Screening for pancreatic lipase inhibitors: evaluating assay conditions using p-nitrophenyl palmitate as substrate

Figures & data

Figure 1. Assay mixture with the addition of sodium deoxycholate (SDC) and Triton X-100 (TX-100).

C: sample without emulsifier; M3-M8: samples with 3-8 mM SDC; T0.1, T0.2, T0.5, T1 and T2: samples with 0.1, 0.2, 0.5, 1.0 and 2.0% TX-100, respectively. Assay conditions: 200 μM pNPP, 10% IPA, 50 mM NaP (pH 8) with and without emulsifier, 1 mg/mL lipase, 25°C. Pictures were taken 30 min after mixing.

Figure 2. Effect of emulsifiers on the rate of hydrolysis.

M5: sample with 5 mM SDC, T2 and T0.5: samples with 2.0% and 0.5% TX-100, Control: sample without emulsifier. Assay conditions: 200 μM pNPP, 10% IPA, 50 mM NaP (pH 8), emulsifier with corresponding concentrations, 1 mg/mL lipase, 25°C. Results were recorded by continuous measurement in 5 min, every 30s. Highest reaction rate was set as 100%. The bars indicate the means ± SD of three independent experiments. ****p < 0.0001 vs. Control (one-way ANOVA followed by Tukey's HSD Post Hoc Test).

Figure 3. Effect of incubation time at 37°C on the rate of hydrolysis.

Assay conditions: 200 μM pNPP, 10% IPA, 50 mM NaP (pH 8), 5 mM SDC, 1.0 mg/mL lipase, 37°C. Results were recorded by continuous measurement in 5 min, every 30s. Rate of reaction without incubation at 37°C was set as 100%. The bars indicate the means ± SD of three independent experiments. ****p < 0.0001 vs. sample without incubation/0 min (one-way ANOVA followed by Tukey's HSD Post Hoc Test).

Figure 4. Relative hydrolysis rates in different buffers and pH.

Assay conditions: 200 μM pNPP, 10% IPA, 50 mM corresponding buffer, 5 mM SDC, 1.0 mg/mL lipase, 25°C. Results were recorded by continuous measurement in 5 min, every 30s. Highest reaction rate was set as 100%. Each data point represents the mean ± SD of three independent experiments. ****p < 0.0001 vs. other samples (one-way ANOVA followed by Tukey's HSD Post Hoc Test).

Figure 5. Effect of organic co-solvent on hydrolysis rates.

Assay conditions: 200 μM pNPP, 10% IPA, 50 mM NaP (pH 8), 5 mM SDC, 1.0 mg/mL lipase, organic solvent at evaluated concentration, 25°C. Results were recorded by continuous measurement in 5 min, every 30s. Rate of control sample or sample without co-solvent was set as 100%. Each data point represents the mean ± SD of three independent experiments.

Figure 6. Effects of NaCl (a) or CaCl₂ (b) on hydrolysis rates.

Assay conditions: 200 μM pNPP, 10% IPA, 50 mM NaP (pH 8), 5 mM SDC, 1.0 mg/mL lipase, NaCl or CaCl2 at evaluated concentration, 25°C, results recorded by continuous measurement in 5 min, every 30s. Sample without NaCl or CaCl2, same volume of buffer was used instead. Rate without the two salts was set as 100%. Each data point represents the mean ± SD of three independent experiments.

Figure 7. Relative hydrolysis rates of lipase solutions stored at –20°C with the addition of different amounts of glycerol as antifreeze.

Assay conditions: 200 μM pNPP, 10% IPA, 50 mM NaP (pH 8), 5 mM SDC, 1.0 mg/mL lipase in buffer with glycerol, 25°C, results recorded by continuous measurement in 5 min, every 30s. Rate without glycerol in day 0 is set 100%. Each data point represents the mean ± SD of three independent experiments.

Table 1. Experimental conditions for in vitro lipase inhibitory assay.

Experimental parameter	Proposed value
Substrate	200 µM p-nitrophenyl palmitate
Buffer (pH)	50 mM sodium phosphate pH 8.0 or 50 mM Tris·HCl pH 9.0
Temperature	room temperature (25°C)
Wavelength	410–415 nm
Emulsifier	5 mM sodium deoxycholate
Ionic salt	1 mM calcium chloride; 5 mM sodium chloride
Co-solvent to dissolve inhibitor	DMSO, MeOH (up to 30% v/v); EtOH (up to 20% v/v) IPA and AcCN (up to 10% v/v)
Enzyme storage condition	Enzyme solution in buffer supplemented with 10% (v/v) glycerol –20°C (up to 1 month)

Table 2. IC₅₀ values determined by proposed condition sets.

Condition set	IC50 (ng/mL)	R^2
NaP-10	12.40 ± 2.15	0.9927
NaP-20	9.32 ± 1.51	0.9929
Tris·HCl-20	12.80 ± 2.24	0.9926

Data availability statement

The data that support the findings of this study are available in figshare [https://figshare.com/] at http://doi.org/10.6084/m9.figshare.16864072.v1.