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Biomedical subjects

B Kirschbaum

Publications and source records attributed to B Kirschbaum.

At least 19 recordsLinked to original sources
PubMed2004

A novel complex regulates cardiac actin gene expression through interaction of Emb, a class VI POU domain protein, MEF2D, and the histone transacetylase p300.

Expression of the mouse cardiac actin gene depends on a distal enhancer (-7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3' AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation.

Acetyltransferases↗
PubMed2002

Serial ferritin concentrations in hemodialysis patients receiving intravenous iron.

BACKGROUND: Treatment of the anemia of chronic renal failure with intravenous iron and erythropoietin is highly effective, but frequently leads to ferritin levels which are much higher than those seen in the general population. High ferritin concentrations raise concern about the potential toxicity of increased body iron stores. PATIENTS AND METHODS: We retrospectively evaluated parameters of iron metabolism over a 4-year period among all our chronic hemodialysis patients who had been receiving intravenous iron and erythropoietin. Initially, patients received intermittent infusions of 300 mg intravenous iron x 3 doses for a low ferritin or low percent saturation of total iron binding capacity (TIBC), but this protocol was subsequently changed to weekly or biweekly infusions of 50-100 mg. RESULTS: We observed an improvement in average hemoglobin values, modest increases in serum iron and saturation of iron binding capacity, and a 125% increase in ferritin levels over 4 years. TIBC decreased. Overall, ferritin values increased 79 microg/l for each 1% increase in TIBC saturation. Ten patients with ferritin concentration greater than 1,000 pg/l received a three month course of vitamin C with no decline in the ferritin concentration. CONCLUSION: Current protocols for iron delivery may result in progressive increases in ferritin levels. Concern about the risks of iron overload should temper the quantity of iron used in dialysis programs.

Anemia↗
PubMed2002

Correlative studies of urine fluorescence and free radical indicators.

AIMS: Inflammation results in the production of free radicals which damage proteins, lipids, and nucleic acids. The products of these reactions are cleared, in part, by the kidney and appear in the urine. In this study, urine fluorescence was measured in individuals with various nephropathies to determine the value of these assays for detecting the excretion of end products of radical-mediated chemical reactions. METHODS: Urine fluorescence was quantified at wavelengths which correspond to the presence of advanced glycosylation endproducts (AGE) and dityrosine (di-TYR). The samples were also tested for isoprostanes, nitrite/nitrate, hydrogen peroxide, and thiobarbituric acid reactive substances (TBARS). RESULTS: Fluorescence values, expressed per ml urine or per mg creatinine (crt), were not normally distributed and covered a wide range. There were significant differences in fluorescence among groups of patients classified by diagnosis, but the differences did not allow sharp distinction of diagnostic categories. Fluorescence assays correlated significantly with TBARS but not with isoprostanes, nitrite/nitrate, or hydrogen peroxide. Fluorescence tended to increase with age. Gender and race did not affect the results. CONCLUSIONS: Because of the many factors which can affect free radical production and tissue injury, the value of urine fluorescence assays to screen for radical-mediated toxicity appears to be limited. Serial studies of patients will be needed to determine whether urine fluorescence will be useful to monitor responses to treatment or be predictive of progression vs. remission of renal disease.

Cross-Sectional Studies↗
PubMed2001

Total urine antioxidant capacity.

Total antioxidant capacity has been determined for several body fluids and provides a convenient means to compare antioxidant defenses among patients with acute or chronic inflammatory illnesses. We have studied urine specimens from a control group and a variety of patients with hypertension and acute and chronic renal diseases using an ABTS antioxidant assay as described for blood. Other urine assays included fluorescence markers for advanced glycosylation end products (AGE) and di-tyrosine (di-tyr), protein, uric acid, and creatinine concentrations. Urine antioxidant activity was standardized against ascorbic acid. We found that both the lag time and the area under the curve (AUC) in the ABTS assay were highly correlated with one another and correlated with the protein and uric acid concentrations, except for those specimens collected from patients with acute renal failure (ARF). The lack of correlation in the ARF group was not associated with significant differences in lag time or AUC. Correlations were seen also between antioxidant parameters and fluorescence for AGE and di-tyr. The results indicate that the predominant antioxidants in the urine of patients with acute renal failure differ from those found in the urine of individuals with hypertension and chronic nephropathies. The ABTS assay provides a convenient marker for the antioxidant content of urine.

Acute Kidney Injury↗
PubMed2001

CBQCA assay of primary amine losses during hemodialysis.

Nitrogen loss during hemodialysis is an important issue since all steps taken to improve clearance of urea will also increase loss of amino acids and other normally recyclable sources of nitrogen. Most dialyzers exclude albumin and other proteins, which react well with dyes commonly employed to measure protein, but do allow passage of mono- and oligopeptides that go undetected in protein assays. The CBQCA reagent provides a highly sensitive assay of primary amines that will detect amino acids, low molecular weight polypeptides, and high molecular weight proteins. We collected dialysate using a split stream technique from 28 chronic dialysis patients. With bovine albumin as the standard, the CBQCA assay reported 24.3+/-9.8 g albumin-equivalents (mean+/-S.D.) per dialysate whereas a Coomassie blue assay measured 1.19+/-0.78 g. The CBQCA assay values were substantially higher than previously reported amino acid losses. The CBQCA fluorescent assay for amines provides a simple assay for quantifying primary amine losses in dialysate fluid.

Amines↗
PubMed2001

Renal regulation of plasma total antioxidant capacity.

Hyperuricemia has been labeled both a risk factor and marker for cardiovascular pathology in addition to being associated with gout and kidney disease. Uric acid in vitro acts as a potent antioxidant capable of scavenging hydroxy radicals and peroxynitrite and reacting with nitric oxide. Some clinical studies have provided evidence that, in vivo, uric acid is oxidized under conditions associated with high oxidant stress and may spare other antioxidants such as ascorbic acid. The plasma level of uric acid is controlled by the rates of production and excretion or degradation of uric acid. Under most circumstances, it is the renal clearance of uric acid which primarily determines the plasma concentration. Many factors of exogenous and endogenous origin can influence renal tubular absorption and secretion of uric acid. We suggest that renal urate clearance is not haphazard but regulated by an unknown signal that is issued in response to the level of oxidative stress. Since much cardiovascular pathology is now believed to have an inflammatory component and is associated with enhanced production of free radicals, the accompanying hyperuricemia may be viewed as a compensatory response of potential benefit.

Antioxidants↗
PubMed2000

Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in mitogen-stimulated primary human T-lymphocytes.

BACKGROUND: Mycophenolate mofetil (MMF) is an effective immunosuppressant developed for use in organ transplantation. It specifically targets lymphocyte purine biosynthesis. However, side effects do occur. Understanding how the active metabolite of MMF, mycophenolic acid (MPA) affects the normally integrated interaction between intracellular purine and pyrimidine pathways might aid the development of improved therapeutic regimes. METHODS: We used a primary human T-lymphocyte model to study how preincubation with MPA (0.1-50 microM) affected normal ribonucleotide pool responses to phytohemagglutinin using radiolabeled precursors. RESULTS: MPA not only restricted the mitogen-induced expansion of GTP pools, but actually induced a severe drop in both GTP (10% of unstimulated cells) and GDP-sugar pools, with a concomitant fall in ATP (up to 50%). These effects were concentration dependent. By contrast, uridine pools expanded whereas CTP pools remained at resting levels. These changes were confirmed by the altered incorporation of [14C]-bicarbonate and [14C]-glycine into nucleotides. Restriction of [14C]-hypoxanthine incorporation and reduction of [14C]-uridine uptake comparable to that of unstimulated cells indicated that MPA also inhibited both salvage routes of nucleotide synthesis. CONCLUSION: MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms underlying these disproportionate changes can best be explained by MPA-related inhibition of amidophosphoribosyltransferase, catalysing the first step in purine biosynthesis. This would increase phosphoribosylpyrophosphate availability, thereby stimulating UTP biosynthesis. Such imbalances, coupled with ATP-depletion, could underlie reported side effects and might be overcome by appropriate combination therapies.

Adenosine Triphosphate↗
PubMed2000

Spurious metabolic acidosis in hemodialysis patients.

Metabolic acidosis with an increased anion gap (AG) is frequently seen among patients with end-stage renal failure that is corrected to a variable degree by chronic hemodialysis. The degree of acidosis is generally interpreted from the concentration of total carbon dioxide (tCO(2)) in blood drawn from the vascular access used for dialysis. As with many dialysis units in the United States, our laboratory studies for outpatients are performed in a central laboratory several hundred miles away and must be shipped there by air freight. We observed a consistent and clinically important difference between the tCO(2) content of samples reported from the central laboratory compared with results reported from a local university hospital chemistry laboratory. The central laboratory readings were always lower, resulting in an increase in the AG. Delays in centrifugation of the blood to separate the clot from the serum and in the initiation of analysis led to an increase in the lactate content of the samples. That increase, however, was insufficient to explain the difference in tCO(2) levels. These data suggest that something happens to the samples in transit to cause an artifactual reduction of the tCO(2) level. For many dialysis patients, the severity of their acidosis may be falsely represented by the tCO(2) content of blood samples reported from central laboratories.

Acidosis↗
PubMed2000

Relationships between the copper and iron systems in hemodialysis patients and variables affecting these systems.

The copper-binding protein ceruloplasmin oxidizes ferrous iron to ferric iron, an action that is critical for the binding of iron to transferrin in plasma. Ceruloplasmin, in common with ferritin and transferrin, is an acute-phase protein that is altered by inflammation. We sought to identify interrelationships between the copper and iron systems by measuring copper, ceruloplasmin, ferroxidase, ferritin, transferrin, iron, and iron-binding capacity in a group of hemodialysis patients. We looked for evidence of inflammation and free-radical injury by assaying for protein carbonyl groups, protein pyrrolation, di-tyrosine, and advanced oxidation protein products. Our findings were compatible with an active inflammatory state that affected both iron and copper metabolism. Transferrin levels were low, whereas ceruloplasmin levels were elevated compared to normal. Copper concentration was increased proportional to ceruloplasmin. Several variables including ceruloplasmin and transferrin were observed to correlate significantly with the level of pyrrolated protein. The data suggest that posttranslational modification of circulating proteins may affect their structural, enzymatic, and ligand-binding properties. Abnormalities in copper metabolism and their influence on iron handling in renal failure are complex and will require additional study before their importance can be defined.

Adult↗
PubMed2000

The anion gap associated with pregnancy-induced hypertension.

OBJECTIVES: To determine if pregnancy-induced hypertension (PIH) is associated with a narrow anion gap. METHODS: Chart review of women with uncomplicated pregnancies and those with PIH and varying degrees of proteinuria. Electrolytes, total protein, albumin, creatinine, uric acid, a qualitative or quantitative measurement of urine protein were required for inclusion into the study. RESULTS: A low anion gap relative to women with normal pregnancies was found only in the group of PIH patients who had the greatest degree of proteinuria. The anion gap correlated directly with the concentration of plasma albumin. Women with PIH, proteinuria, and elevated liver transaminases had the lowest plasma albumin values and the smallest anion gap. CONCLUSION: The degree of hypoalbuminemia determines the magnitude of the anion gap in women with PIH and proteinuria.

Acid-Base Equilibrium↗
PubMed1999

Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid.

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [14C]carbamoyl aspartate from [14C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

Amidophosphoribosyltransferase↗
PubMed1999

Urine electrolytes and the urine anion and osmolar gaps.

Urine ammonia concentration is crucial to understanding and quantifying the kidney's response to metabolic acidosis. This test is generally not performed by clinical laboratories. The urine anion gap and osmolar gaps have been proposed as surrogate measures of urine ammonia in patients with hyperchloremic acidosis. We measured ammonium and other electrolytes in the urine of patients attending our renal disease clinic who did not have severe metabolic acidosis and compared the results with those calculated by standard formulae for the anion and osmolar gaps. We found no correlation between measured ammonium values and the anion gap and attributed this lack of agreement to the presence in urine of substantial amounts of unmeasured inorganic anions, which the formula fails to consider. There was significant correlation between measured ammonium and the osmolar gap but not good agreement between the absolute values provided by the 2 methods. Solutes including sulfate and phosphate were quantified in 24-hour urine collections and showed great variability with respect to measured chloride and estimated protein catabolism. We conclude from these studies that there is no substitute for the direct determination of urine ammonium when an accurate concentration is desired.

Acid-Base Equilibrium↗
PubMed1999

Identification and characterization of potential new therapeutic targets in inflammatory and autoimmune diseases.

The isoxazole derivative Leflunomide (HWA 486) is a novel immunoregulatory and anti-inflammatory drug. Affinity chromatography was used to purify and identify Leflunomide binding proteins, which might play a role as potential cellular targets in the molecular mode of action. The Leflunomide derivative A 0273 was covalently coupled to a Fractogel(R) matrix. This column was used to separate a cytosolic protein extract of the macrophage cell line RAW 264.7 by several selected and specific gradient elution steps. Proteins that were specifically eluted through the active metabolite of Leflunomide, A 1726, were identified by subsequent protein sequence analysis. This allowed us to specify 10 cytosolic proteins, which bind with high affinity to this matrix. Three of them, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and phosphoglycerate mutase belong to the second part of the glycolytic pathway. The binding specificity of these protein/drug interactions was further evaluated using BIAcore(R) analysis. Kd values of glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase and lactic dehydrogenase were similar to the Kd value of a known Leflunomide target protein, dihydroorotate dehydrogenase. In order to elucidate the features as well as the overall relevance of these results, cytosolic fractions of three additional cell lines MOLT-4, A20.2J, HeLa were compared using the same chromatographic protocol. The elution profiles as well as subsequent Western blot analyses confirmed the data obtained previously for the macrophage cell line RAW 264.7.

Amino Acid Sequence↗
PubMed1999

Hypotransferrinemia of chronically hemodialyzed patients.

Increasingly, the iron needs of hemodialysis patients receiving erythropoietin are being met by infusions of intravenous iron guided by laboratory tests to measure body iron availability. The interpretation of assays based on ferritin and transferrin must take into account the effect of inflammation on both proteins and malnutrition on the latter. In our chronic hemodialysis population, hypotransferrinemia was present in greater than 90% of the patients. Using statistical methods and laboratory studies, we sought to identify the principal reasons for the high prevalence of hypotransferrinemia. We observed that transferrin levels were disproportionately low relative to albumin and prealbumin and correlated inversely with ferritin levels. There was no correlation between transferrin and the soluble transferrin receptor. After the infusion of 900 mg of iron, transferrin saturation increased and total transferrin decreased so that unsaturated iron bonding capacity decreased as well. Ferritin concentrations increased significantly after iron loading. Attempts to demonstrate activation of the patients' antioxidant mechanisms associated with iron infusion were negative. We concluded that the low transferrin may be principally the result of diminished synthesis related to the chronic inflammatory status of hemodialysis patients, which favors production of ferritin, but iron and nutritional status may also influence the blood transferrin concentration. These factors make interpretation of transferrin-dependent assessment of body iron stores unreliable and can result in inadequate or overly aggressive iron-replacement therapy.

Adult↗
PubMed1998

Leflunomide inhibits pyrimidine de novo synthesis in mitogen-stimulated T-lymphocytes from healthy humans.

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic-acid dehydrogenase. Leflunomide (25 and 50 microM), like Brequinar (0.5 and 1 microM), a demonstrated dihydroorotic-acid dehydrogenase inhibitor, was cytostatic, not cytotoxic, with proliferation being halted in the G1 phase. Both drugs restricted the normal 4-8-fold mitogen-induced expansion of pyrimidine pools over 72 h to concentrations found in nonstimulated T-cells and [14C]bicarbonate incorporation into UTP, ATP, and GTP. Uridine (50 microM) restored expansion of all pools, but [14C]bicarbonate incorporation into ATP and GTP only, not UTP. [14C]Hypoxanthine salvage was also restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors. [14C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 microM Leflunomide. 100 microM Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidine-related effects reported for Leflunomide at 100 microM and above.

Adenosine Triphosphate↗