Mohammad Kutub Ali

Calcium-induced bilirubin-dependent hemolysis of human erythrocytes.

Human erythrocytes, preincubated with different concentrations of calcium chloride (0.17-1.67 mM) showed hemolysis after addition of bilirubin (72 microM). Hemolysis was observed only when cells were incubated first with calcium followed by bilirubin and not vice versa. This hemolysis was found to be dependent upon several factors such as concentration of bilirubin, time of incubation of erythrocytes with calcium and time of incubation of bilirubin with the calcium-loaded erythrocytes. Inclusion of EDTA in the incubation medium reduced the percentage hemolysis to a significant extent. Involvement of activated oxygen species in hemolytic process seems to be unlikely as inclusion of sodium azide and catalase did not prevent hemolysis. A comparison of other bivalent cations such as Ba2+, Mg2+, Mn2+ and Cu2+ with Ca2+ for their ability to hemolyse cells in presence of bilirubin shows that Ba2+ and Mg2+ are ineffective, whereas both Mn2+ and Cu2+ induce hemolysis both in the absence as well as in the presence of bilirubin. However, their mechanism of hemolysis is different from that of calcium-induced hemolysis. Formation of calcium-induced hydrophobic aggregates of phospholipid molecules in erythrocyte membrane may open the new binding sites for bilirubin on these membranes which may perturb the membrane conformation.

A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods.

In this study, we used three different methods for the extraction of membrane-bound bilirubin (EMB) from erythrocyte membranes. Use of 2.5% albumin, pH 7.4, for elution of EMB resulted in only 34% of the total EMB which was estimated after the solubilization of bilirubin-loaded erythrocyte membranes (BLEMs) with 1% SDS. On the other hand, incubation of BLEMs with 38 mM sodium carbonate solution containing 5 mM EDTA, pH 11.0, yielded 77% of the total EMB. Application of Fogs reaction for the estimation of EMB directly on the BLEMs resulted in the estimation of 75% of the total EMB. These results suggest that either of the above methods, i.e. use of albumin or high pH, or direct Fogs reaction cannot estimate the total EMB correctly. Increase in ionic strength from 0.15 to 0.45 did not release any EMB from erythrocyte membranes. Therefore, the best method for the estimation of total EMB is the solubilization of membrane with 1% SDS followed by Fogs reaction method.

Binding of bilirubin to mammalian erythrocytes.

Binding of bilirubin to erythrocytes of various mammalian species in the presence of their respective plasma albumins was studied at pH 8.0, ionic strength 0.41 and at 37 degrees C. In human, buffalo, goat, and sheep, the amount of erythrocyte-bound bilirubin increased with the increase in both the bilirubin/albumin molar ratio (B/A) and the total bilirubin concentration. In all species, the binding patterns were qualitatively similar. However, at any given B/A, goat erythrocytes bound the highest amount of bilirubin, followed by buffalo and human erythrocytes; sheep erythrocytes bound the lowest amount of bilirubin. Increase in erythrocyte-bound bilirubin per unit increase in bilirubin concentration at a constant B/A as obtained from the values of the slope of the plot between erythrocyte-bound bilirubin and total bilirubin in the incubate, was found to be highest for goat erythrocytes, followed by buffalo, human, and sheep erythrocytes. At given bilirubin concentration, percentage fractional binding of bilirubin between any two B/As was found to be highest between 1.5 and 2.0, suggesting that a greater amount of bilirubin was transferred from plasma to cells between B/As 1.5 and 2.0. Percentage fractional binding of bilirubin was highest in goat erythrocytes, followed by buffalo, human, and sheep erythrocytes. These differences in the amount of erythrocyte-bound bilirubin were not due to the effect of various plasma albumins. These results suggest that the difference in bilirubin binding by different mammalian erythrocytes can be attributed to the difference in either the affinity of the erythrocyte receptors or their number or both.

Binding of bilirubin to erythrocytes from different mammalian species

The binding of bilirubin to erythrocytes of several mammalian species, i.e. human, buffalo, goat and sheep was studied. In all cases, curves between bilirubin desorbed from erythrocytes and bilirubin in the incubate followed Michaelian saturation kinetics. The dissociation constants of the bilirubin-receptor complex and saturable binding sites were calculated using double reciprocal plots. Goat erythrocytes had the highest dissociation constant (265.7 mumol/l) and highest saturation (125.9 microM), whereas sheep erythrocytes had the lowest dissociation constant (115.6 mumol/l) and lowest saturation (62.5 microM). Buffalo and human erythrocytes bound bilirubin in a similar fashion, and the values of interaction parameters were midway between those obtained with goat and sheep erythrocytes. Differences in the affinity and number of saturable binding sites can be attributed to the different make-up of the erythrocyte membranes of these species.

Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes.

Washed erythrocytes from human, buffalo, sheep and goat preincubated with different concentrations of calcium chloride (16.7-1830 microM) showed significantly different rates of hemolysis (up to 62%) after addition of bilirubin (72 microM). Goat erythrocytes displayed marked resistance to hemolysis with only 11% hemolysis observed at the highest calcium concentration. Similar trend in hemolysis was also observed when the concentration of CaCl2 was fixed (330 microM) and bilirubin concentration varied (0-72 microM). (Ca(2+)-Mg(2+)-ATPase levels were found significantly lower in goat and sheep erythrocyte membranes compared to human and buffalo erythrocyte membranes. This was correlated well with the observed hemolysis in various mammalian erythrocytes.

Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes

Binding of bilirubin to erythrocyte membranes of human, buffalo, sheep and goat was studied after phospholipase C, trypsin and neuraminidase treatment. Phospholipase C and trypsin treatment of membranes greatly enhanced the bilirubin binding in all mammalian species, whereas, neuraminidase treatment resulted into a small increase in the membrane-bound bilirubin. Human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human>sheep>buffalo>goat; the order was: human>buffalo>sheep>goat; in phospholipase C- and trypsin-treated erythrocyte membranes. These binding results indicate that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes as the differences observed in the membrane-bound bilirubin among mammalian species were directly correlated with the sum of choline phospholipids, especially phosphatidylcholine and sphingomyelin content of the erythrocyte membranes. The negatively charged phosphate moiety of phospholipids of the membranes appears to inhibit a large amount of bilirubin binding to the membrane as its removal by phospholipase C greatly enhanced the binding. Furthermore, membrane proteins and carbohydrate also seem to play a significant regulatory function on the binding as their degradation and/or removal in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites.

Generalized Theory and Analysis of Scalar Modulation Techniques for a

The basic requirement of all the scalar modulation strategies employed for matrix converter, is to define the desired output voltages. The output phase voltages, if not optimized, results in underutilization of semiconductor device ratings. For better utilization, the output reference phase voltages are modified such that the sinusoidal nature of line voltages is not altered. Multiphase drives are in consideration and much work is being done and reported on multiphase matrix converter-based system. New modulation strategies for such systems are being formulated. Most of these new strategies are based on scalar approach; which in contrast to the space vector pulse width modulation, require addition of friendly harmonics in order to achieve optimized output reference phase voltages and maximum utilization of the matrix converter semiconductor switches. On the other hand, in space vector pulse width modulation, the maximum voltages are inherently achieved. This paper presents a generalized theory, explained in mathematical as well as graphical manner, defining the optimized output reference phase voltages for any number of input and output phases, whether odd or even, for m × n matrix converter. Further, this paper also explores the Venturini method for m × n case. It is found that the method is extendable to 3 × n matrix converters only. Finally, a simple and generalized algorithm, applicable to all m × n matrix converter configurations, is discussed. Results for 3 × 5 matrix converter are validated by simulation and experimental results.

m {\;\times\;} n

This paper analyzes a three phase fixed input to five phase variable amplitude/frequency output matrix converter topology to feed a five phase induction motor. Less spacious applications like aeroplanes, ships etc. are employing multiphase machines more frequently. Front-end converters have been so far employed to interface the load with the supply. These converters employ dc-link capacitor - which is disadvantageous for low space applications. The alternative is seen in matrix converters. The maximum limit on attainable output voltage in a matrix converter with a three phase input is found to be 50 % of the input voltage. It is found that this limit can be increased to 78.86 % for three-to-five phase system, if input and output voltages are optimized. A simplified modulation method for five phase output is defined by employing direct transfer function approach (DTFA). The performance of a five phase motor is then analyzed. It is found that lower voltage level is required at the input of the matrix converter to run a motor at rated voltage with voltage optimization than without voltage optimization. This aspect is again beneficial for low space applications.

Matrix Converter

This paper analyzes a three-phase fixed input to five-phase variable amplitude/frequency output matrix converter topology to feed a five phase system. Conventionally, back-to-back topology is employed, which requires dc-link capacitor and needs large energy storage elements. This is disadvantageous for low space applications like aeroplanes, electric vehicles etc. The alternative is seen in matrix converters. The maximum limit on attainable output voltage in a three-phase input matrix converter is found to be 50 % of the input voltage. It is found that this limit can be increased to 78.86 % for three-phase input to five-phase output matrix converter. To achieve this, input and output waveforms are optimized. Coefficients are accordingly derived. A simplified modulation method for five-phase output is defined. The simulation results that are obtained by applying input filter show practical feasibility.

Analysis of a three-to-five-phase matrix converter using DTFA

Interaction of bilirubin with different types of erythrocyte membrane vesicles such as unsealed, heterogeneous, sealed and inside-out membrane vesicles prepared from human and goat erythrocytes was studied. Out of various types of membrane vesicles, in both species, unsealed membrane vesicles bound quantitatively higher amounts of bilirubin followed by heterogeneous and sealed membrane vesicles whereas inside-out membrane vesicles bound the lowest amount of bilirubin. These differences in the amount of bound bilirubin to different membrane vesicles were correlated well with the percentage accessibility of sialic acid to neuraminidase in these membranes suggesting that bilirubin bound preferentially to the outer layer of erythrocyte membranes than the inner layer. Further, membrane vesicles prepared from human erythrocytes bound higher amounts of bilirubin than those prepared from goat erythrocytes. This can be ascribed to different phospholipid composition of these membranes.