How Does Naf Inhibit Glycolysis-Related Enzymes?

NaF is not an effective agent to prevent glycolysis, as it inhibits enolase, a late enzyme in the glycolytic pathway. Enzymes upstream of enolase remain active and continue to metabolize glucose until substrates are exhausted, causing the antiglycolytic action of fluoride to be delayed for up to 4 hours. NaF inhibits enolase, an enzyme acting late in the glycolytic pathway, and has no effect on enzymes that act early in the glycolytic pathway. Acidification of blood below a pH of 6.0 stops glycolysis immediately.

In the past, sodium fluoride (NaF) exerted its action on enolase, which catalyzed a down-stream step of glycolysis. However, the lack of rapid inhibition by NaF on glycolysis is due to its mechanism of action: fluoride inhibits enolase, which is a late enzyme in the glycolytic pathway. The inhibitory species is the fluorophosphate ion, which when bound to magnesium forms a complex with enolase and inactivates the enzyme.

Acidification inhibits hexokinase and phosphofructokinase enzymes that act early in the glycolytic pathway. Glycolysis is instantly inhibited in erythrocytes, leukocytes, and other cells. The inhibitory species is the fluorophosphate ion, which when bound to magnesium forms a complex with enolase and inactivates the enzyme.

It has been known that fluoride ions can interrupt glycolysis by inhibiting the activities of the glycolytic enzyme, enolase. An inhibition by Na+ of one or more of the first four reactions would decrease the glycolytic rate by lowering the rate of ATP formation or utilization. The inhibitory species is the fluorophosphate ion, which when bound to magnesium forms a complex with enolase and inactivates the enzyme.

Addition of NaF or MFP to rat hepatocytes results in a decrease in lactate and an increase in glucose, 3- and 2-phosphoglycerate production.

What does sodium fluoride prevent?

What is this medication?. SODIUM FLUORIDE (SOE dee um FLOOR ide) prevents tooth decay. It may also be used to reduce gum and tooth sensitivity. It works by making your teeth stronger. Fluoride is a mineral that plays an important role in the health of your teeth and bones.

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This medicine may be used for other purposes; ask your health care provider or pharmacist if you have questions.

How does fluoride inhibit enzymes?

Fluoride Inhibits Enzymes through Competition with Magnesium. Fluoride in 0. 01% concentration has an appreciable effect in inhibiting the anaerobic production of energy from glucose. Of the salts examined only fluoride inhibits glycolysis in low concentrations in various tissues.

The Institute of Technology and Business, Okružní 517/10, 370 01 České Budějovice, Czech Republic.

Submission received: 7 September 2020 / Revised: 29 September 2020 / Accepted: 30 September 2020 / Published: 13 October 2020.

What can inhibit the process of glycolysis?

Citrate, the first product of the citric acid cycle, can also inhibit PFK. If citrate builds up, this is a sign that glycolysis can slow down, because the citric acid cycle is backed up and doesn’t need more fuel.

Why does NaF inhibit glycolysis?

NaF is not an effective agent to prevent glycolysis, as it inhibits enolase, a downstream enzyme in the glycolytic pathway. Enzymes upstream of enolase remain active and continue to metabolize glucose until substrates are exhausted, causing the antiglycolytic action of fluoride to be delayed for up to 4 hours. NaF alone is not an effective antiglycolytic agent, and time and temperature are critical variables.

A simple and effective replacement for NaF is needed, and a US patent (no. 4 780 419) was applied for in 1986 and issued in 1988 to K. Uchida, S. Okuda, and K. Tanaka, assigned to Terumo Corporation, Tokyo, Japan. After filing the US patent, Uchida et al. published a detailed study on how acidification quickly inhibits glycolysis. Acidification inhibits hexokinase and phosphofructokinase, enzymes that act early in the Embden–Meyerhof pathway. Glycolysis is instantly inhibited in erythrocytes, leukocytes, and platelets when blood pH is maintained between 5. 3 and 5. 9 with a citrate buffer. The inhibitory effect of acidification is sustainable for approximately 10 hours at 25°C.

Terumo Medical Corporation manufactures a blood-collection tube (Venosafe® Glycaemia) that contains the ingredients identified in the patent. However, Terumo does not specify the ratios of the ingredients and there is no published study of this acidified Terumo blood-collection tube for measuring blood glucose concentrations. Samples of the tube containing the acidification reagents were obtained for experimental use only through a UK laboratory.

What are the inhibitors of glycolysis?

This study investigates the inhibition of five glycolysis pathway molecules (GLUT1, HKII, PFKFB3, PDHK1 and LDH) using nine inhibitors (Phloretin, Quercetin, STF31, WZB117, 3PO, 3-bromopyruvate, Dichloroacetate, Oxamic acid, NHI-1) in panels of breast and ovarian cancer cell line models. All compounds tested blocked glycolysis, as indicated by increased extracellular glucose, decreased lactate production, and increased apoptosis. Sensitivity to several inhibitors correlated with the proliferation rate of the cell lines. Seven compounds had IC 50 values associated with each other, consistent with a shared mechanism of action. A synergistic interaction was revealed between STF31 and Oxamic acid when combined with the antidiabetic drug metformin. Sensitivity to glycolysis inhibition was also examined under a range of O 2 levels (21 O 2, 7 O 2, 2 O 2 and 0. 5 O 2), and greater resistance to the inhibitors was found at low oxygen conditions (7 O 2, 2 O 2 and 0. 5 O 2 ) relative to 21 O 2 conditions. These results indicate growth of breast and ovarian cancer cell lines is dependent on all the targets examined in the glycolytic pathway with increased sensitivity to the inhibitors under normoxic conditions. Targeting aerobic glycolysis is a promising strategy to preferentially kill cancer cells dependent on this pathway, and in recent years, multiple glycolytic inhibitors have been developed.

What does sodium fluoride inhibit?

Fluoride ions can interrupt glycolysis by inhibiting the activities of the glycolytic enzyme, enolase, and the gluconeogenetic enzyme, phosphoenolpyruvate synthetase. However, the reprogramming of gene expression underlying cellular responses to fluoride, especially under anaerobic conditions, is still poorly understood. This study compares the genome-wide transcriptomic profiles of E. coli grown in the absence or presence of sodium fluoride (NaF) under anaerobic conditions and assesses the impact of fluoride-dependent ATP depletion on RNA turnover. Tiling array analysis revealed transcripts displaying altered abundance in response to NaF treatments. Quantile-based K-means clustering uncovered a subset of genes that were highly upregulated and then downregulated in response to increased and subsequently decreased fluoride concentrations, many of which (~40) contained repetitive extragenic palindromic (REP) sequences. Northern blot analysis confirmed their considerably enhanced abundance in response to NaF treatment. An mRNA stability analysis of osmC and yghA transcripts demonstrated that fluoride treatment slows down RNA degradation, enhancing RNA stability and steady-state mRNA levels. The turnover of these transcripts depends on RNase E activity and RNA degradosome. The study shows that NaF exerts significant effects at the whole-transcriptome level under hypoxic growth, mimicking the host environment, and fluoride can impact gene expression posttranscriptionally by slowing down ATP-dependent degradation of structured RNAs.

How does fluoride inhibit glycolysis?

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 5585 g 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 ≤0. 1 mmol/L even at 90 min (data omitted for clarity).

Changes in lactate and glucose concentrations over time.

What does sodium fluoride do to glucose?

Evidently sodium fluoride takes effect slowly but effectively in preserving glucose in blood for at least three days. Its use, however, is unnecessary if the concentration of glucose is to be measured within the first hour after sampling.

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What enzyme does sodium fluoride inhibit?

This is not a new discovery. Seventy years ago, Ernest Bueding and Walter Goldfarb 4 reported that NaF did not prevent a decrease in glucose concentrations when blood was assayed four hours after collection, having been kept at 208C. Glucose concentrations fell by 2–12% in samples preserved with 1% NaF. Only samples containing iodoacetate, in addition to NaF, were found to have minimal loss of glucose ( 5 The patent application described why NaF alone failed to inhibit glycolysis for the first few hours. NaF inhibits enolase, an enzyme acting late in the glycolytic pathway, and has no effect on enzymes that act early in the glycolytic pathway. In contrast, acidification of blood below a pH of 6. 0 stops glycolysis immediately.

In 1988, Uchida et al. 6 published a full description of the experimental studies upon which the patent was based. Acidification inhibits hexokinase and phosphofructokinase, enzymes that act early in the Embden–Meyerhof pathway. Glycolysis is instantly inhibited in erythrocytes, leukocytes and platelets when the blood pH is maintained between 5. 3 and 5. 9 with a citrate buffer.

In 1989, Chan et al. 7 confirmed the 1941 findings of Bueding and Goldfarb and recommended that physicians not depend on NaF to inhibit glycolysis. Chan’s well-designed study had no impact, at the time, on manufacturing practices or the recommendations of organizations such as the American Diabetes Association (ADA).

How does NaF inhibit fermentation?

For instance, it has been demonstrated that NaF inhibits the activity of enzymes involved in the glycolysis pathway, such as pyruvate kinase and enolase, in yeast fermentation. The generation of ATP and ethanol, the main products of yeast fermentation, may be reduced as a result of this.