A guide for measurement of circulating metabolic hormones in rodents: Pitfalls during the pre-analytical phase

Abstract

Researchers analyse hormones to draw conclusions from changes in hormone concentrations observed under specific physiological conditions and to elucidate mechanisms underlying their biological variability. It is, however, frequently overlooked that also circumstances occurring after collection of biological samples can significantly affect the hormone concentrations measured, owing to analytical and pre-analytical variability. Whereas the awareness for such potential confounders is increasing in human laboratory medicine, there is sometimes limited consensus about the control of these factors in rodent studies. In this guide, we demonstrate how such factors can affect reliability and consequent interpretation of the data from immunoassay measurements of circulating metabolic hormones in rodent studies. We also compare the knowledge about such factors in rodent studies to recent recommendations established for biomarker studies in humans and give specific practical recommendations for the control of pre-analytical conditions in metabolic studies in rodents.

Keywords: Hormone measurement; Immunoassay; Mouse; Pre-analytical variability; Rat; Sample processing.

Fig. 1

Variables determining the measurement of circulating analytes in biological fluids.

Fig. 2

Example illustrating inter-assay variability of rodent immunoassays. Serum IGF-I was measured in serum of mice with 2 commercially available immunoassays (IDS and DSL). Passing–Bablok regression; slope: 0.831; intercept: 162.4).

Fig. 3

Experimental setup scheme illustrating the different handling and processing strategies for blood samples which were used for the analyses of metabolic hormones in rats.

Fig. 4

Measurement of circulating metabolic hormones ((A) leptin; (B) total and acylated ghrelin; (C) insulin; (D) total glucose-dependent insulinotropic peptide (GIP); (E) total glucagon-like peptide 1 (GLP-1)) in rats under different pre-analytical conditions. The first graph for each hormone shows absolute concentrations under different pre-analytical conditions separately for each rat. For better comparison of the different pre-analytical conditions, a second bar chart for each hormone depicts the effects in relation to the concentration obtained from serum (n=9/condition). Therefore, the serum concentration has been set to 100% and other conditions are expressed as a percentage thereof (*p<0.05, **p<0.01, ***p<0.001). Abbreviations: “RT”: room temperature; “Prot.”: addition of a general protease inhibitor; “Prot.+DPP-4”: addition of a general and a specific DPP-4 inhibitor; “Prot.+HCl”: addition of a general protease inhibitor and acidification of the sample with HCl. Data in bar charts are expressed as means±SEM.

Fig. 4

Measurement of circulating metabolic hormones ((A) leptin; (B) total and acylated ghrelin; (C) insulin; (D) total glucose-dependent insulinotropic peptide (GIP); (E) total glucagon-like peptide 1 (GLP-1)) in rats under different pre-analytical conditions. The first graph for each hormone shows absolute concentrations under different pre-analytical conditions separately for each rat. For better comparison of the different pre-analytical conditions, a second bar chart for each hormone depicts the effects in relation to the concentration obtained from serum (n=9/condition). Therefore, the serum concentration has been set to 100% and other conditions are expressed as a percentage thereof (*p<0.05, **p<0.01, ***p<0.001). Abbreviations: “RT”: room temperature; “Prot.”: addition of a general protease inhibitor; “Prot.+DPP-4”: addition of a general and a specific DPP-4 inhibitor; “Prot.+HCl”: addition of a general protease inhibitor and acidification of the sample with HCl. Data in bar charts are expressed as means±SEM.

Fig. 5

Impact of repeated freeze–thaw cycles on circulating metabolic hormones ((A) leptin; (B) total ghrelin; (C) insulin; (D) total glucose-dependent insulinotropic peptide (GIP); (E) total glucagon-like peptide 1 (GLP-1)) in rats. The first bar chart for each hormone shows hormone concentrations in plain serum after 0, 1, 2, 5 (n=5 each) and 10 (n=9) consecutive freeze–thaw cycles. Data are presented as a percentage of the fresh (untouched) serum aliquot which has been set to 100%. The second graph for each hormone (not available for leptin) depicts the effects of 10× freezing and re-thawing (“freeze/thaw”) in native EDTA plasma and in EDTA plasma samples which were pre-treated with one or two protease inhibitors. Again, values are expressed in relation to the fresh (untouched) plasma aliquot without the addition of any protease inhibitors (n=5/condition). Hormone concentrations of the fresh aliquots have been set to 100% (*p<0.05, **p<0.01, ***p<0.001). Data are expressed as means±SEM. Abbreviations: “Plasma+Prot.”: EDTA blood (plasma) including a general protease inhibitor; “Plasma+Prot.+DPP-4”: EDTA blood (plasma) including a general protease inhibitor and a specific DPP-4 inhibitor.

Fig. 5

Impact of repeated freeze–thaw cycles on circulating metabolic hormones ((A) leptin; (B) total ghrelin; (C) insulin; (D) total glucose-dependent insulinotropic peptide (GIP); (E) total glucagon-like peptide 1 (GLP-1)) in rats. The first bar chart for each hormone shows hormone concentrations in plain serum after 0, 1, 2, 5 (n=5 each) and 10 (n=9) consecutive freeze–thaw cycles. Data are presented as a percentage of the fresh (untouched) serum aliquot which has been set to 100%. The second graph for each hormone (not available for leptin) depicts the effects of 10× freezing and re-thawing (“freeze/thaw”) in native EDTA plasma and in EDTA plasma samples which were pre-treated with one or two protease inhibitors. Again, values are expressed in relation to the fresh (untouched) plasma aliquot without the addition of any protease inhibitors (n=5/condition). Hormone concentrations of the fresh aliquots have been set to 100% (*p<0.05, **p<0.01, ***p<0.001). Data are expressed as means±SEM. Abbreviations: “Plasma+Prot.”: EDTA blood (plasma) including a general protease inhibitor; “Plasma+Prot.+DPP-4”: EDTA blood (plasma) including a general protease inhibitor and a specific DPP-4 inhibitor.

Fig. 6

Effect of different centrifugation protocols on circulating metabolic hormones (leptin, total ghrelin, insulin, total GIP and total GLP-1) in rat serum (n=5/centrifugation protocol). Concentrations measured in aliquots centrifuged with the standard centrifugation protocol (3000g for 10 min at 4 °C) have been set to 100%, the concentrations from aliquots centrifuged with the “fast” protocol (10,000g for 3 min at room temperature) are expressed as a percentage thereof. Data are expressed as means±SEM.