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The authors' hypothesis is that the members of the tricarboxylic acid cycle (TCA cycle) such as citrate decrease in association with increased ketone body formation. To prove this hypothesis the connection between ketone bodies and citrate formation of milk was studied. A fluorimetric method was used to determine citrate and a headspace sampling gas chromatographic (GC) method was developed for determination of ketone bodies. Under real conditions of milk sampling, transport and storage, preserved milk samples of 119 clinically healthy dairy cows obtained in the 48 hours after milking were investigated. A low level of acetoacetate (ACAC) was found in all samples. This fact can be explained by the spontaneous decarboxylation of acetoacetate during sample storage (previously decarboxylised acetoacetate = pdACAC) and, consequently, the majority of the amount of acetoacetate in the samples (AC+pdACAC) appeared in the measured acetone concentrations. Based on the measured acetone concentration of milk samples two groups were formed retrospectively: HA (high-acetone) group (n = 41) with an AC+pdACAC concentration of ≯ 0.4 mmol/l and a LA (low-acetone) group (n = 78) with an AC+pdACAC level of ≤ 0.4 mmol/l. In the milk of cows of Group HA a positive correlation (r = +0.623) and linear connection between acetone (AC+pdACAC) and β-hydroxybutyrate (BOHB) levels was found [BOHB = 2.491 + 0.586 × (pdAC + ACAC)]. Furthermore, in this group a negative correlation between citrate and BOHB and AC+pdACAC was also established (r = -0.579). Focusing on the results of this group the authors found a significant drop of AC+pdACAC and citrate during the metabolically critical first 1-4 weeks of lactation. For this reason they suggest that simple, easy, automated methods (i.e. flow injection analysis, Fourier transformation infrared analysis) should be introduced for the simultaneous determination of acetone and citrate concentration in milk to make the evaluation of the energy status of high-producing dairy cows easier and more certain.

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