitochondrial function in digitonin-permeabilized mononuclear cells and cultured skin fibroblasts of patients with chronic progressive external ophthalmoplegia.

For quantitative elucidation of maximal mitochondrial oxidation capacities in human mononuclear cells, cultured human skin fibroblasts and human thrombocytes the optimal amount of digitonin for plasma membrane permeabilization was determined to be 5, 10, and 0.1 micrograms/10(6) cells, respectively. Using these concentrations the rate of respiration of permeabilized cells with the mitochondrial substrates succinate (+ rotenone) or glutamate + malate can be stimulated between two- and fourfold by ADP and inhibited by carboxyatractyloside. The maximal respiratory activities of well-characterized preparations of permeabilized mononuclear cells of five patients with chronic progressive external ophthalmoplegia were compared to healthy controls and a 30 to 50% decrease of the ADP-stimulated respiration rates with glutamate + malate and succinate + rotenone was detected. This is an indication for the presence of the mitochondrial defect in respiratory active blood cells. Additionally, for two of these patients the mitochondrial defects were proven to be detectable by the determination of maximal oxygen consumption rates of digitonin-permeabilized cultured skin fibroblasts. Therefore, the determination of maximal oxidation capacities of a well-defined cell population using strictly standardized conditions of digitonin permeabilization is judged as a useful and sensitive method for the elucidation of mitochondrial function in extramuscular tissue.

Adolescent↗
PubMed1995

Chronic consumption coagulopathy due to an occult splenic haemangioma: Kasabach-Merritt syndrome.

UNLABELLED: We report an 11-year-old girl with a 2-year history of bruising associated with thrombocytopenia and dysfibrinogenaemia. On admission she presented with a large subcutaneous haematoma and splenomegaly and was severely anaemic. Laboratory investigations revealed signs of consumption coagulopathy. Radiological examination showed splenic, retroperitoneal and intra-ossal haemangiomas. After splenectomy, platelet count and coagulation parameters returned to normal. CONCLUSION: Contrary to widely held views, occult visceral haemangioma can lead to Kasabach-Merritt syndrome beyond infancy and is not necessarily associated with visible cutaneous haemangioma. It should be included in the differential diagnosis of chronic thrombocytopenia at any age. Early determination of fibrinogen degradation product levels is advised in order to detect an underlying chronic consumption coagulopathy prompted by an extensive search for multifocal haemangioma.

Afibrinogenemia↗
PubMed1995

Quantitative determination of high-, low-, and very-low-density lipoproteins and lipoprotein(a) by agarose gel electrophoresis and enzymatic cholesterol staining.

Quantification of lipoprotein cholesterol was performed by enzymatic staining of cholesterol in a new agarose gel electrophoresis method that allows the separation of LDL, VLDL, HDL, and lipoprotein(a) [Lp(a)]. Lp(a) shows an electrophoretic mobility clearly distinct from VLDL and HDL. The total CVs of lipoprotein cholesterol varied between 2.7% and 3.9% for LDL, 7.8% and 23.2% for VLDL, 5.2% and 9.5% for HDL, and 6.8% and 16.4% for Lp(a). Comparison of LDL-, VLDL-, and HDL-cholesterol concentrations with the results of a combined ultracentrifugation and precipitation technique gave correlation coefficients of 0.961, 0.947, and 0.918, respectively; comparison of Lp(a)-cholesterol values with those of a nephelometric Lp(a) assay gave r = 0.906. The new electrophoretic assay has several advantages: It allows the quantification of Lp(a)-cholesterol; VLDL-cholesterol is not affected by Lp(a)-cholesterol; and the LDL-cholesterol fraction does not contain Lp(a)-cholesterol, as happens with LDL-cholesterol determined by ultracentrifugation and precipitation.

Adult↗
PubMed1995

Direct determination of lipoprotein(a) cholesterol by ultracentrifugation and agarose gel electrophoresis with enzymatic staining for cholesterol.

Lipoprotein(a) [(Lp(a)], a low-density lipoprotein (LDL)-like particle, contains in addition to LDL a specific protein component, apolipoprotein(a) [apo(a)]. Conventionally, Lp(a) has been measured by immunological methods that distinguish between Lp(a) and LDL by dealing with apo(a) as an antigen. We describe a new method to determine Lp(a) on the basis of its cholesterol content. Very-low-density lipoproteins were removed from serum by preparative ultracentrifugation at a density of 1.006 kg/L. The infranate was subjected to agarose gel electrophoresis to separate Lp(a) and LDL. Lp(a) cholesterol was then determined by direct enzymatic staining for cholesterol. On electrophoresis of the > 1.006 kg/L (bottom) fraction, Lp(a) migrates to the pre-beta position, regardless of the genetic apo(a) isoform. The interassay CVs of Lp(a) cholesterol determinations ranged from 6.9% to 11.5%, and the results correlated well with the Lp(a) concentrations measured by immunonephelometry (r = 0.937). There was an inverse relation between the molecular mass of the genetically determined apo(a) isoforms and Lp(a) cholesterol concentrations. Patients with angiographically proven coronary artery disease (CAD) had significantly more Lp(a) cholesterol than healthy controls did. The ratio of Lp(a) cholesterol to immunologically determined Lp(a) tended to be lower in CAD patients, suggesting that Lp(a) particles contained less cholesterol than apo(a). In addition, the new method allows determination of LDL cholesterol without contamination by Lp(a).

Adult↗
PubMed1995

Determination of triglycerides in lipoproteins separated by agarose gel electrophoresis.

We developed a simple method for the quantitation of triglycerides in electrophoretically separated lipoproteins by specific enzymatic staining. After electrophoresis, glycerol is liberated from triglycerides by the action of cholesterol esterase. Glycerol is oxidized by a sequence of enzymatic reactions. Due to the presence of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in the reaction mixture, two moles of the precipitating dye formazane are generated per mole glycerol. The relative amounts of alpha, pre-beta, and beta lipoproteins are determined by densitometric scanning at 570 nm. Absolute triglyceride concentrations of the respective lipoprotein fractions are calculated from total triglycerides. When tested with purified very low density lipoproteins, the electrophoresis assay was linear between 0.08 and 6.5 g/l pre-beta lipoprotein triglycerides. The intra-assay and inter-assay coefficients of variation were between 5.2% and 9.8%, and between 3.2% and 12.9%, respectively. Comparison of the electrophoresis method with a combined ultracentrifugation/precipitation method in 172 sera resulted in the following correlation coefficients: alpha lipoprotein versus high density lipoprotein triglycerides, r = 0.847; pre-beta lipoprotein versus very low density lipoprotein triglycerides, r = 0.989; beta lipoprotein versus low density lipoprotein triglycerides, r = 0.815. This method is easy to perform, and is a precise and accurate technique for the determination of lipoprotein triglycerides. It is the first reliable method that allows the direct quantification of LDL triglycerides without ultracentrifugation.

Analysis of Variance↗
PubMed1995

Deteriorating free radical-trapping capacity and antioxidant status in plasma during bone marrow transplantation.

Organ toxicity in BMT may in part be due to free radical damage. Therefore the 'Total Radical-trapping Antioxidant Parameter of plasma' (TRAP), individual plasma antioxidants, serum iron and linoleic acid, a main substrate of lipid peroxidation, were monitored before and after BMT, and they were compared with values obtained from healthy controls. Seven patients (3 AML, 3 CML, 1 multiple myeloma) receiving 16 mg/kg busulfan, 30-45 mg VP-16 and 120 mg/kg cyclophosphamide were investigated. TRAP values declined during chemotherapy by about 40% (day -9: 1019 +/- 245 mumol/l, mean +/- s.d.; day 0: 660 +/- 164 mumol/l; P < 0.05). The concentration of uric acid, one of the main antioxidants in plasma, decreased markedly (day -9: 339 +/- 108 mumol/l, day 0: 148 +/- 61 mumol/l; P < 0.05) and paralleled TRAP values. Vitamin E and bilirubin did not change from day -9 to 0 whereas vitamin C increased (day -9: 46 +/- 16 mumol/l, day 0: 89 +/- 44 mumol/l; P < 0.05). Serum iron rapidly increased within the pre-transplantation period, reaching values normally seen only in iron overload (day -9: 11.8 +/- 5.2 mumol/l, day 0: 40.6 +/- 6.5 mumol/l; P < 0.05). Linoleic acid levels were normal at the start and decreased substantially (27.0 +/- 1.6 wt% at day -9; 15.7 +/- 4.9 wt% at day 0; P < 0.05), indicating possible lipid peroxidation during high-dose chemotherapy. In conclusion, complex monitoring of the antioxidant status before and after BMT revealed a breakdown of plasma antioxidant defence and of radical-vulnerable lipids, which was associated with high circulating levels of iron.

Adult↗
PubMed1995

Pseudo-pseudohypertriglyceridemia: a case of increased free glycerol without evidence for glycerol kinase deficiency.

High concentrations of glycerol in plasma may result in overestimation of triglycerides in chemical and enzymatic assays. Known causes of increased glycerol concentrations in blood include glycerol-contaminated blood-collector tubes and pseudohypertriglyceridemia, a condition caused by genetic defects in the gene for glycerol kinase. We report a patient with sudden hearing loss who presented with an increased blood glycerol concentration after undergoing an oral glycerol test.

Adult↗
PubMed1994

Origin of human mast cells: development from transplanted hematopoietic stem cells after allogeneic bone marrow transplantation.

Although mast cells are hematopoietic cells, little is known about the origin of their precursors in vivo. In this study, the origin (donor v recipient genotype) of human mast cells (MCs) was analyzed in a patient who underwent allogeneic bone marrow transplantation (BMT). The patient presented with secondary acute myeloid leukemia (French-American-British classification, M2) arising from refractory anemia with excess of blast cells and bone marrow (BM) mastocytosis. Transplantation was performed in chemotherapy-induced complete remission. On days 88, 126, 198, and 494 after BMT, mast cells were enriched to homogeneity from bone marrow mononuclear cells (BM MNCs) by cell sorting for CD117+/CD34- cells. Purified mast cell populations were CD117(c-kit)+ (> 95%), CD34- (< 1%), CD3- (< 1%), CD14- (< 1%), and virtually free of contaminating cells as assessed by Giemsa staining. The genotype of MCs was analyzed after amplification by polymerase chain reaction (PCR) of a variable number tandem repeat (VNTR) region within intron 40 of the von Willebrand factor (vWF) gene. Unexpectedly, on days 88 and 126 after BMT, sorted MCs displayed recipient genotype as shown by vWF.VNTR-PCR. However, on days 198 and 494, PCR analysis showed a switch to donor genotype in isolated mast cells. Peripheral blood (PB) and BM MNC as well as highly enriched (sorted) CD3+ T cells (PB, BM), CD4+ helper T cells (PB), CD8+ T cells (PB), CD19+ B cells (PB), CD14+ monocytes (PB, BM), and CD34+ precursor cells (BM) showed donor genotype throughout the observation period. Together, these results provide evidence that human MCs developed in vivo from transplanted hematopoietic stem cells. Engraftment and in vivo differentiation of MCs from early hematopoietic progenitor cells may be a prolonged process.

Bone Marrow Transplantation↗
PubMed1994

Measurement of fluorescence changes of NAD(P)H and of fluorescent flavoproteins in saponin-skinned human skeletal muscle fibers.

Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of alpha-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the alpha-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed.

Adenosine Diphosphate↗
PubMed1994

[Osteosarcoma--apoptosis and proliferation. Study of bcl-2 expression].

The relationship between the growth of tumors and the expression of the protooncogene Bcl-2 could be shown in epithelial tumors. A bcl-2 expression leads to a prolonged cell survival due to an inhibition of apoptosis. The potential meaning of bcl-2 expression in mesenchymal tumors remains still unknown. The fact, that the heterogenous group of osteosarcoma is not sufficiently characterized at present, suggested to investigate the bcl-2 expression in osteosarcoma. Thus, immunohistochemistry was used to analyze 47 specimens of different osteosarcomas of 36 patients. Sixteen cases (46%) showed a strong expression of bcl-2 and 13 cases (35%) were moderately positive for bcl-2. Seven cases (19%) were negative for bcl-2. The heterogenous, negative up to strong expression of bcl-2 yield clues, that the Bcl-2 controlled regulation of programmed cell death could be an important factor of cellular kinetics. Additionally the cellular proliferation rate was determined with the monoclonal antibody MIB 1, directed against the Ki-67 epitope. The data of bcl-2 expression and cellular proliferation rate lead to a classification correlating with the histological classification. To verify the importance of apoptosis in the genesis of mesenchymal tumors and whether Bcl-2 may play an important role as a predictive factor for the prognosis of osteosarcoma, further investigations will be needed.

Adolescent↗
PubMed1994

Quantification of liver fat using magnetic resonance spectroscopy.

Localized proton MR spectroscopy using stimulated echoes was used to quantify the liver fat concentration in patients with various degrees of fatty liver due to alcohol abuse. Ten patients underwent a liver biopsy followed by chemical triglyceride estimation of the fatty content. A statistically significant correlation was found between the fat concentration measured in the liver biopsies, and the concentration calculated from the spectroscopic experiments (r = 0.9, p < .001). Quantitative assessment of liver fat concentrations using localized spectroscopy is superior to methods based on differences in relaxation times, and can be used to estimate the fat concentration over the full range of fat content in contrast to the spectroscopic imaging methods. Localized spectroscopy may replace liver biopsy in the diagnosis of diffuse fatty infiltrations, and can be used for follow-up, due to its noninvasive nature.

Adult↗
PubMed1994

Short-term effect of recombinant human growth hormone in patients with alcoholic cirrhosis.

As growth hormone possesses anabolic properties that are active on protein metabolism, and thus of potential benefit to patients with chronic liver disease, we determined the metabolic effects of recombinant human growth hormone on insulin-like growth factor-I (IGF-I) its specific binding proteins, and liver function. Twenty consecutive patients with cirrhosis were randomized to recombinant human growth hormone (Norditropin, 4 I.U. twice daily) subcutaneously for 6 weeks (n = 10) or conventional medical treatment (n = 10). The serum concentrations of insulin-like growth factor-I in the recombinant human growth hormone group increased after 3 (p < 0.01) and 6 weeks (p < 0.02), whereas no significant changes were observed in the control group. The change in insulin-like growth factor-I during the treatment period was expressed as area under the curve (AUC). The AUCIGF-I was significantly larger in the recombinant human growth hormone group (median AUCIGF-I: 12.1, range: 0.0-54.7 weeks.nmol/l) than in the control group (median AUCIGF-I: 0.2, range: -10.6-9.9 weeks.nmol/l) (p < 0.007). Insulin-like growth factor binding protein-3 concentrations increased in the recombinant human growth hormone treated patients as well as in controls, whereas no change in insulin-like growth factor binding protein-1 concentrations was found. No significant changes were seen in the area under the curve for biochemical liver function tests. We conclude that administration of recombinant human growth hormone induces an increase in very low levels of insulin-like growth factor-I, even in patients with cirrhosis with advanced disease, but the clinical benefits remain to be demonstrated.

Adult↗
PubMed1994

Methotrexate pharmacokinetics and prognosis in osteosarcoma.

PURPOSE: The influence of methotrexate (MTX) pharmacokinetic parameters on the efficacy of high-dose MTX (HDMTX) in osteosarcoma was analyzed. PATIENTS AND METHODS: MTX serum peak values from 198 patients in 1,703 treatment courses and more detailed pharmacokinetic data from 185 patients in 1,045 treatment courses from the Cooperative Osteosarcoma Study Group (COSS) studies COSS-80, COSS-82, and COSS-86 were investigated. RESULTS: A mean threshold peak level of > or = 1,000 mumol/L for the repeated MTX courses of individual patients was found to correlate significantly to prognosis in study COSS-80 (18% v 64% actuarial 10-year disease-free survival [DFS], P = .0001). Six courses of HDMTX per patient who achieved peak values > or = 1,000 mumol/L were found to be sufficient for a full effect to be seen in DFS in COSS-80. The MTX peak level was found to correlate closely to the area under the curve (AUC). However, AUC was a less powerful determinator of prognosis than the mean threshold MTX peak value. In patients who received cisplatin (DDP) as one of the additional drugs to MTX, the peak values and AUC were significantly increased (1,396 v 1,276 mumol/L, P = .011; 6,684 v 5,820 h.mumol/L, P < or = .002) and only a few patients (6%) did not achieve mean threshold MTX peak values. In addition, following restriction of hydration fluid after the MTX infusion from 4.5 to 3.0 L/m2 per 24 hours, the early MTX half-life (t1/2) and the AUC, but not the MTX peak value, were significantly increased (3.4 v 3.05 hours, and 6,760 v 5,998 h.mumol/L, respectively, P < or = .002). CONCLUSION: MTX pharmacokinetics significantly influence the efficacy of MTX in osteosarcoma. Individual adaptation of the MTX dose to ensure a threshold peak serum level > or = 1,000 mumol/L does not seem necessary at a fixed dose of 12 g MTX/m2, restriction of hydration fluid to 3 L/m2 per 24 hours, and concomitant use of DDP within the drug regimen.

Actuarial Analysis↗
PubMed1994

Treatment of nongerminomatous germ-cell tumors of the pineal region.

Germ-cell tumors can be subdivided into germinoma, embryonal carcinoma, choriocarcinoma, endodermal sinus tumor (yolk-sac tumor), and teratoma. They are also distinguished by their production of secreted markers such as alpha-fetoprotein produced in endodermal sinus tumors and embryonal carcinoma or beta-human chorionic gonadotropin, produced by choriocarcinoma and embryonal carcinoma. Germinoma and teratoma produce none of the markers. Because it has been proposed that teratomas may differentiate from multipotent stem cells contained in embryonal carcinoma and are thus lineage related, the presence of markers indicates the presence of a nongerminomatous germ-cell tumor. Nongerminomatous germ-cell tumors are an invariably fatal subgroup within the pediatric pineal region germ-cell tumors. There is no effective, established therapeutic regimen. We report the treatment regimen for three children diagnosed with this highly aggressive tumor entity. The children were first given a course of chemotherapy with bleomycin, etoposide, and cisplatin. This resulted in the normalization of markers and the shrinkage of tumors. These were then removed by the infratentorial supracerebellar approach. Removal was followed by a second course of chemotherapy with vinblastine, ifosfamide, and cisplatin; after which the children underwent radiotherapy. All three children are well and without evidence of residual or recurrent disease 20, 30, and 32 months after surgery, respectively. We propose this therapy regimen for children in whom the markers are positive.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1994

Growth hormone (GH) treatment increases serum insulin-like growth factor binding protein-3, bone isoenzyme alkaline phosphatase and forearm bone mineral content in young adults with GH deficiency of childhood onset.

Recent studies have demonstrated that growth hormone (GH)-deficient adults have a markedly decreased bone mineral content compared to healthy adults. However, there are conflicting results regarding the effects of GH treatment on bone mineral content in GH-deficient adults. Therefore, we evaluated the effect of GH treatment on a marker of bone formation (bone alkaline phosphatase), hepatic excretory function and distal forearm bone mineral content in GH-deficient adults. Growth hormone was administered subcutaneously in 21 adults (13 males and 8 females) with GH deficiency of childhood onset for 4 months in a double-blind, placebo-controlled GH trial, while 13 of the patients then received further GH for an additional 14 months. Serum insulin-like growth factor I (IGF-I) increased significantly from 100 to 279 micrograms/l and IGF binding protein-3 (IGFBP-3) from 1930 to 3355 micrograms/l after 4 months of GH treatment (p < 0.0001). In addition, the molar ratio between IGF-I and IGFBP-3 increased significantly from 0.22 to 0.33 after GH treatment (p < 0.0001). Bone alkaline phosphatase increased significantly from 38.6 to 92.9 U/l during GH therapy in male patients (p < 0.0001), whereas liver-derived alkaline phosphatase was unaltered by GH. In the females, the increase in bone alkaline phosphatase did not reach statistical significance (19.1 vs 40.0 U/l, p = 0.06). The GH-induced increase in bone alkaline phosphatase correlated significantly with the increase in serum IGFBP-3 (r = 0.46, p = 0.04) but not with the increase in serum IGF-I (p = 0.16).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Intraarterial chemotherapy for osteosarcoma: does the result really justify the effort?

The use of multiagent chemotherapy has led to improved survival for osteosarcoma patients. Infusion of cisplatin by the intraarterial route in order to devitalize the primary tumor more effectively has been advocated in this context. While systemic drug levels do not seem to be compromised by this regional approach, the envisioned increase in platinum content of osteosarcoma tissue has not been substantiated. As far as clinical results are concerned, neither a comparison of various protocols including cisplatin by either intraarterial or intravenous application, nor the only controlled study dealing with the subject (COSS 86), were able to demonstrate a correlation between the route of cisplatin administration and tumor response to chemotherapy. Therefore, the use of the intraarterial route for cisplatin treatment of osteosarcoma cannot be considered standard therapy but is still an investigational approach without proven benefits. In addition, the risk of side effects and the increased cost of this procedure should be considered before deciding to use intraarterial treatment for osteosarcoma.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Treatment of osteosarcoma: experience of the Cooperative Osteosarcoma Study Group (COSS).

Using high-dose methotrexate, doxorubicin, and cisplatinum (or BCD) for adjuvant chemotherapy in osteosarcoma of the extremities, we achieved 8-year metastasis-free survival rates of 60-70%. No relapse has been observed after that time. A dose of 12 g/m2 of high-dose methotrexate seems superior to 6 g; doxorubicin was found to be indispensable for efficient therapy and administering ifosfamide in addition seemed to be beneficial. Primary chemotherapy appeared to be safe and to facilitate surgery. The response on chemotherapy provided valuable prognostic information. Salvage of poor responders by alternative postsurgical chemotherapy was unsuccessful. Intraarterial, as opposed to intravenous, use of cisplatinum, in addition to systemic three-drug chemotherapy, did not improve the local tumor response rate. The local failure rate was low (4.7%); it was higher, however, after limb-salvage procedures than after amputation and rotationplasty (11.1% vs. 2.2%, p < 0.05). The outcome after local failure was almost universally fatal. The most intriguing late sequelae of chemotherapy were cardiomyopathy due to doxorubicin and hearing loss due to cisplatinum. Given the limited number of effective drugs, it might be difficult to further improve the cure rate and also to diminish late toxicity. Exploration of the most effective but least toxic mode of drug administration might be one possibility. Another might be reduction of the cumulative doses and therapy duration, while simultaneously increasing the dose rate.

Antineoplastic Combined Chemotherapy Protocols↗