Table 4.
Characteristics of currently available enzymatic assays to quantify PPi in plasma.
| Silcox et al. 1973[42] | Cook et al. 1978[28] | MgGuire et al. 1980[30] | Current study[32] | |
|---|---|---|---|---|
| enzymology | 1. Yeast inorganic pyrophosphatase | 1. Pyrophosphate-fructose-6-phosphate 1-phosphotransferase 2. Fructose-bisphosphate aldolase 3. Triosephosphate isomerase 4. Glycerol-3-phosphate dehydrogenase |
1. UDP-glucose pyrophosphorylase 2. Phosphoglucomutase 3. Glucose-6-phosphate dehydrogenase |
1. ATP-sulfurylase 2. Firefly luciferase |
| Sample preparation | Protein precipitation and removal of inorganic phosphate | Perchloric acid extraction with subsequent pH adjustment to 5–6 with potassium hydroxide | Platelet removal by filtration1) | Platelet removal by filtration1) |
| Radionuclides required? | Yes, [32P]PPi | no | Yes, [14C] UDP-glucose or [3H] UDP-glucose2) | no |
| Detection | Colorimetric detection of inorganic phosphate | NADPH generation/fluorescence | Liquid scintillation counting (LSC) | Bioluminescence |
| Sample volume 3) | 5 ml | 2 ml | 80 μl4) | Max. 10 μl |
| Time/sample | >1 hour (30 samples/week) | ~5 minutes + time needed for protein removal and pH adjustment | ~ 1 hour + time needed for LSC | ~5 minutes |
| Sensitivity | ND | ~ 250 pmol5) | 10 pmol | < 50 fmol6) |
1)
Platelets are a source of contaminating PPi and ATP and their removal provides more accurate estimations plasma PPi concentrations [32].
2)
The same enzymes can also be used to quantify PPi in plasma in an assay that follows NADPH reduction fluorometrically. The fluorometric variant of this assay has a reported sensitivity of ~ 50 pmol [24, 29, 30].
3)
Plasma volume needed to complete the assay.
4)
Includes an inorganic pyrophosphatase treated control.
5)
Estimation based on figure 2 of Cook et al. [28].