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I have some samples of whole blood that are a little bit expensive and I want to significantly reduce the concentration of PPi in the samples by causing a reaction. I don't have any experience in practical chemistry, but I understand that in theory, there are enzymes that can hydrolyze pyrophosphate into orthophosphate (e.g. inorganic pyrophosphatase).

I see that I can purchase inorganic pyrophosphatase suspended in 3.2 M ammonium sulfate solution, (pH approximately 6), from yeast, on Sigma-Aldrich and in theory converts 1 micromol pyrophosphate to orthophosphate per minute at room temperature (in the presence of Mg-ions). For my purpose, the reduction in pH is not a problem. I am more concerned about whether PPi concentration would actually go down, or if I could expect cell lysis or other effects to occur, causing PPi to rise.

Can I use this reagent to reduce the measurable PPi concentrations in the blood samples? And if so, how would I do it?

EDIT: I am doing an experiment with bacterial growth in plasma, and comparing with serum deficient in pyrophosphate. I want to have a rough idea of the appropriate way to hydrolyse the PPi.

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  • $\begingroup$ I guess it might be helpful to know the reason you are removing pyrophosphate? Like this paper suggests, pyrophosphate activity might be inhibited by compounds present in blood, and it would help if you tested on a different sample while monitoring the pyrophosphate level as they do in this paper $\endgroup$ May 16, 2022 at 5:10
  • $\begingroup$ @PrashantBharadwaj I added the information that you requested. $\endgroup$
    – Mikkel Rev
    May 16, 2022 at 18:56
  • $\begingroup$ @MikkelRev, you mention whole blood, plasma, and serum. Which of these are you ultimately using as your bacterial growth medium? This will affect the steps of your protocol. $\endgroup$
    – acvill
    May 17, 2022 at 20:47

1 Answer 1

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Bernhard et al. 1 describe a method for the treatment of human plasma with yeast inorganic pyrophosphatase. The PPase-treated plasma is used as both a diluent and a negative control in their PPi conversion assay. The use of a salt solution as a secondary negative control confirms the depletion of PPi from PPase-treated plasma samples. I've quoted their protocol and validation steps below, but here is my summary:

  1. Centrifuge whole blood and transfer supernatant. This serves to remove most of the cells from the plasma fraction.
  2. Filter plasma with 300 kDa filter. This will remove any remaining cells so the amount of PPi contributed by any cell lysis is negligible.
  3. Add PPase and mix by inversion. The authors do not supplement the plasma with Mg2+ nor any buffers.
  4. Incubate for 30 minutes at room temperature.
  5. Filter plasma with 30 kDa filter. The recombinant PPase from NEB has a mass of 71 kDa 2, so this step likely serves to remove PPase from the plasma, which would confound the use of PPase-treated plasma as a diluent for PPi measurement.
  6. Aliquot and freeze PPase-treated plasma.

Generation of Pyrophosphatase-treated Plasma

For human plasma, blood drawn from donors (everything at room temperature) was spun down at 3500 rpm for 15 min, and plasma was then transferred to a 15-mL tube. Then, 2 mL of plasma was filtered through a Centrisart® I 300 kDa mass cutoff filter at 2500 × g for 30 min. The filtered plasma was transferred to a fresh 1.5 mL Eppendorf AG tube. Yeast inorganic pyrophosphatase (PPase; New England Biolabs Inc) was added at 1:1000 plasma, mixed well by inverting 4 times, and incubated for 30 min at room temperature. Five hundred microliters of PPase-treated plasma was filtered through a Nanosep Centrifugal Device With Omega Membrane 30K (Pall Corp) at 13,200 × g for 5 to 10 min. The PPase-treated, filtered plasma was frozen at −80°C in 500 µL aliquots. A new stock was made every 2 months.

Assay Description and Validation

The assay utilized ATP sulfurylase to convert PPi to ATP, which was then detected by a luciferase/luciferin luminescence detection kit. The luminescent signal is directly proportional to the amount of ATP present; the assay is a semiquantitative assay (regulation 493.1256(d)(iii) of Clinical Laboratory Improvement Amendments of 1988). Target analyte of assay validation was PPi. The specimens were human platelet-free plasma samples. The low limit of detection was 0.15 µM; the quantitative range was 0.15 to 10 µM. Normal ranges, determined by testing PPi levels of 50 healthy adult volunteers (50% male), were 1.43 to 7.40 µM (unpublished data). For validation and clinical testing, a standard curve was generated for each run by titrating measured amounts of PPi in PPase-treated pooled human plasma. The PPi standard curve was prepared by diluting powder PPi in water; thus, concentration was equal to 1 mM PPi final. One millimolar of PPi was diluted 1:100 in PPase-treated donor plasma and mixed well. Defined concentrations were achieved by serial 1:2 dilutions in PPase-treated donor plasma. A negative control of Hank’s balanced salt solution alone and PPase-treated pooled human plasma were included on each assay plate. Each concentration was run in triplicate.


References

  1. Bernhard E, Nitschke Y, Khursigara G, Sabbagh Y, Wang Y, Rutsch F. A Reference Range for Plasma Levels of Inorganic Pyrophosphate in Children Using the ATP Sulfurylase Method. J Clin Endocrinol Metab. 2022 Jan 1;107(1):109-118.
  2. https://www.neb.com/products/m2403-pyrophosphatase-inorganic-yeast
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