Fluorimetric methods for the measurement of intermediate metabolites (lactate, pyruvate, alanine, β-hydroxybutyrate, glycerol) using a COBAS FARA centrifugal analyser

Intermediate products of the metabolism of glucose, fat and amino-acid are important in the evaluation of such metabolic disorders as diabetes mellitus, liver disease and metabolic acidosis. In the present study, methods for the measurement of intermediate metabolites (lactate, pyruvate, alanine, β-hydroxybutyrate and glycerol) have been adapted to a fast centrifugal analyzer: the COBAS FARA. Correlation coeffcients rangedfrom 0.90 to 0.99, compared to established manual spectrophotometric methods. Within-run coeffcients of variation (CVs) ranged between 2.9 and 8.8% at low levels, between 1.5 and 5.7% at medium levels and between 1.2 and 5.6% at high levels. Between-run CVs were between 4.0 and 15.0% at low levels, between 1.7 and 7.0% at medium levels and between 1.3 and 2.7% at high levels. These fluorimetric assays for the determination of intermediate metabolites on COBAS FARA (Roche) have a good sensitivity and precision, are less costly than manual methods and can be used on a routine basis.


Introduction
The measurement of intermediate metabolism products was used in the past to understand such metabolic disorders as diabetes mellitus, liver disease and metabolic acidosis [1][2][3][4][5]. They are now measured to evaluate early metabolic alterations in certain classes of patients, for example obese patients and relatives of diabetic subjects who are at risk for developing metabolic disorders. Enzymatic assays for intermediate metabolites were devised in the 1950s [6], and manual methods for the determination of intermediate metabolites (lactate, pyruvate, alanine, fl-hydroxybutyrate and glycerol) were rapidly developed for the study of physiological and pathological states [7-1 1], however, these methods are laborious and have little sensitivity. They are also expensive, as they use relatively large quantities of enzymes with little substrate to enable the reaction to proceed rapidly to equilibrium. In order to analyse large numbers of samples rapidly in small volumes of blood or plasma, automated photometric [12] or fluorimetric techniques [13 and 14] have been developed utilizing either continuous flow methods [15] or centrifugal analysers with a fluorimetric attachment [16][17][18]. These methods are more precise and sensitive and use very little sample, thus making metabolic studies possible in children and neonates. The present study started from the need to use fluorimetric methods with assays particularly suited to routine work. The measurement of lactate, fl-hydroxybutyrate, alanine, pyruvate and glycerol was developed on a fast centrifugal analyser (COBAS FARA).

Patients
Fifty subjects were subdivided into three groups: normal; impaired glucose tolerant; and diabetic patients attending a diabetic clinic. Each patient had a random blood sample for the determination of intermediate metabolites. They were chosen because they had a wide concentration range of all metabolites. Blood samples were processed simultaneously using the COBAS FARA methods and manual methods described in the literature [7][8][9][10][11].

Sample preparation
Lactate, fl-hydroxybutyrate, alanine, pyruvate and glycerol are unstable in whole blood and an immediate and correct processing of the sample is necessary at the time of venepuncture [15]. Plastic tubes containing 3 ml of 5 (0"5 mol/1)perchloric acid (PCA), previously cooled to 0C, were weighed; 1-1.5 ml of blood was added immediately after sampling and the tube was weighed again, thus giving an accurate measurement of the amount of blood used. In a few pathological cases, whether the levels of metabolites were over the highest calibrator points, dilutions were made adding adequate: amounts of 5% PCA. All these changes were taken into account in the final calculation of the data.

Comparison of methods
The methods of measurement for lactate, pyruvate, alanine, fl-hydroxybutyrate and glycerol were adapted for use on the COBAS FARA II (Roche, Basle, Switzerland) with a fluorimetric attachment by making a number of modifications in the composition of buffers, and coenzyrne/enzyme reagents used in previous methods [ 16] and modifying the reaction modes available on the analyser to take the improved calculation programs into account.
The PM voltage was set using 1200 gmol/1 calibrator.
The PM voltage was set using water as sample. The program was set by introducing the following variables: calibration mode: linear regression; sample volume: 10 gl; reagent solution: 170 lal; starter solution: 10 l.tl; incubation time: 300 s; number of readings: 10; interval between readings: 30 s; calculation step: endpoint (first 1, last 10).
The PM voltage was set using 150 gmol/1 calibrator. The program was set by introducing the following variables: calibration mode: linear regression; sample volume: 10gl; reagent solution: 270gl; number of readings: 12; interval between readings: 30 s; calculation step: endpoint (first 1, last 12).

Statisticl analysis
Data are presented as mean +_SD. Data were processed by least-squares regression analysis.
Enzyme reagent" 20 mg NAD and 100 lal alanine dehydrogenase in 10 ml 0"1 mol/1 phosphate buffer pH 7"4. The reagent solution is prepared by adding 0"7 ml of this solution to 8"8 ml of the buffer.
The methods correlated well with manual methods (see table 3); a very small positive intercept, not significantly different from zero, was obtained in all cases except for -hydroxybutyrate. This is probably because manual spectrophotometric methods are less sensitive than fluorimetric methods at low concentrations of analytes. calibration curves; better values were found at the highest points of the calibration curves (table 1).
The high sensitivity of the methods is illustrated by the fact that the reaction sample volumes usually varied from 5 to 10 gl of PCA precipitates with an even smaller volume of blood.
Continuous flow methods [15] were also used for the determination of the intermediate metabolites, but these methods presented some disadvantages when compared to fast centrifugal analyser: for example, the need to run 'blank fluorescence' and to draw calibration graphs and compare large numbers of peaks which can generate gross errors [16]. The present methods on COBAS FARA II are similar to a few spectrophotometric or fluorimetric methods adapted for previous fast centrifugal analysers [16,19]. Major innovations are in the ability to create new reaction modes with mathematical modeling of the data and particular care in the handling of samples. In conclusion, the fluorimetric assays for the determination of intermediate metabolites on COBAS FARA II have a good sensitivity and precision, are less costly than manual methods and can be used on a routine basis.