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. 1968 Dec;47(12):2630–2638. doi: 10.1172/JCI105946

Incorporation of orthophosphate-32P into erythrocyte phospholipids in normal subjects and in patients with hereditary spherocytosis

Claude F Reed 1
PMCID: PMC297434  PMID: 4302178

Abstract

The in vitro incorporation of inorganic 32P into erythrocyte phospholipids has been studied in normal subjects and in splenectomized patients with hereditary spherocytosis (HS). Phosphatidic acid (PA) was the only lipid measurably labeled in both kinds of cells. The actual turnover rate of PA phosphate was determined by simultaneously isolating inorganic phosphate (Pi) and adenosine triphosphate (ATP) and determining their specific activities. This turnover is very small: 1.3 μmoles P/liter of erythrocytes per hr in normal cells and 4.0 μmoles P in HS erythrocytes when either ATP or cellular Pi is considered the immediate precursor. This value represents less than 0.1% of the total membrane lipid phosphate. Incorporation of added 32Pi into the other phosphatides, including phosphatidyl serine, was essentially zero in both kinds of cells.

The effects of stimulation and inhibition of active cation transport, metabolic depletion, and extracellular phosphate concentration on both the degree of labeling and the actual turnover of PA phosphate were studied. In any given experiment, the degree of labeling of PA depended on the specific activities of the other intracellular phosphates (Pi and ATP). The actual turnover rate of PA phosphate, however, did not vary with active transport or metabolic depletion. The greater turnover of PA phosphate in HS erythrocytes may be due to the somewhat younger age of these cells. The results suggest that the very low turnover of PA phosphate in erythrocytes is mediated by nonspecific enzyme reactions, and that it is quantitatively insignificant in both normal and HS erythrocytes. The results also emphasize the importance of measuring intracellular phosphate precursors in any study evaluating cellular phospholipid turnover from added 32Pi.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARTLETT G. R. Methods for the isolation of glycolytic intermediated by column chromatography with ion exchange resins. J Biol Chem. 1959 Mar;234(3):459–465. [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. GLYNN I. M., SLAYMAN C. W., EICHBERG J., DAWSON R. M. THE ADENOSINE-TRIPHOSPHATASE SYSTEM RESPONSIBLE FOR CATION TRANSPORT IN ELECTRIC ORGAN: EXCLUSION OF PHOSPHOLIPIDS AS INTERMEDIATES. Biochem J. 1965 Mar;94:692–699. doi: 10.1042/bj0940692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Grossman C. M., Horky J., Kohn R. In vitro incorporation of 32P-orthophosphate into phosphatidyl ethanolamine and other phosphatides by mature human erythrocyte ghosts. Arch Biochem Biophys. 1966 Oct;117(1):18–27. doi: 10.1016/0003-9861(66)90120-2. [DOI] [PubMed] [Google Scholar]
  5. HANAHAN D. J., OLLEY J. N. Chemical nature of monophosphoinositides. J Biol Chem. 1958 Apr;231(2):813–828. [PubMed] [Google Scholar]
  6. HOKIN L. E., HOKIN M. R. Diglyceride kinase and other path ways for phosphatidic acid synthesis in the erythrocyte membrane. Biochim Biophys Acta. 1963 Mar 12;67:470–484. doi: 10.1016/0006-3002(63)91852-3. [DOI] [PubMed] [Google Scholar]
  7. HOKIN L. E., HOKIN M. R., MATHISON D. Phosphatidic acid phosphatase in the erythrocyte membrane. Biochim Biophys Acta. 1963 Mar 12;67:485–497. doi: 10.1016/0006-3002(63)91853-5. [DOI] [PubMed] [Google Scholar]
  8. HOKIN L. E., HOKIN M. R. THE INCORPORATION OF 32P FROM TRIPHOSPHATE INTO POLYPHOSPHOINOSITIDES (GAMMA-32P)ADENOSINE AND PHOSPHATIDIC ACID IN ERYTHROCYTE MEMBRANES. Biochim Biophys Acta. 1964 Oct 2;84:563–575. doi: 10.1016/0926-6542(64)90126-x. [DOI] [PubMed] [Google Scholar]
  9. JARNEFELT J. Some aspects of the physiological significance of the adenosinetriphosphatase of brain microsomes. Biochim Biophys Acta. 1962 Jun 4;59:655–662. doi: 10.1016/0006-3002(62)90645-5. [DOI] [PubMed] [Google Scholar]
  10. Jacob H. S., Karnovsky M. L. Concomitant alterations of sodium flux and membrane phospholipid metabolism in red blood cells: studies in hereditary spherocytosis. J Clin Invest. 1967 Feb;46(2):173–185. doi: 10.1172/JCI105520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jacob H. S. Membrane lipid depletion in hyperpermeable red blood cells: its role in the genesis of spherocytes in hereditary spherocytosis. J Clin Invest. 1967 Dec;46(12):2083–2094. doi: 10.1172/JCI105695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Maizels M. Cation control in human erythrocytes. J Physiol. 1949 May 15;108(3):247–263. [PMC free article] [PubMed] [Google Scholar]
  13. Mulder E., van Deenen L. L. Metabolism of red-cell lipids. 3. Pathways for phospholipid renewal. Biochim Biophys Acta. 1965 Oct 4;106(2):348–356. doi: 10.1016/0005-2760(65)90043-3. [DOI] [PubMed] [Google Scholar]
  14. NAGANO K., NAKAO M. Cation carrier in the erythrocyte membrane. J Biochem. 1962 Aug;52:99–102. doi: 10.1093/oxfordjournals.jbchem.a127588. [DOI] [PubMed] [Google Scholar]
  15. PAYSANT-DIAMENT M., POLONOVSKI J. [In vitro incorporation of P32 into the globular phospholipids of human blood]. Bull Soc Chim Biol (Paris) 1960;42:337–350. [PubMed] [Google Scholar]
  16. PAYSANT M., MAUPIN B., POLONOVSKI J. [Phospholipid metabolism of the blood. VII. In vitro incorporation of P32 phosphat into the phospholipids of the erythrocytes of human blood]. Bull Soc Chim Biol (Paris) 1963;45:247–252. [PubMed] [Google Scholar]
  17. RAPPORT M. M., ALONZO N. Photometric determination of fatty acid ester groups in phospholipides. J Biol Chem. 1955 Nov;217(1):193–198. [PubMed] [Google Scholar]
  18. REED C. F., SWISHER S. N., MARINETTI G. V., ENEN E. G. Studies of the lipids of the erythrocyte. I. Quantitative analysis of the lipids of normal human red blood cells. J Lab Clin Med. 1960 Aug;56:281–289. [PubMed] [Google Scholar]
  19. Reed C. F. Phospholipid exchange between plasma and erythrocytes in man and the dog. J Clin Invest. 1968 Apr;47(4):749–760. doi: 10.1172/JCI105770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reed C. F., Swisher S. N. Erythrocyte lipid loss in hereditary spherocytosis. J Clin Invest. 1966 May;45(5):777–781. doi: 10.1172/JCI105392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reed C. F., Young L. E. Erythrocyte energy metabolism in hereditary spherocytosis. J Clin Invest. 1967 Jul;46(7):1196–1204. doi: 10.1172/JCI105613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. WESTERMAN M. P., JENSEN W. N. IN VITRO INCORPORATION OF RADIOPHOSPHORUS INTO THE PHOSPHATIDES OF NORMAL HUMAN BLOOD CELLS. Proc Soc Exp Biol Med. 1965 Feb;118:315–319. doi: 10.3181/00379727-118-29830. [DOI] [PubMed] [Google Scholar]
  23. Weed R. I., Bowdler A. J. Metabolic dependence of the critical hemolytic volume of human erythrocytes: relationship to osmotic fragility and autohemolysis in hereditary spherocytosis and normal red cells. J Clin Invest. 1966 Jul;45(7):1137–1149. doi: 10.1172/JCI105420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wieneke A. A., Woodin A. M. The polyphosphoinositide content of the leucocyte, erythrocyte and macrophage. Biochem J. 1967 Dec;105(3):1039–1045. doi: 10.1042/bj1051039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. ZILVERSMIT D. B. The design and analysis of isotope experiments. Am J Med. 1960 Nov;29:832–848. doi: 10.1016/0002-9343(60)90117-0. [DOI] [PubMed] [Google Scholar]
  26. de Graeff J., Dempsey E. F., Lameyer L. D., Leaf A. Phospholipids and active sodium transport in toad bladder. Biochim Biophys Acta. 1965 Jul 7;106(1):155–170. doi: 10.1016/0005-2760(65)90104-9. [DOI] [PubMed] [Google Scholar]

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