High-flux dialysis lowers plasma leptin concentration in chronic dialysis patients.

Leptin is a protein produced by fat cells and involved in body weight regulation. Plasma leptin is significantly higher in some hemodialysis (HD) patients than in normal controls. We examined the influence of dialyzer membrane biocompatibility and flux on elevated plasma leptin concentrations in hemodialysis patients. Employing a crossover design, leptin and tumor necrosis factor-alpha (TNF-alpha) levels were serially determined in eight chronic dialysis patients. Patients were dialyzed sequentially on low-flux cellulosic (TAF) dialyzers, low-flux (F8) polysulfone, high-flux (F80B) polysulfone, then low-flux polysulfone and cellulosic dialyzers again. Mean leptin concentrations were similar when low-flux polysulfone or cellulosic dialyzers were employed (141.9+/-24.2 microg/L versus 137.8+/-18.4 microg/L, respectively (P=NS). In contrast, leptin fell significantly on the high-flux polysulfone dialyzer (99.4+/-16.2 microg/L) compared with cellulosic (P < 0.005), and low-flux polysulfone dialyzers (P < 0.02). Leptin clearance by the high-flux polysulfone dialyzer was significantly higher than the low-flux dialyzers (50.4+/-21.5 v -9.6+/-10.3 mL/min; P=0.043), but did not account fully for the 30% decline in plasma leptin during the high-flux arm of the study. Concentrations of TNF-alpha were lower when high-flux polysulfone dialyzers were employed, but there was no correlation of individual TNF-alpha levels with leptin concentrations. High-flux dialysis lowers plasma leptin concentrations an average of 30%, but biocompatibility does not influence leptin levels. The decrease in plasma leptin on high-flux dialysis cannot be explained solely by enhanced clearance.

Increased plasma leptin concentration in end-stage renal disease.

Leptin is a 16-kDa protein recently identified as the obese gene product involved in body weight regulation. Administration of recombinant leptin to ob/ob mice, which have a genetic defect in leptin production, reduces food intake and increases energy expenditure. Leptin is synthesized by fat cells, and in normal humans, plasma concentrations are proportional to adiposity. The physiological actions and the degradation pathways of leptin in humans are unknown. We investigated renal elimination of leptin by comparing plasma leptin concentrations in end-stage renal disease (ESRD) patients with normal controls. Our hypothesis was that if renal filtration is a significant route of elimination, the hormone would accumulate in ESRD patients. Mean plasma levels in 141 ESRD patients (26.8 +/- 5.7 and 38.3 +/- 5.6 micrograms/L for males and females, respectively) were significantly higher (P < 0.001) than mean values obtained in normal controls (11.9 +/- 3.1 and 21.2 +/- 3.0 micrograms/L for males and females, respectively). Leptin concentrations in ESRD patients correlated directly with body mass index (BMI; r = 0.77 for men and 0.78 for women). The rate of increase in leptin concentrations with BMI was significantly greater in ESRD patients (5.5 and 6.6 micrograms/L/U BMI for men and women, respectively) than in normal controls (1.4 and 2.6 micrograms/L/U for men and women, respectively). Pre- and postdialysis leptin levels in hemodialysis patients were similar. Western blot of plasma from ESRD patients with high leptin levels showed bands corresponding to the intact protein (16 kDa) with no lesser or greater molecular mass species observed. Leptin concentrations in patients with ESRD did not correlate with measures of residual renal function (serum creatinine, beta 2-microglobulin, PTH, or GH levels). Similarly, we found no correlation between leptin levels and the number of years patients had been on dialysis or with recent weight changes. We conclude that intact leptin is increased in ESRD patients, but does not appear to cause decreased weight. As leptin levels did not correlate with residual renal function, increased production may account for the high levels observed.

Calorimetric analysis of lambda cI repressor binding to DNA operator sites.

Enthalpies and heat capacities were determined by isothermal titration calorimetry for bacteriophage lambda cI repressor binding to DNA containing various combinations of the three operator sites OR1, OR2, and OR3 (each comprising a consensus half-site and a specific nonconsensus half-site). Differential scanning calorimetry was employed to evaluate the effects of specific DNA binding on thermal melting of the N-terminal and C-terminal repressor domains. Principal findings of this study are as follows: (1) Binding of repressor to each of the DNA operators is dominated by a large negative enthalpy, in agreement with earlier van't Hoff analyses of quantitative footprint titration data [Koblan & Ackers (1992) Biochemistry 31, 57-65]. The calorimetric data also revealed negative heat capacities for cI binding that are of comparable magnitude with many other systems [Spolar & Record (1994) Science 263, 777-784]. However, this feature in combination with the large negative values of binding enthalpies leads to an enthalpic dominance throughout the physiological temperature range. The resulting thermodynamic profile is opposite to the entropically dominated binding observed for many systems, including lambda cro repressor which binds to the same sites as cI and also employs a helix-turn-helix binding domain [Takeda et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8180-8184]. It is suggested that these thermodynamic differences may arise from interactions of the cI repressor's N-terminal "arm" with the DNA. (2) Repressor monomers do not bind significantly to DNA containing either a consensus half-site or a nonconsensus half-site. Binding affinity to the double-consensus operator is much weaker than to any of the natural full-site operators. The same was found with other combinations of half-sites. A mutant repressor (PT158) which is severely defective in dimerization [Burz et al. (1994) Biochemistry 33, 8399-8405] was also found to bind only full-site operators and showed dimeric stoichiometry. (3) The thermal melting unit for N-terminal domains in the absence of DNA was found to reach values of 6-8 (monomer units) at concentrations where high-order oligomers of wild-type protein are formed [Senear et al. (1993) Biochemistry 32, 6179-6189]. However, in the presence of DNA operator sites, the cooperative unit for thermal unfolding was reduced to precisely two monomers, indicating that the N-terminal domain binds strictly as a dimer. (4) Significant nonadditivity was observed for the repressor binding enthalpies and heat capacities determined with multiple combinations of full-site operators.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential scanning calorimetric study of 5-enolpyruvoyl shikimate-3-phosphate synthase and its complexes with shikimate-3-phosphate and glyphosate: irreversible thermal transitions.

The thermal denaturation of native Escherichia coli 5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase, its binary complex with shikimate-3-phosphate (S3P) and its ternary complex with S3P and glyphosate have been studied using highly-sensitive differential scanning calorimetry (DSC). All observed transitions are strongly scanning-rate-dependent and irreversible. Consistent with these observations, the data were better fit by a simple irreversible model than by the controversial reversible model more commonly employed. The results obtained provide additional support for the application of irreversible models to the thermal denaturation of proteins. The calculated parameters, activation energy (Ea), enthalpy of denaturation (delta H) and transition temperature (Tm), obtained from fitting to an irreversible model agree well with values obtained from approximation techniques. Further, the results show that the formation of the ternary complex greatly enhances the thermal stability of the enzyme (delta Tm = 10.6 degrees C), while the binding of S3P alone increases the transition temperature only slightly (delta Tm = 3 degrees C). The heat of binding calculated at the transition temperature also demonstrates the greater stability of the ternary complex (delta H = -70 kcal/mol) versus the binary complex (delta H = -10 kcal/mol).