Effect of intravenous iron supplementation on erythropoiesis in erythropoietin-treated premature infants.

OBJECTIVE: To test the efficacy and safety of combining intravenous iron in amounts approximating the in utero iron accretion rate and the postnatal iron loss with erythropoietin (EPO) in very low birth weight (VLBW) infants. METHODS: A prospective, controlled, randomized, unmasked trial lasting 21 days was performed in 29 clinically stable VLBW infants <31 weeks' gestation and <1300 g birth weight not treated with red blood cell transfusions during the study period. Mean (+/- standard error of the mean) age at study entry was 23 +/- 2.9 days. After a 3-day run-in baseline period in which all participants received oral supplements of 9 mg/kg/day of iron polymaltose complex (IPC), participants were randomized to receive 18 days of treatment with: 1) oral IPC alone (oral iron group); 2) 300 U of recombinant human EPO (r-HuEPO) kg/day and daily oral IPC (EPO + oral iron group); 3) 2 mg/kg/day of intravenous iron sucrose, r-HuEPO, and oral iron (intravenous iron + EPO group). To assess efficacy of the 3 treatments, serial blood samples were analyzed for hemoglobin (Hb), hematocrit (Hct), reticulocyte count, red blood cell indices and plasma levels of transferrin, transferrin receptor (TfR), ferritin, and iron. Oxidant injury was assessed before and after treatment by plasma and urine levels of malondialdehyde (MDA) and o-tyrosine. RESULTS: At the end of treatment, Hb, Hct, reticulocyte count, and plasma TfR were markedly higher in both of the EPO-treated groups, compared with the oral iron group. At study exit a trend toward increasing Hb and Hct levels and significantly higher reticulocyte counts were observed in the intravenous iron + EPO group, compared with the EPO + oral iron group. During treatment, plasma ferritin levels increased significantly in the intravenous iron + EPO group and decreased significantly in the other 2 groups. By the end of treatment, ferritin levels were significantly higher in the intravenous iron + EPO group compared with the other 2 groups. Although plasma and urine MDA or o-tyrosine did not differ among the 3 groups, plasma MDA was significantly greater in the subgroup of intravenous iron + EPO participants sampled at the end of the 2-hour parenteral iron infusion, compared with values observed immediately before and after parenteral iron-dosing. CONCLUSIONS: In stable VLBW infants receiving EPO treatment, parenteral supplementation with 2 mg/kg/day of iron sucrose results in a small, but significant, augmentation of erythropoiesis beyond that of r-HuEPO and enteral iron alone. However, to reduce the potential adverse effects of parenteral iron/kg/day on increasing plasma ferritin levels and on causing oxidative injury, we suggest that the parenteral iron dose used should be reduced and/or the time of infusion extended to maintain a serum iron concentration below the total iron-binding capacity.

Growth promoting effect of human plasma ultrafiltrate bioactive fraction (TBP) for human non-functioning pituitary adenoma cells in vitro.

We described before that the chromatographically purified "human plasma ultrafiltrate bioactive fraction" (humoral factor tentatively denoted as tumor basic protein--TBP) regulates in vitro release of ACTH from pituitary adenomas stimulating the hormone release from the tumors showing low hormonal activity in vitro and inhibiting ACTH production in vitro by highly hormonally active pituitary tumors. In this study we describe growth promoting effects (determined by 3H-TdR incorporation assay) of TBP (5 microg/l, i.e. 10% w/v plasma equivalent concentration) for 10 non-functioning pituitary tumors. The effects of TBP appeared to negatively correlate with the in vitro growth abilities of the tumors that were otherwise dependent on the duration of the clinical symptoms of the tumor presence. Hence, similar to its effects on hormonal activity of the pituitary tumors, TBP stimulated the growth of the tumors which did not express high spontaneous 3H-TdR intensity, but did not stimulate the cells with high capacity of spontaneous 3H-TdR incorporation. Moreover, all the tumors that were stimulated by TBP were nononcocytic adenomas while oncocytoma cells were not stimulated at all. Thus, TBP shows activity of humoral (plasma) factor involved in the growth regulation of pituitary adenomas that might be used to define the growth abilities of these tumors, especially in case of null cell adenomas and oncocytomas as were the tumors used in this study.

4-Hydroxynonenal prevents NF-kappaB activation and tumor necrosis factor expression by inhibiting IkappaB phosphorylation and subsequent proteolysis.

Extensively oxidized low density lipoprotein (ox-LDL), a modulator of atherogenesis, down-regulates the lipopolysaccharide (LPS)-induced activation of transcription factor NF-kappaB. We investigated whether 4-hydroxynonenal (HNE), a prominent aldehyde component of ox-LDL, represents one of the inhibitory substances. NF-kappaB activation by stimuli such as LPS, interleukin (IL)-1beta, and phorbol ester, but not tumor necrosis factor (TNF), was reversibly inhibited by HNE in a dose-dependent manner in human monocytic cells, whereas AP-1 binding was unaffected. Using similar HNE concentrations, LPS-induced kappaB- and TNF or IL-8 promoter-dependent transcription was prevented. Furthermore, pretreatment with HNE suppressed TNF production but not lactate dehydrogenase levels. Under these conditions the binding of LPS to monocytic cells was not significantly affected. However, induced proteolysis of the inhibitory proteins IkappaB-alpha, IkappaB-beta, and, at a later time point, IkappaB-epsilon was prevented. This is not due to inhibition of the proteasome, the major proteolytic activities of which remain unaffected, but rather to a specific prevention of the activation-dependent phosphorylation of IkappaB-alpha. This is the first report which demonstrates that HNE specifically inhibits the NF-kappaB/Rel system. Down-modulation of NF-kappaB-regulated gene expression may contribute at certain stages of atherosclerosis to low levels of chronic inflammation and may also be involved in other inflammatory/degenerative diseases.

Plasma antioxidants and cognitive performance in middle-aged and older adults: results of the Austrian Stroke Prevention Study.

OBJECTIVES: To study the association between cognitive status and plasma concentrations of various antioxidants in middle-aged and older individuals without neuropsychiatric disease. DESIGN: Evaluation of cross-sectional data from a cohort study. SETTING: The Austrian Stroke Prevention Study. PARTICIPANTS: A total of 1769 subjects aged 50 to 75 years, with no history or signs of neuropsychiatric disease, selected randomly from the community register. MEASUREMENTS: The score on the Mattis Dementia Rating Scale (MDRS) was dichotomized according to age-and education-specific lowest quartile cut-off points. Reversed-phase high performance liquid chromatography measurements of the plasma concentrations of lutein/zeaxanthin, cryptoxanthin, canthaxanthin, lycopene, alpha-carotene, beta-carotene, retinol, gamma-tocopherol, alpha-tocopherol, and ascorbate were measured. RESULTS: Individuals with MDRS results below the lowest quartile cut-off point had lower levels of beta-carotene and alpha-tocopherol than their counterparts with test performance above this limit (0.44+/-.33 micromol/L vs 0.51+/-.48 micromol/L, P < .001; and 29.50+/-7.98 micromol/L vs 30.93+/-11.10 micromol/L, P < .001, respectively). Only alphatocopherol remained significantly associated with cognitive functioning when logistic regression analysis was used to adjust for possible confounders including age, sex, month of blood sampling, years of education, smoking, lipid status, and major risk factors for stroke (P = .019). CONCLUSION: These observations are compatible with the view that some dietary antioxidants may protect against cognitive impairment in older people.

Increased stress parameter synthesis in the yeast Saccharomyces cerevisiae after treatment with 4-hydroxy-2-nonenal.

The metabolism of glutathione (GSH), a marker of oxidative stress and trehalose, a rather general physiological stress marker, was examined in exponentially growing Saccharomyces cerevisiae cells after treatment with 4-hydroxynonenal (HNE). GSH was entirely depleted within a 2 h incubation with 250 microM HNE. After removal of the aldehyde it was replenished by de novo synthesis leading to an overshooting GSH level, which later decreased to the basal level. In addition, trehalose was elevated 4-fold in HNE-treated yeast cells compared to control cells. We conclude that increased GSH levels upon HNE treatment are a general phenomenon of eukaryotic cells to ensure protection and survival during further harsh conditions. Furthermore, we have discovered a new indication for the stress marker trehalose in S. cerevisiae.

Effect of supplementation of smoking men with plain or slow release ascorbic acid on lipoprotein oxidation.

OBJECTIVE: To study the effects of two month ascorbic acid supplementation on in vitro lipoprotein oxidation resistance and on in vivo lipid peroxidation, and to compare the absorption of two ascorbic acid preparations. DESIGN: Randomized, single blinded and placebo-controlled clinical trial. SETTING: Men, aged 36-65 y, smoking 11-40 cigarettes daily. SUBJECTS: Sixty-two subjects were recruited by newspaper advertisements and randomized. Fifty-nine subjects completed the study. INTERVENTION: Subjects were randomized into three groups to receive 250 mg BID of plain or slow release ascorbic acid tablets or placebo daily for two months. In the pharmacokinetic part of the study, the absorption of the ascorbic acid preparations was followed for 12 h. MAIN OUTCOME MEASURES: Plasma malondialdehyde (MDA) concentration and the oxidation resistance of VLDL + LDL. For the pharmacokinetic study, the area under the plasma concentration curve (AUC) of ascorbic acid. RESULTS: Plasma reduced ascorbic acid increased by 32% in the plain ascorbate group and by 54% in the slow release group during a two month supplementation. Plasma MDA increased in the plain ascorbic acid group compared with placebo group (P < 0.05), but there were no significant differences in the changes in lipoprotein oxidation reactions induced by copper or by hemin and H2O2. Plasma reduced and total ascorbic acid AUC values were significantly higher in both plain and slow release ascorbate groups compared with placebo group. CONCLUSIONS: Oral supplementation of 500 mg of ascorbic acid daily for two months alone without any other antioxidant does not appear to have protective effect on either in vitro lipoprotein oxidation resistance or in vivo lipid peroxidation in smoking men, but might even promote the formation of MDA.

Effect of oral coenzyme Q10 supplementation on the oxidation resistance of human VLDL+LDL fraction: absorption and antioxidative properties of oil and granule-based preparations.

Coenzyme Q10 (Q10) is supposed to be an important endogenous lipid-soluble antioxidant. We studied 60 healthy 46 +/- 7 (mean +/- SD)-year-old smoking men. They were randomized into three groups to receive oil-based or granular Q10 (90 mg/d) or placebo for 2 months. Oil-based capsule elevated Q10 in plasma by 178% and in VLDL+LDL fraction by 160%. The granular preparation increased Q10 in plasma by 168% and in VLDL+LDL by 127%. However, the 2-month Q10 supplementation did not increase the oxidation resistance of VLDL+LDL fraction, as assessed by copper induced VLDL+LDL oxidation, haemin+H(2)O(2)-induced VLDL+LDL oxidation, total antioxidative capacity of LDL, and plasma malondialdehyde measurements. The first and the last dose was used to carry out a 12 h pharmacokinetic study (five subjects per group), which indicated that only a small part of supplemented Q10 was absorbed to the circulation in 12 h and that the absorption varied extensively between subjects. Our results suggest that at least among smoking men, 90 mg of orally supplemented Q10 daily does not increase the oxidation resistance of VLDL+LDL. Bioavailability of both the granular and the oil-based Q10 preparation was similar during the long-term supplementation, but one dose of 30 mg had only a marginal effect on the plasma levels of Q10.

[Prevalence and risk factors in the population of Graz (Austrian Stroke Prevention Study)]. / Prävalenz von Risikofaktoren in der Grazer Bevölkerung (Austrian Stroke Prevention Study).

The Austrian Stroke Prevention Study recruited 1960 randomly selected subjects aged 50 to 75 years during a 3-year period of enrollment. The response rate of the study was 32.4%. A telephone interview with 200 randomly selected non-responders yielded no differences to responders regarding the frequency of major vascular risk factors known to the subjects. Besides demographics, the study assessed arterial hypertension, diabetes mellitus, cardiac disease, smoking, a complete lipid status including the apolipoprotein-E genotype, serum fibrinogen and anticardiolipin antibodies as well as various natural antioxidants such as vitamins A, C, E and beta-carotene. Arterial hypertension, diabetes mellitus, cardiac disease and hypercholesterolemia > 200 mg/dl were strikingly common and occurred in 38%, 7.6%, 32% and 76%, respectively. Suboptimal plasma concentrations of vitamin A, E, and beta-carotene were noted in 77.2%, 56.1% and in 53.2% of study participants. The rate of treatment of major risk factors known to the subjects prior to study entry were 60.3% and 70% for arterial hypertension and diabetes mellitus, but only 37.1% and 6.3% for cardiac disease and hypercholesterolemia > 250 mg/dl. Diet was commonly used to treat diabetes but was almost neglected in the treatment of other vascular risk factors. These data provide an orientation on the prevalence of risk factors and the use of primary preventive measures for stroke treatment in our community.

Preparation of fatty acid methyl esters from lipoprotein and macrophage lipid subclasses on thin-layer plates.

A simple, accurate, and fast procedure for quantitative analysis of fatty acids (FA) in simple lipid subclasses from different biological specimens is presented. Lipid extracts of isolated plasma lipoproteins (very low, low, and high density lipoproteins; VLDL, LDL, and HDL, respectively) and permanent J774 mouse macrophages were fractionated into lipid subclasses by thin-layer chromatography (TLC) on silica gel 60 plates. Bands comigrating with authentic lipid standards were scraped off under argon and subjected to direct, in situ transesterification with BF3/MeOH in the presence of the TLC adsorbent. Fatty acid methyl esters were subsequently quantitated by capillary gas chromatography. A comparison of the FA content present in total lipid extracts and in lipid subclasses separated by TLC revealed recoveries ranging from 93 (J774 cell extracts) to 99.7% (LDL). The method described is applicable for the measurement of FA in individual lipid subclasses and was successfully applied to quantitatively analyze the FA composition of the phospholipid, triacylglycerol, and cholesteryl ester fraction derived from VLDL, LDL, and HDL. In J774 lipid extracts, the FA composition of the phospholipid-, monoacylglycerol-, diacylglycerol-, free fatty acid-, triacylglycerol-, and cholesteryl ester fraction was quantitated. In addition we have analyzed the time-dependent loss of the major HDL polyunsaturated fatty acids (18:2, 20:4) in the phospholipid and cholesteryl ester fraction during copper-dependent peroxidation of HDL. We have not encountered analytical problems concerning low FA recoveries from CE-rich lipid extracts as indicated by almost quantitative recoveries of FA in LDL, HDL, and J774 extracts.

Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus.

A thermostable xylanase from the filamentous fungus Thermomyces lanuginosus (DSM 5826) was purified. This enzyme has an apparent molecular weight of 24-26 kDa as determined by SDS polyacrylamide gel electrophoresis. cDNA and genomic DNA fragments coding for this enzyme were cloned and sequenced. The cDNA contains an open reading frame encoding a polypeptide of 225 amino acids and was functionally expressed in E. coli as a LacZ fusion protein. Comparison of the cDNA sequence with the genomic DNA sequence showed that the xylanase was encoded by two exons interrupted by an intron of 106 bp. Comparison of the deduced amino acid sequence to other published xylanases revealed high homology to xylanases of the family G glycanases.

Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.

Analysis of the in vitro secretory activity of human pituitary adenomas: modification of corticotropin release from adenoma tissue explant cultures by addition of a human plasma ultrafiltrate bioactive fraction.

The lack of control of tumour behaviour is manifested in different ways, depending primarily on the type of tumour. This results in numerous problems of tumour diagnosis and therapy. In the case of "benign" tumours, like pituitary adenomas, in vitro studies are often used for evaluation of the tumour. The use of tissue explant cultures of human pituitary adenomas and the comparison of the feature of cultured tumours with their behaviour in vivo showed that corticotropin is released not only from the tumours associated with Cushing's disease, but also from clinically non-functioning tumours. Hence, it was supposed that the release of corticotropin in vivo from non-secreting tumours is probably under the influence of certain neuroendocrine and/or systemic humoral factors. To test this possibility, samples of 22 tumours were cultured in plain culture medium or in the presence of the "human plasma ultrafiltrate bioactive fraction" (tentatively termed as TBP) prepared by anion-exchange chromatography. In the presence of TBP the release of corticotropin was strongly inhibited in adenomas showing relatively high spontaneous secreting activity in vitro (> 200 ng/l in 24 hours), while immunohistochemistry of these tumours indicated accumulation of corticotropin inside the cells. In contrast, TBP stimulated corticotropin release from tumours that showed relatively low basic corticotropin release (< 200 ng/l in 24 hours), with no obvious change in cellular corticotropin immunoreactivity. Such a dual activity of TBP was not observed for 8 samples of adenomas cultured in the presence of surrounding pituitary tissue, probably because TBP did not affect corticotropin secretion by the normal pituitary cells (as indicated by immunohistochemistry). From these results, it appears that TBP could be one of the humoral factors involved in the regulation of corticotropin release from pituitary adenoma tissue. Its possible involvement in the regulation of corticotropin release from normal pituitary tissue, however, is uncertain.

Determination of fatty acids in the main lipoprotein classes by capillary gas chromatography: BF3/methanol transesterification of lyophilized samples instead of Folch extraction gives higher yields.

The amount of individual fatty acids contained in the main human lipoproteins VLDL, LDL, lipoprotein (a), HDL2, and HDL3 were determined by two different methods. In Method I, the lipids were first extracted by the classical Folch procedure and then transesterified with BF3/methanol and separated by capillary GC. In Method II the lipoprotein solution was freeze dried prior to transesterification with BF3/methanol. In all lipoproteins except VLDL significantly more fatty acids were found with Method II as compared to Method I. For total fatty acids the increase was up to 17.5%, for polyunsaturated fatty acids up to 24.5%. The total fatty acid content determined by Method II resembled closely the content independently derived from the enzymatically determined lipid composition. The results indicate that in case of lipoproteins quantification of fatty acids should be made with freeze-dried samples rather than with Folch extracts.