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Clinical Chemistry 52: 453-457, 2006. First published January 19, 2006; 10.1373/clinchem.2005.063412
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2006;52:453-457.)
© 2006 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Semiautomated Method for Determination of Serum Paraoxonase Activity Using Paraoxon as Substrate

Valentine Charlton-Menysa, Yifen Liu and Paul N. Durrington

1 Division of Cardiovascular and Endocrine Sciences, Department of Medicine, Manchester Royal Infirmary, Manchester, United Kingdom.

aAddress correspondence to this author at: Division of Cardiovascular and Endocrine Sciences, Department of Medicine, Manchester Royal Infirmary, Manchester M13 WL, United Kingdom. Fax 44-0-161-272-6410; e-mail valentine.menys{at}cmmc.nhs.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Discussion
 References
 
Background: Serum paraoxonase (PON1) is an enzyme associated with HDL, and its ability to protect LDL from oxidation is one mechanism by which HDL protects against atherosclerosis. Low concentrations of PON1 are found in patients with type 2 diabetes or coronary heart disease. Serum PON1 activity may also be important in avoidance of organophosphate toxicity in industry.

Methods: The generally accepted method for determining PON1 activity requires use of a recording spectrophotometer and is not suited to large numbers of samples; in addition, automation presents particular problems because of the extreme toxicity of substrates such as paraoxon. We established a relatively safe microtiter plate method that facilitates the determination of PON1 activity at a rate of 120 samples per hour.

Results: PON1 activity was determined by the generally accepted method (x) and the new method (y); results correlated with a slope close to unity (y = 0.93x + 8; r = 0.97; P <0.0001; n = 101). Examination of differences by Bland–Altman plots showed a weak concentration-dependent difference (r = 0.33; P <0.0001; n = 101). The intra- and interassay sample CVs, obtained with samples with PON1 activities ranging from 41 to 348 nmol · min–1 · mL–1, were 3.5% and 2.7%, respectively (n = 16).

Conclusion:The proposed method for determination of PON1 activity is simple, relatively safe, and inexpensive and is suitable for analysis of large numbers of samples.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Discussion
 References
 
Serum paraoxonase (PON1) is a calcium-dependent esterase associated exclusively with HDL (1). Oxidative modification of LDL plays a central role in atherogenesis (2), and PON1 directly protects LDL from oxidative modification (3). Furthermore, measurement of serum PON1 activity may be important for minimizing the exposure of susceptible individuals in, for example, the agricultural industry to organophosphates (4). Organophosphates are still the most widely used insecticides throughout the world, although their use is decreasing in most prosperous countries. Serum PON1 may also be important for assessing the severity of organophosphate poisoning (5)(6). Although relatively nontoxic substrates, such as phenylacetate, can be used to determine PON1 activity, the hydrolysis of paraoxon seems most closely related to the inverse relationship with coronary heart disease (7). In addition, different forms of the enzyme have different substrate specificities toward organophosphates, which they do not exhibit toward phenylacetate. Thus, any industrial application of the PON1 activity assay will require the use of an organophosphate. The use of an organophosphate substrate such as paraoxon, however, presents particular problems for automation, for example, in conventional analyzers such as the Cobas Mira (8). Removal of traces of the toxic substrate from the equipment requires the use of high concentrations of NaOH, which could damage the equipment and would make it difficult to perform assays for other analytes on the same instrument.

The most widely used method for determining PON1 activity in serum is the method using a continuous recording spectrophotometer (9)(10)(11)(12)(13)(14). An automated method for serum PON1 has also been developed (8)(15)(16), as have semiautomated microtiter plate methods (17)(18). However, no direct comparison of the automated methods with the widely used spectrophotometric method has been performed. Although Richter and Furlong (17) used both the standard spectrophotometric method and the microtiter plate method to measure PON1 activity, they did not report a direct comparison of the results obtained with the 2 methods, and the distribution of results in their report indicates that there were differences.

When an organophosphate substrate is added to serum, for example, there is an early burst of hydrolytic activity that is not the result of PON1 activity but of type B esterase activities, which are subsequently blocked by irreversible substrate binding but which, if included in the activity measurement, do not represent PON1 activity. With the growing interest in PON1 in cardiovascular disease and recognizing the need to assay PON1 activity in large numbers of samples, we felt it was timely to reassess the microtiter plate method and to compare the results directly with those obtained with the recording spectophotometric method.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Discussion
 References
 
We obtained flat-well (0.4 mL) polystyrene NUNC microtiter platelets from Scientific Lab Supplies Ltd., and semimicro acrylic cuvettes from Sarstedt Ltd. Paraoxon (O,O-diethyl-O-p-nitrophenylphosphate), 4-nitrophenol (p-nitrophenol), and other reagents were from Sigma Chemical Co. A 2 mol/L solution of NaOH was prepared fresh each day to inactivate paraoxon. We used serum samples that had been obtained for a variety of studies and had been stored at –20 °C; we thawed these samples at ambient temperature. The HDL used was isolated from fresh serum by preparative ultracentrifugation at a density range of 1.063–1.21 kg/L. Paraoxon substrate (3.3 mmol/L) was prepared fresh each day by addition of paraoxon to a buffer containing 2 mmol/L CaCl2 and 100 mmol/L Tris, pH 8.0. A solution of 4-nitrophenol in buffer was used to determine the molar absorptivity of the nitrophenol produced by the hydrolysis of paraoxon; the molar absorptivity at 405 nm (pH 8.0) was 17 600. The molar absorptivity of the paraoxon substrate solution was 331 at 405 nm (pH 8.0) and did not change over 1 h at ambient temperature, indicating minimal spontaneous hydrolysis of paraoxon.

determination of pon1 activity
The pH used to assess PON1 activity is very important because other paraoxon hydrolytic activities occur at pH >8.5. This was clearly demonstrated in the study by Furlong et al. (9), which showed that esterase activity associated with albumin causes hydrolysis of paraoxon at pH >8.5. Accordingly, we assayed PON1 activity at pH 8.0. For the standard recording spectrophotometric method (Beckman DU68 spectrophotometer), 0.5 mL of paraoxon substrate was added to 4 cuvettes, followed by 25 µL of serum, with mixing, before recalibration of the spectrophotometer (within 0.42 min) (10). The absorbance at 405 nm was recorded over a period of 3 min from recalibration. For the manual microtiter plate method, 10-µL serum samples were pipetted into the wells, the paraoxon substrate (0.2 mL) was added sequentially to single rows (n = 8) by use of a multitip pipettor, and the absorbance at 405 nm was measured immediately and at 0.42, 3, and 3.42 min. Rates of hydrolysis of paraoxon were calculated, and serum PON1 activity was expressed as follows: for the DU68 spectrophotometer, absorbance (A)/min x 1193 = nmol · min–1 · mL–1; and for the Multiskan microtiter plate reader, A/min x 1193 x 1.724 nmol · min–1 · mL–1. The pathlength correction factor was calculated as the ratio of the results obtained with the microtiter plate method to the values obtained with the DU68 spectrophotometer, the latter being obtained with a pathlength of 1 cm. The mean temperature at which PON1 activities were determined was the same for both methods, 25 °C (range, 24.5–25.5 °C). Serum pools with low and high PON1 activity were prepared, and aliquots were stored at –20 °C. PON1 activity was determined in aliquots from each pool on each day, and the results were used to correct for interassay changes caused by alterations in temperature, although these changes were minimal.

semiautomated microtiter plate method
The procedure for the microtiter plate method was as follows: We set the Multiskan Instrument Parameter to step mode. For measurement mode, we selected the following: kinetic mode; filter, 405 nm; lag time, 0.42 min; interval time, 0.5 min; total time, 3 min; no-reagent blank; and end results. We added 10 µL of the sample to microwells (columns 1, 3, 5, 7, 9, and 11; 8 wells/column). The microtiter plate was then loaded on the Multiskan reader. With a multitip pipettor, we added 0.2 mL of paraoxon substrate to all 8 wells in column 1 and started the assay. At 4-s intervals, we added paraoxon substrate to columns 3, 5, 7, 9, and 11. Results were printed out in sequence with A/min calculated. Columns 2, 4, 6, 8, and 10 could then be used for a second series of assays with the same plate. The contaminated tips and used plates were decontaminated in 2 mol/L NaOH.

method comparison
For the method comparison we prepared 2-fold dilutions of a standardized preparation of HDL that had been isolated by preparative ultracentrifugation of serum and used those dilutions to measure PON1 activity by both the DU68 and manual plate methods. The agreement between the 2 methods was very good, with slopes near unity and parallel dilution curves (Fig. 1A ). We also used the generally accepted DU68 method and the manual microtiter plate method to determine PON1 activity in 101 serum samples, calculating the rates by subtracting the reading at 0.42 min for each sample from the reading at 3.42 min. The correlation between the results for PON1 activity in the serum samples was very high (Fig. 1B ). For the manual microtiter plate method, we also calculated the rates by subtracting the reading taken immediately after substrate addition from the reading at 3 min, and the results agreed closely with the results for the plate method calculated from the change in absorbance between 0.42 and 3.42 min: y = 0.92x + 11 (r = 0.96) vs y = 0.93x + 8 (r = 0.97; P <0.001 for both). Regarding rates of change in absorbance, the initial rate (0 to 0.42 min) was higher (0.085 absorbance units/min) than the rate calculated from 0 to 3 min (0.060 absorbance units/min; P <0.0001), and the rate calculated from 0.42 to 3.42 min, as in the established method, was lower still (0.048 absorbance units/min; P <0.001 vs 0 to 3 min). A Bland–Altman difference plot showed a weak but significant PON1 activity-dependent difference between results for the 2 methods (Fig. 2A ) that was not significant if PON1 activities >300 nmol · min–1 · mL–1 were excluded. The intraassay sample CV was 3.5% (n = 16), and the interassay sample CV was 2.7% (n = 16).


Figure 1
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  		Figure 1. Relationship between HDL protein concentration and PON1 activity as determined by the established spectrophotometric method ({circ}) and the new microtiter plate method (•; A), and serum PON1 activities as determined by the spectrophotometric method and the new microtiter plate method (B).

(A), equations for the lines: for the spectrophotometric method ({circ}), y = 162x – 9.6 nmol · min–1 · mL–1 (r = 0.997; n = 16); for the microtiter plate method (•), y = 163x – 6.3 (r = 0.999; n = 16). (B), equation for the line: y = 0.93x + 8 nmol · min–1 · mL–1 (r = 0.97; P <0.0001; n = 101).


Figure 2
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  		Figure 2. Bland–Altman difference plot for PON1 activity determined by the DU68 spectrophotometric method and the new microtiter plate method (A), and PON1 activities as determined by the DU68 method and a semiautomated kinetic microtiter plate method (B).

We next determined PON1 activity in 50 serum samples by the DU68 method and the semiautomated plate method and calculated the rates with correction to 0.42 min. The agreement between results was very good (Fig. 2BUp ). On average, PON1 activities obtained with the DU68 method were ~11% higher than those obtained with the semi-automated microtiter plate method [104 (22–433) vs 94 (14–410) nmol · min–1 · mL–1; P <0.001], but we could use the equation derived from the Bland–Altman plot (Fig. 2AUp ) to make corrections.

calcium dependence of pon1 activity
Using the plate method proposed in this study, we assessed the calcium dependence of PON1 activity. After adding disodium EDTA (final concentration, 3 mmol/L) to paraoxon substrate, we measured PON1 activity in 8 serum samples; PON1 activities ranged from 23 to 223 nmol · min–1 · mL–1 (the activity measured in the absence of EDTA). Addition of EDTA to the substrate thus effectively abolished PON1 activity. Addition of up to 2 mmol/L disodium EDTA to the serum samples (final EDTA concentration in the reaction mixture, 0.01 mmol/L) before addition of the paraoxon substrate (which contained 2 mmol/L calcium) had little effect, but addition of 4 mmol/L EDTA to the serum samples (final EDTA concentration in reaction mixture, 0.01 mmol/L) before addition of the substrate led to a 60% decrease in PON1 activity. Addition of EDTA to final concentrations up to 0.02 mmol/L did not further inhibit the PON1 activity. This experiment showed that despite the great excess of calcium in the substrate buffer (2 mmol/L), addition of the paraoxon substrate to serum samples already containing 4 mmol/L EDTA could not overcome the ~60% inhibition of total PON1 activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Discussion
References
 
We measured serum PON1 activity by manual and semiautomated methods, and the results agreed closely with those obtained by the recording spectrophotometric method. Regarding the rate of paraoxon hydrolysis, we found that the calculated rate decreased when rates were determined between different time points. The calculated rate decreased in the order 0–0.42 min > 0–3 min > 0.42–3.42 min. This has significant impact on the calculated PON1 activity, such that calculations based on the above time points yielded PON1 activities of 164 (34–480) nmol · min–1 · mL–1 vs 101 (19–357) nmol · min–1 · mL–1 (P <0.001) vs 74 (13–290) nmol · min–1 · mL–1 (P <0.001; n = 54). Thus, it is essential that corrections are made for readings at an early time point—up to 0.5 min but not at zero time—before calculation of the rate of paraoxon hydrolysis. In studies using Cobas (Roche Diagnostics) automated analyzers, readings were taken at 10 and 90 s (8)(15), and this may have led to overestimation of PON1 activity. We found that the semiautomated method gave higher results than the DU68 method, but this was significant only at higher PON1 activities. This difference might be explained by more effective mixing of sample with substrate in the DU68 method. Mixing in the semiautomated method occurs by dilution of the serum sample with a 20-fold larger volume of substrate, whereas in the DU68 method, serum is added to substrate by physical mixing.

The calcium dependence of the paraoxon hydrolytic activity of PON1 is well known (19). Our results show that recalcification of serum to which sufficient EDTA was added to abolish PON1 activity can restore only ~40% of the paraoxonase activity. The recently reported structure of PON1 (20) suggests the location of the 2 calcium ions associated with each molecule. One is close to the surface of the molecule, but the other is deeply situated at the bottom of the cleft in which the active site is located. The practical implication is that PON1 activity, certainly with respect to organophosphate hydrolysis, must be measured in serum and not EDTA plasma with the currently reported methods or with earlier ones.

We further validated the microtiter plate method by assaying PON1 activity in dilutions of HDL protein isolated at a density of 1.063–1.21 kg/L. Both the spectrophotometric and microtiter plate methods gave excellent correlations between PON1 activity and HDL protein concentration, and the slopes, which approximated unity, were directly superimposable. These results also confirm that PON1 activity, as determined by both methods, is associated with HDL protein.

In conclusion, the semiautomated method is suitable for assaying PON1 activity in large numbers of serum samples. Microtiter plates and pipette tips are inexpensive, and paraoxon contamination may be readily removed by immersion of plates and pipette tips in 2 mol/L NaOH. Microtiter plate readers are also considerably less expensive than automated analyzers such as the Cobas Mira.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Discussion
 References
 

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