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Stabilization of glucose in blood specimens: mechanism of delay in fluoride inhibition of glycolysis.

To the Editor:

The recent report by Gambino (1) brought attention to the often overlooked fact that fluoride does not prevent loss of plasma glucose during the first 30-90 min (or longer) after blood collection (2). Although fluoride is effective in preventing later loss of glucose (1,2), the mechanism of the delay in its action is a matter of some interest.

Fluoride acts primarily by inhibiting enolase in the glycolytic pathway. Fluoride strongly inhibits the enzyme in the presence of inorganic phosphate. The inhibitory species is the fluorophosphate ion, which when bound to magnesium forms a complex with enolase and inactivates the enzyme. The delay in fluoride's prevention of glucose loss in blood samples is sometimes attributed to a postulated delay in the entry of fluoride ion into the blood cells in which the glycolytic enzymes reside. Several observations cast doubt on this explanation, however.

As part of a project for quality improvement of sample-handling requirements, we collected blood from a volunteer into four 5-mL Vacutainer (BD) tubes, 2 containing sodium fluoride/potassium oxalate, 10 mg/8 mg, and 2 with lithium heparin, 51 U, and plasma separating tube gel. The tubes were not centrifuged. After sample collection, 1 tube of each type was kept at room temperature, and another was put immediately into an ice water bath. The tubes were kept upright and mixed immediately before removing aliquots at 0, 15, 30, 45, 60, and 90 min. Each aliquot was centrifuged immediately for 1 min at 5585g in a microcentrifuge. The plasma was removed and lactate and glucose were measured on an Architect Analyzer (Abbott). The study was repeated on blood samples obtained from a second volunteer on a different day. The results for tubes at room temperature are summarized in Fig. 1. In the tubes of blood stored in ice water, with or without fluoride, decreases in glucose and increases in lactate were [less than or equal to] 0.1 mmol/L even at 90 min (data omitted for clarity).

If fluoride does not enter blood cells rapidly, then it cannot rapidly inhibit the production of lactate, which is produced from pyruvate, the final product of glycolysis. By contrast, as shown in Fig. 1, fluoride completely blocked the production of lactate for >30 min during a time when consumption of glucose was brisk in the presence of fluoride. In portions of the blood that were incubated without fluoride (Fig. 1), the quantity of lactate produced was as expected, that is, almost 2 mol of lactate were produced for each 1 mol of glucose consumed (e.g., the concentration of lactate increased at 90 min by 1.1 mmol/L in samples from each volunteer, whereas the concentration of glucose decreased by 0.6 mmol/L, Fig. 1). These data are similar to those reported by Feig et al. (3) for washed human erythrocytes, which showed consumption of glucose at 10 min after addition of fluoride, but negligible production of lactate at that time point. Moreover, Astles et al. (4), although focusing on somewhat later time points after blood collection, also reported that fluoride rapidly inhibited production of lactate in whole blood.

A parsimonious explanation of these findings is that after fluoride is mixed with blood it rapidly blocks enolase (within <5 min), and that enzymes upstream of enolase in the glycolytic pathway remain active. Thus glucose continues to be metabolized to glucose 6-phosphate, which is further metabolized to other phosphorylated metabolites of glucose, all of which accumulate in the cells. Thus the glucose concentration continues to decrease in the plasma. By contrast, lactate is stable because, with enolase inhibited, no phosphoenolpyruvate is formed and thus there is no substrate for pyruvate kinase and there is no production of pyruvate or lactic acid.

[FIGURE 1 OMITTED]

With the glycolytic pathway blocked, other pathways may also metabolize phosphorylated sugars. Such metabolism will continue until equilibrium states are reached for the several reactions involved. In particular, the rate of phosphorylation of glucose to glucose 6-phosphate will decrease because this rate depends on the supply of ATP, the concentration of which decreases in erythrocytes by almost 90% at 60 min after addition of fluoride (3).

The proposed explanation for delay in the action of fluoride does not require postulating the existence of a barrier to the movement of fluoride into erythrocytes and leukocytes. In fact, studies of lactate transport into human erythrocytes indicate that fluoride exchange across the erythrocyte membrane is rapid (5). We conclude that the delay in fluoride's ability to stop the use of glucose reflects continuing metabolism of glucose despite inhibition of the downstream target enzymes inhibited by fluoride.

Grant/Funding Support: LMM's postdoctoral training in clinical chemistry and laboratory medicine is supported by a Past Presidents' Scholarship from the Van Slyke Foundation of the American Association for Clinical Chemistry.

Financial Disclosures: None declared.

Acknowledgments: The authors thank Judy Hundley, Bob Miller, Victoria Reynolds, and the staff of the clinical chemistry laboratory at the University of Virginia for excellent technical support.

References

(1.) Gambino R. Glucose: a simple molecule that is not simple to quantify. Clin Chem 2007;53: 2040-1.

(2.) Chan AYW, Swaminathan R, Cockram CS. Effectiveness of sodium fluoride as a preservative of glucose in blood. Clin Chem 1989;35:315-7.

(3.) Feig SA, Shohet SB, Nathan DG. Energy metabolism in human erythrocytes, I: effects of sodium fluoride. J Clin Invest 1971;50:1731-7.

(4.) Astles R, Williams CP, Sedor F. Stability of plasma lactate in vitro in the presence of antiglycolytic agents. Clin Chem 1994;40:1327-30.

(5.) Chapman BE, Kuchel PW. Fluoride transmembrane exchange in human erythrocytes measured with 19F NMR magnetization transfer. Eur Biophys J 1990;19:41-5.

DOI: 10.1373/clinchem.2007.102160

Leann M. Mikesh David E. Bruns *

Department of Pathology University of Virginia Charlottesville, VA

* Address correspondence to this author at:

Department of Pathology

P O Box 800168

University of Virginia School of Medicine

Charlottesville, VA 22908

email dbruns@virginia.edu
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Title Annotation:Letters
Author:Mikesh, Leann M.; Bruns, David E.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:May 1, 2008
Words:991
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