Quantitation of Routine Chemistry Analytes | Blood & Stool

Chemical Analysis

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Photometric Measurements

Light may be absorbed by a substance dissolved in solution (absorbance), or may be scattered or reflected by particles suspended in solution (turbidimetry). Specific wavelengths of light are chosen for each analysis based on the properties of the substance being measured. A typical light source generates a broad range of wavelengths of light. Our lamp produces light of wavelengths from 405 nm (violet light) to 620 nm (green light). A device called a monochromator or filters are used to select the desired wavelength from the spectrum of light produced by the light source. A monochromator disperses the light much like a prism disperses light, and allows selection of a narrow band of wavelengths to be directed through the cuvette containing the sample. If an analyte has an intrinsic color or generates a color upon chemical reaction, visible light is absorbed when it passes through a solution containing the analyte or reaction products. The selective absorbance of certain wavelengths of light from the spectrum of white light gives the solution its color. Compounds that have no visible color often absorb light in the ultraviolet region and this absorbance can be used in the same way as absorbance of visible light. The specific wavelength of light chosen is based on the absorption properties of the compound being measured. As the amount of a substance in solution increases, the relative amount of light that passes through the solution and reaches the detector decreases. The decrease in light is termed absorbance.

Determining the Concentrations of Light Absorbing Substances in Solution

Absorption Photometry

For a given method, the coefficient of absorbance and the length of the cell are constants, so the change in absorbance is directly proportional to the concentration. The figure above shows the difference between the emitted light (lO) and the light that reaches the photodetector (lS). Beer's Law describes the relationship between concentration and absorbed light.

Endpoint Reactions

When an analyte is detected using a chemical reaction, there are two options for assessing its concentration. One is to wait until the reaction is complete and the total amount of analyte is converted to product — called an endpoint reaction. Endpoint reactions are especially suitable for chemical reactions which are completed in a relatively short time and are "stoichiometric" meaning that they produce one product molecule or complex for each molecule of analyte. For example, a reaction of albumin with the dye bromocresol purple (BCP) produces a colored complex. If the reaction is allowed to continue until all the albumin present in solution has reacted and the maximum amount of colored product has formed, the color at the end of the reaction reflects the total amount of albumin as the albumin-dye complex. The measurement of serum albumin is based on its quantitative binding to the indicator 3, 3', 5, 5'- tetrabromo-m-cresol sulphophthalein (bromocresol green, BCG). The albumin-BCG complex absorbs maximally at 600 nm. Endpoint reactions can measure the creation of a product or the loss of reactant. If the method measures the creation of a product, the absorbance is higher at the endpoint than at the start point — called an end-up reaction. If the method measures the disappearance of a reactant, the absorbance is lower at the endpoint than at the start point — called an end-down reaction.

One Product Molecule or Complex is Produced for Each Molecule of Analyte

The Endpoint Colorimetric Method

End point reaction methods determine the total amount of analytes consumed during the progression of a reaction. This method measures the total amount of analytes that participate in the reaction.

Rate Reactions

If the analyte is an enzyme, i.e., a molecule which can catalyze the conversion of unlimited numbers of reagent molecules (termed substrates) to product, the amount of product at the endpoint would not reflect the amount of enzyme. Instead, the endpoint would reflect the amount of substrate that was present. For this reason, enzyme activity is determined by a rate reaction rather than an endpoint reaction. In such cases, determination of the enzyme concentration is based on how fast a fixed amount of substrate is converted to product. The more enzyme present, the faster the conversion. Examples of enzymes that are often measured in the clinical laboratory include lipase (a digestive enzyme measured in pancreatic diseases) and alanine aminotransferase (an enzyme responsible for interconversion of amino acids measured in liver diseases). Rate reactions can measure the appearance of a product or the disappearance of a substrate. If measuring the appearance of a product, the absorbance increases with time (called a rate-up reaction). If measuring the disappearance of a substrate, the absorbance decreases with time (called a rate-down reaction). Rate reactions may also be used for measurement of analytes that are not enzymes. For example, if a reaction is very slow to reach an endpoint, a rate method may be more practical in order to obtain a result in a shorter timeframe. A typical example is ammonia — a waste product of protein metabolism.

Enzyme Concentration is Expressed in Terms of Activity

Kinetic Enzyme Assays

Kinetic reaction methods measure the difference in absorbance between two points during the progression of the reaction. Moreover, kinetic assays allow an optimal and more precise determination of enzyme activity because a constant amount of product is formed during the time interval that is being monitored — from T1 to T2.

Blanking for Endpoint Reactions

Blanking is a term that describes a correction for background constituents that contribute directly to the signal being measured. In the case of a colorimetric reaction, blanking measures the innate background color in the sample. Subtraction of the background absorbance from the final absorbance ensures that the background color is not inappropriately attributed to the analyte. For example, in the measurement of albumin using bromcresol green (BCG), the amount of albumin is calculated from the absorbance of light at wavelength 620 nm — the light absorbed by the green-colored albumin-dye complex. The absorbance is used to compute the amount of albumin present based on a calibration curve. However, if other substances in the blood sample also absorb light at 620 nm, their absorbance reading could be incorrectly attributed to albumin and the resultant albumin concentration will appear to be higher than it actually is. To correct for these other substances, the absorbance of the solution may be measured prior to the addition of the dye and only the change in absorbance above that initial value is used to compute the albumin concentration. Alternatively, the sample may be diluted with a nonreactive solution, such as saline, in a second cuvette and the absorbance of the diluted sample can be used to correct the result. Three common interfering substances that are found in plasma and serum are hemoglobin (from red blood cells), lipids (such as triglycerides) that in high concentration result in a turbid (cloudy) solution, and bilirubin (a yellow-orange colored product formed from the breakdown of hemoglobin). These three substances are so commonly found in samples that a special approach is used to assess their presence and correct for their interference in optical analyses.

Time Windows for Rate Reactions

Sometimes several substances present in the sample would react with the reagents to produce products that absorb light at the same wavelength as the product from the analyte. In such a case, blanking before addition of the reactive reagent will not correct for the interfering substances since the color does not form until the reagent is added. However, in many cases, reaction conditions (such as pH of the solution or concentration of reagents) can be chosen, so that the interfering substance reacts at a different time than the target analyte. The interfering substance may react faster and be consumed before the target analyte, or may react more slowly and contribute little or no signal in the early timeframe of the reaction. If the interferent reacts more rapidly, measurement is taken at time points late in the reaction course when the rate of color change reflects only the target analyte. If the interferent reacts more slowly, then measurement is taken at time points early in the reaction when the color change is primarily due to the target analyte. An example of the value of using a timed window in a rate reaction is seen with the Jaffe method for creatinine. In the Jaffe reaction, creatinine reacts with a solution of alkaline picrate to form a red-orange product. Unfortunately, many other substances found in biologic samples also react with alkaline picrate to form red-orange products. Some of these include acetoacetate and protein. It was found that acetoacetate reacts completely within the first 20 seconds and protein demonstrates a lag time, reacting only after two minutes. So, a time window that begins sometime after 60 seconds and ends within the first two minutes will reflect product formed from creatinine with little interference from either acetoacetate or protein.