Chemical Reactions as a Cause of Bilirubin Interference

Một phần của tài liệu Endogenous Interferences in Clinical Laboratory Tests (Trang 41 - 45)

The previous mechanisms of interference caused by bilirubin described above have all depended on the physical chemical properties of bilirubin or its breakdown product, biliverdin . The mechanism dealt with light absorption or elution in chromatography.

In addition to its physical chemical properties, bilirubin may cause interference by means of its chemical reaction with reagents involved in the methods for determina- tion of analytes.

3.4 Chemical Reactions as a Cause of Bilirubin Interference       29

3.4.1 Bilirubin Reaction with Creatinine Methods

One well-studied interference is bilirubin’s effect on creatinine determinations. Cre- atinine, a breakdown product of creatine, is an important analyte for the assess- ment of renal function. It is widely used and one of the most frequently ordered tests among clinical chemistry test panels. Creatinine measurement, as determined by the Jaffe reaction (alkaline picrate), suffers from interferences from many different com- pounds, especially those with strong ketone groups or cepha rings [13].

Before the kinetic method for the determination of creatinine was instituted, the protein of serum was separated from the filtrate and the creatinine was determined as an endpoint procedure. The effects of bilirubin on creatinine determinations by the kinetic method have always been mixed, with some methods not demonstrating an interference , while others showing a strong negative interference [14].

Bilirubin is a very reactive compound with a relatively strong absorbance near the wavelengths used in the Jaffe reaction [14]. In the kinetic method, an absorbance reading is taken immediately after mixing of the sample with picric acid and NaOH, followed by at least one more absorbance reading taken sometime later in the reac- tion. The advantage of the kinetic method is that the first absorbance reading can act as a blank for the reaction and one does not need to wait until an endpoint is reached to calculate the creatinine concentration. If bilirubin interfered simply by its absorb- ance alone, the first kinetic reading would subtract out this absorbance; further, an early absorbance reading would not cause a negative interference , thus there must be some reaction occurring with bilirubin that would give rise to a negative interfer- ence  [14].

In the alkaline picrate method for the determination of creatinine, NaOH mixed with picric acid makes a strongly alkaline solution. The actual chromogen in the reac- tion is the picrate, which when it reacts with creatinine, shifts the picrate absorbance band approximately 10 nm, which is sufficient to measure an absorbance change [15]. The concentration of creatinine is determined by the change in absorbance near 500 nm and this change in absorbance is a positive number. In an alkaline medium, bilirubin can be oxidized to form biliverdin, which demonstrates a major decrease in the change in absorbance as measured at 510 nm (biliverdin absorbs more light at 630 nm than does bilirubin, which absorbs more at 510 nm) [14]. In the initial part of the reaction, bilirubin absorbs light near 500 nm, the wavelength for the assay for creatinine. As the reaction proceeds in time, creatinine reacts with picrate and the absorbance for that species increases, but bilirubin is converted to biliverdin and that absorbance decreases. The net change in absorbance decreases and a depression in the value for creatinine is reported.

The effect of bilirubin depends on its form. Bilirubin can be either conjugated or unconjugated. The conjugated bilirubin has glucuronide sugars attached to it, which make it soluble in water and it is typically measured as direct bilirubin. Unconjugated bilirubin is not soluble in water and is attached to albumin in the plasma. Unconju-

gated bilirubin is typically measured as the difference between total bilirubin and direct bilirubin. Unconjugated bilirubin is liable to oxidation to biliverdin in alkaline solutions and gives rise to a negative interference for creatinine. Conjugated bilirubin is less likely to oxidize to biliverdin, and thereby less likely to cause a negative inter- ference [16]. The choice of method also has an effect. If one were to dialyze or ultrafil- trate the sample first, and perform the reaction on the filtrate (as done with some older methods), then there would not be any unconjugated bilirubin present in the filtrate and therefore no negative interference [17, 18]. The concentration of NaOH, temperature and timing kinetics, i.e., the time between the first and last absorbance readings will affect the bilirubin interference as well, and is a source for the varied results seen with this analyte; a greater decrease is seen with increasing concentrations of NaOH [16].

One would think that a simple solution to the bilirubin interference with the Jaffe method for creatinine would be to replace the Jaffe method with an enzymatic method for the determination; however, the enzymatic method for creatinine has demon- strated negative interference for creatinine [19].

There are three major reactions for the determination of creatinine utilizing an enzymatic reaction [4]. The first two methods use creatininase .

(3.4)

This method has not been widely accepted.

The second method to use creatininase produces hydrogen peroxidase . Its reactions are as follows:

(3.5)

This method is fairly popular as is the next one, based on the use of creatinine imi- nohydrolase:

(3.6) Creatinine+H2O→Creatine

Creatine+ATP →Creatine phosphate+ADP ADP+Phosphoenolpyruvate →Pyruvate+ATP Pyruvate+NADH+H+→Lactate+NAD+

Creatinine+H2O →Creatine Creatine+H2O→Sarcosine+Urea

Sarcosine+O2+H2O→Formaldehyde+Glycine+H2O2

Phenolderivative+4-Aminoantipyrine+H2O2→H2O+Colored product

Creatinine+H2O→N-Methylhydantoin

N-Methylhydantoin+ATP+H2O→N-Carbamoylsarcosine+ADP+Phosphate N-Carbamoylsarcosine+H2O→NH3+CO2+Sarcosine

Sarcosine+O2+H2O→Formaldehyde+Glycine+H2O2

Aniline dye+4-Aminoantipyrine+H2O2→H2O+Colored product

3.4 Chemical Reactions as a Cause of Bilirubin Interference       31

These two methods using hydrogen peroxide and peroxidase as the detection system experience a negative bilirubin interference [20]. The magnitude depends on both the concentrations of creatinine and of bilirubin. The absorbance spectrum of biliru- bin, with a peak near 460 nm, overlaps the absorbance band of the colored product (Trinder chromophore ) [20]. In addition, it has been speculated that bilirubin may react with one of the peroxidase reaction intermediates, decreasing the concentration of chromophore produced and its net absorbance [20].

3.4.2 Bilirubin Reactions with Peroxidase Methods

Other evidence suggests that bilirubin may cause an interference with peroxidase methods by five possible mechanisms: as a substrate for peroxidase; absorbance at the same wavelengths as the product; intercepting and removing an oxidase interme- diate; reacting with a peroxidase intermediate, such as hydrogen peroxide; bleach- ing of the final reaction color [21]. Bilirubin does not appear to have an high enough Km to displace creatinine from peroxidase. The Trinder chromophore does appear to be stable in the presence of bilirubin. Bilirubin does absorb at 505 nm, the absorp- tion band for the Trinder chromophore, but in this case, it could potentially provide a positive interference , but even more important, the coupled enzyme reaction follows a kinetic reaction and proper blanking of the reaction would eliminate this interfer- ence . It is most likely that bilirubin reacts with the hydrogen peroxide. Hydrogen peroxide is a strong oxidizer and bilirubin is readily oxidized to biliverdin, bilirubin acting as a strong anti-oxidant. Bilirubin could then remove hydrogen peroxide from the detection system. Control of the bilirubin interference often uses bilirubin oxidase or potassium ferricyanide.

Other common chemistry methods utilize the Trinder chromophore, or a hydro- gen peroxide intermediate, in their detection system. Lipase acts on triglycerides to produce fatty acids and glycerol; in turn, glycerol oxidase acts on glycerol to produce dihydroxyacetone and hydrogen peroxide [22]. Uricase acts on uric acid to produce allantoin and hydrogen peroxide [4]. Cholesterol oxidase acts on cholesterol to produce hydrogen peroxide as well [23]. These three methods share the production of hydrogen peroxide as an intermediate species with detection of hydrogen peroxide through peroxidase-coupled assays as a common step with the enzymatic determina- tion of creatinine [23]. Bilirubin interferes with these methods by means of the same mechanisms as described for creatinine. The sensitivity of commercial methods utiliz- ing peroxidase-coupled assays varies depending on the choice of wavelength, substi- tution of an aniline rather than phenol derivative, the presence of ferrocyanide in the reagent mix, and other reaction conditions [23]. Some assays are highly sensitive to bilirubin, the interference becoming significant at concentrations below 43 μmol/L, ranging up to high concentrations of bilirubin, e.g., 684 μmol/L [23]. The interfer- ences are mixed as well, some showing negative interference and others showing

positive interference , again suggesting that more than one mechanism of interference may be operating at a time [23].

Not only endogenous substances, but also exogenous analytes suffer from biliru- bin interference . One method for measuring salicylate is to use the Trinder method, in which ferric ions (Fe3+) react with salicylate to form a purple color which can be meas- ured at 540 nm. There is a positive interference related to an increase in absorbance at the chosen wavelength with bilirubin and is caused by the direct reaction of the ferric ions with bilirubin [24]. Use of dual wavelength blanking at 700, 750 and 800 nm did not have an effect on the interference [24].

Một phần của tài liệu Endogenous Interferences in Clinical Laboratory Tests (Trang 41 - 45)

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