Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy.
Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-13C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13 +/- 0.02 mumol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21 +/- 0.02 mumol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59 +/- 0.10 mumol/h per ml erythrocytes, compared to normal erythrocytes, 1.64 +/- 0.10 mumol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16 +/- 0.16 mumol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to stimulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.
Schrader
M C
MC
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
Simplaceanu
V
V
Ho
C
C
D000755:Anemia, Sickle Cell / Q000378:metabolism*; D002247:Carbon Isotopes; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008751:Methylene Blue / Q000494:pharmacology; D010427:Pentose Phosphate Pathway; D010732:Phosphofructokinase-1 / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D013997:Time Factors
D002247:Carbon Isotopes; D010732:Phosphofructokinase-1; D005947:Glucose; D008751:Methylene Blue