PRACTICAL 2

Determination of sodium and potassium ion concentration: Theory

2.1 Introduction

The aims of this practical are:

to demonstrate safe laboratory working procedures

to demonstrate two simple but different techniques for making the clinically important measurements of sodium and potassium ion concentration

to introduce the simple concept of quantitation using a calibration curve

to highlight the difference between accuracy and precision and illustrate the importance of these factors in clinical assays

2.2 Theory and background

Sodium and potassium

The major cation of the extracellular fluid is sodium. The typical daily diet contains 130-280 mmol (8-15 g) sodium chloride. The body requirement is for 1-2 mmol per day, so the excess is excreted by the kidneys in the urine.

Reference range (intervals) for sodium
Serum	136-145 mM
Cerebrospinal fluid	130-150 mM
Sweat	10-40 mM
Urine (varies with intake)	40-220 mmol/day

Hyponatraemia (lowered plasma [Na⁺]) and hypernatraemia (raised plasma [Na⁺]) are associated with a variety of diseases and illnesses and the accurate measurement of [Na⁺] in body fluids is an important diagnostic aid.

Potassium is the major cation found intracellularly. The average cell has 140 mM K⁺ inside but only about 10 mM Na⁺. K⁺ slowly diffuses out of cells so a membrane pump (the Na⁺/K⁺-ATPase) continually transports K⁺ into cells against a concentration gradient. The human body requires about 50-150 mmol/day.

Reference range (intervals) for potassium
Serum	3.5-5.1 mM
Cerebrospinal fluid	about 70% of serum
Sweat	4.0-9.7 mM (men) 7.6-15.6 mM (women)
Urine (varies with intake)	25-125 mmol/day
Erythrocytes (intracellular)	105 mM

Hypokalaemia (lowered plasma [K⁺]), hyperkalaemia (increased plasma [K⁺]) and hyperkaluria (increased urinary excretion of K⁺) are again indicative of a variety of conditions and the clinical measurement of [K⁺] is also of great importance.

The flame photometer

A traditional and simple method for determining sodium and potassium in biological fluids involves the technique of emission flame photometry. This relies on the principle that an alkali metal salt drawn into a non-luminous flame will ionise, absorb energy from the flame and then emit light of a characteristic wavelength as the excited atoms decay to the unexcited ground state. The intensity of emission is proportional to the concentration of the element in the solution. You are probably familiar with the fact that if you sprinkle table salt (NaCl) into a gas flame then it glows bright orange (KCl gives a purple colour). This is the basic principle of flame photometry. A photocell detects the emitted light and converts it to a voltage, which can be recorded. Since Na⁺ and K⁺ emit light of different wavelengths (colours), by using appropriate coloured filters the emission due to Na⁺ and K⁺ (and hence their concentrations) can be specifically measured in the same sample. One drawback of flame photometers, however, is that they respond linearly to ion concentrations over a rather narrow concentration range so suitable dilutions usually have to be prepared. They are also rather complex and relatively expensive machines, as you will see.

A flame photometer can also be used to measure the element lithium in serum or plasma in order to determine the correct dosage of lithium carbonate, a drug used to treat certain mental disturbances, such as manic-depressive illness (bipolar disorder).

In this practical (Experiment A) you will calibrate a flame photometer using standard sodium and potassium solutions then measure the Na⁺ and K⁺ concentrations in a redissolved oral rehydration sachet.

Ion-selective electrodes (ISEs)

You should already be familiar from A-level studies with the pH electrode, the most essential component of which is a sensitive glass membrane which permits the passage of hydrogen ions, but no other ionic species. A small potential difference develops across the membrane which is proportional to the logarithm of the H⁺ concentration (pH) and which can be measured on a millivoltmeter. Ion-selective electrodes (ISEs) which respond relatively specifically to other ions (both anions and cations) operate on the same principle. In clinical laboratories they can be used to measure Ca²⁺, K⁺ and Cl^- in body fluids (blood, plasma, serum, sweat) and F^- in skeletal and dental studies. They are also used to measure a wide variety of other ions in, for example, environmental studies.

When compared to many other analytical techniques, ISEs are inexpensive and simple to use and, unlike the flame photometer, have a linear response over a wide concentration range. However, they have some disadvantages that require attention if good results are to be obtained:

First, despite their name, many of them are not entirely ion-specific. For example, the sodium electrode you will use also responds to potassium ions , although not with the same sensitivity. This means that Na⁺ will be overestimated if a high concentration of K⁺ is present. Mathematical techniques have been devised to compensate for this.

Secondly, they underestimate high concentrations because of "crowding" of the ions at the membrane — some just don't get "seen". The activity coefficient is a measure of this: activity equals concentration at low values, but is less than concentration at high values. ISEs measure activity.

For those of you with a keen desire to learn more about ion-selective electrodes, a comprehensive, though somewhat technical, "beginner's guide" has been posted on the Web by Dr. Chris Rundle of the manufacturing company Nico2000.

In this practical (Experiment B) you will use a sodium ISE to measure the apparent Na⁺ concentration in a redissolved oral rehydration sachet without compensating for the presence of K⁺ ions.

Again, if you are interested, an article comparing the use of flame photometry and ISE for the measurement of sodium and potassium in clinical samples can be found in the library: "A comparison of the measurement of sodium and potassium by flame photometry and ion-selective electrode". Worth H.G. Ann. Clin. Biochem. (1985)22, 343-50.

Accuracy and precision

Accuracy (i.e. how close the measured value is to the true value) and precision (how close a series of measurements on the same sample are to each other) are two distinct factors in determining the usefulness of a clinical chemical assay. If the sodium ion concentration in a biological fluid is, say, 140 mM and method A gives an averaged value of 137 mM while method B gives a value of 125 mM, then method A is more accurate. However, if repeated measurements by A gives values of 140, 147, 134, 127, 130 and 144 mM while B gives 124, 125, 125, 125, 126, and 125 mM, then B is clearly more reproducible, i.e. it is more precise. The ideal clinical assay should have high accuracy AND precision.

The concept of quality assurance

Quality assurance is the process that is performed to ensure that an analytical method produces reliable results. The performance of any assay can be monitored by measuring the concentration of the analyte (i.e. the substance being analysed) in samples that contain a known quantity of the analyte. These samples are called internal quality controls (IQC) and are analysed every time a batch of (patient) samples is processed. The actual values obtained are compared with the expected values and by cumulating these internal QC results the precision and accuracy of the method can be defined. As stated above, precision reflects the reproducibility of the method and accuracy reflects how close the measured value is to the actual value. Acceptable limits can be set for any analysis by determining the precision and accuracy using multiple measurements of the internal QC samples (n=20-30). The accuracy of any assay depends on the availability of standard material of the highest quality with an assigned concentration established by a gold standard analytical method.

At the end of the practical you will compare the results you obtained for Na⁺ measurement by the two methods with the known value of Na⁺ in the redissolved sachet and also compare the accuracy and precision of the two methods using the class data.

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