Carlos A.
Penno
*abe,
Denis
Arsenijevic
ce,
Thierry
Da Cunha
ae,
Gerd A.
Kullak-Ublick
de,
Jean-Pierre
Montani
ce and
Alex
Odermatt
*ae
aDivision of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland. E-mail: alex.odermatt@unibas.ch; Fax: +41 61 267 1515; Tel: +41 61 267 1530
bNovartis Institute for Biomedical Research, Novartis, CH-4009 Basel, Switzerland. E-mail: carlos.penno@unibas.ch; Fax: +41 61 267 1515; Tel: +41 61 267 1484
cDepartment of Medicine/Physiology, University of Fribourg, Fribourg, Switzerland
dDepartment of Clinical Pharmacology and Toxicology, University Hospital Zurich, Zurich, Switzerland
eThe Swiss National Center of Competence in Research (NCCR) Kidney Control of Homeostasis (Kidney.CH) Web: http://www.nccr-kidney.ch/
First published on 14th January 2013
In order to study the roles of individual BAs and due to limited blood sample volumes available from experimental animals, improved methods for the simultaneous quantification of multiple BAs are needed. We developed and validated an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantification of 24 BAs, including 11 unconjugated, 6 glycine-conjugated and 7 taurine-conjugated BAs, in 50 μL of rat serum or plasma. The UPLC-MS/MS method, operated in negative and positive ion mode, allows quantification of BAs using multiple-reaction monitoring (MRM), with specific fragmentation of BAs. The method showed acceptable intra- and inter-day accuracy, precision, extraction recovery and high sensitivity, with a lower limit of quantification (LLOQ) in the pM range for several taurine-conjugated BAs. We applied the established method to investigate potential time-dependent changes of BAs in plasma from sham-operated and uninephrectomized male Sprague-Dawley rats. The levels of several primary and secondary BAs were transiently elevated one week after uninephrectomy, followed by normalization thereafter. In contrast, several conjugated BAs were slightly increased after the second week post-surgery. The established UPLC-MS/MS method, employing specific fragmentation of free and conjugated BAs by MRM, allows the simultaneous quantification of multiple BAs in 50 μL serum or plasma samples, and can be used to assess BA profiles in patho-physiological situations.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been considered the gold standard for quantification of BAs in biological fluids and tissues, due to several advantages over traditional techniques such as gas chromatography (GC)-MS, including ease of sample preparation and no need for hydrolysis of conjugated BAs or complex derivatization reactions.10 However, despite the technological advance in MS to increase sensitivity, several problems still remain to be overcome such as the requirement of large sample volumes depending on the analyte to be quantified,11 the need for derivatization depending on the availability of sample amount,12 interference with contaminating endogenous BAs in biological matrices,11 and limited specificity when using selective ion monitoring (SIM) for quantification.10 Although there is a consensus in the literature regarding the use of multiple reaction monitoring (MRM) for the quantification of taurine- and glycine-conjugated BAs, MRM has not yet been widely used for quantification of unconjugated BAs.11,13–25 In the present study, we applied specific fragmentation using MRM for both conjugated and unconjugated BAs in order to increase the specificity of detection and sensitivity for quantification of BAs in complex biological matrices such as serum and plasma.
Primary BAs are synthesized and conjugated in hepatocytes, followed by excretion into bile and the intestinal tract. Gut microorganisms generate secondary BAs by deconjugation and dehydroxylation. Upon reuptake by intestinal transporters, BAs are reconjugated in the liver to complete the enterohepatic cycle. BAs can also be filtered in the kidney through the glomerulus, followed by urinary excretion. Most BAs undergo reuptake by renal tubular transporters and, under normal conditions, the amount of excreted BAs is low. However, impaired hepatorenal function can lead to increased urinary BA excretion.
The kidney has a key role in the control of whole body homeostasis, including electrolyte balance and blood pressure, production and utilization of systemic glucose, degradation of hormones and excretion of waste metabolites.26 Recent observations unraveled the importance of the kidney in the regulation of lipid metabolism, fat distribution and adipocyte differentiation.27 In rats reduced renal function upon uninephrectomy has been linked with several aspects of the metabolic syndrome such as lipodystrophy of subcutaneous and visceral adipose depots, with lipid depletion, adipocyte dedifferentiation, lipid peroxidation, hypercholesterolemia and hypertriglyceridemia.28 Similarly, nondiabetic patients on hemodialysis manifested fat redistribution with increasing visceral fat and altered serum lipid profiles.29 These findings indicate that reduced renal function can cause disturbances of lipid homeostasis. Due to the close association between BA signaling and metabolic homeostasis30–33 and the observed impact of reduced kidney function on lipid homeostasis, we investigated the impact of uninephrectomy in rats on plasma BA profiles by applying the validated UPLC-MS/MS method.
The mobile phase consisted of water–acetonitrile–formic acid (A) (95/5/0.1; v/v/v) and (B) (5/95/0.1; v/v/v). The eluent gradients were set from 25%–35% of B during 0–8 min, 35%–70% eluent B during 8–18 min and 95% of B at 18.1 min onwards. The run was stopped at 20 min, followed by re-equilibration of the column. The flow rate was set to 0.75 mL min−1. Ionization was performed using an ESI source operated in the positive and negative ion modes. Fragmentation was tuned for each compound using Optimizer software (Agilent Technologies). Optimized conditions are shown in Table 1. The source parameters were set to gas temperature 350 °C, gas flow 15 L min−1, nebulizer pressure 20 psi, sheath gas temperature 250 °C, sheath gas flow 11 L min−1, capillary voltage 3000 V (positive and negative), nozzle voltage 2000 V and cell accelerator voltage 5 V.
BA | Precursor Ion (m/z) | Product ion (m/z) | Collision energy (V) | Polarity | Internal Standard |
---|---|---|---|---|---|
CA | 373.3 | 355.2 | 48 | Positive | CA-d4 |
CDCA | 357.2 | 95.1 | 40 | Positive | CDCA-d4 |
DCA | 357.2 | 95.1 | 40 | Positive | DCA-d4 |
7-oxoDCA | 371.3 | 353.2 | 8 | Positive | DCA-d4 |
HDCA | 357.2 | 95.1 | 40 | Positive | UDCA-d4 |
LCA | 359.3 | 135.1 | 24 | Positive | LCA-d4 |
7-oxoLCA | 373.3 | 355.2 | 8 | Positive | UDCA-d4 |
α-MCA | 373.3 | 355.2 | 8 | Positive | UDCA-d4 |
β-MCA | 373.3 | 355.2 | 8 | Positive | UDCA-d4 |
ω-MCA | 373.3 | 355.2 | 8 | Positive | UDCA-d4 |
UDCA | 357.2 | 95.1 | 40 | Positive | UDCA-d4 |
G-CA | 464.2 | 74 | 37 | Negative | G-CA-d4 |
G-CDCA | 448.2 | 74 | 41 | Negative | G-CDCA-d4 |
G-DCA | 448.2 | 74 | 41 | Negative | G-CA-d4 |
G-LCA | 432.2 | 74 | 41 | Negative | G-UDCA-d4 |
G-7-oxoLCA | 446.2 | 74 | 37 | Negative | G-UDCA-d4 |
G-UDCA | 448.2 | 74 | 37 | Negative | G-UDCA-d4 |
T-CA | 480.3 | 126 | 24 | Positive | G-UDCA-d4 |
T-CDCA | 464.2 | 126 | 28 | Positive | G-CDCA-d4 |
T-DCA | 464.2 | 126 | 28 | Positive | DCA-d4 |
T-LCA | 466.2 | 126 | 28 | Positive | G-UDCA-d4 |
T-7-oxoLCA | 480.3 | 126 | 20 | Positive | G-UDCA-d4 |
T-β-MCA | 480.3 | 126 | 24 | Positive | G-DCA-d4 |
T-UDCA | 464.2 | 126 | 28 | Positive | G-UDCA-d4 |
CA-d4 | 377.3 | 359.2 | 48 | Positive | — |
CDCA-d4 | 361.2 | 95.1 | 40 | Positive | — |
DCA-d4 | 361.3 | 95.1 | 40 | Positive | — |
LCA-d4 | 363.3 | 135.1 | 24 | Positive | — |
UDCA-d4 | 361.2 | 95.1 | 40 | Positive | — |
G-CA-d4 | 468.2 | 74 | 37 | Negative | — |
G-CDCA-d4 | 452.2 | 74 | 41 | Negative | — |
G-UDCA-d4 | 452.2 | 74 | 41 | Negative | — |
Values of the lower limit of quantification (LLOQ) were calculated by assessing the signal to noise ratio (SNR) (baseline noise determined on an interval before and after the peak of interest and using the peak height as signal definition). Five replicates were extracted and analyzed for each concentration. A signal equal or higher than ten times that of the baseline was considered the LLOQ, with accuracy between 80 and 120% of the true value and coefficient of variation (CV) of 15%. Due to the persistence of trace amounts of G-CA, G-CDCA and G-DCA after charcoal treatment, their LLOQs were determined as being the lowest concentration at which these analytes could be quantified with sufficient precision (CV of 15%) and accuracy (between 85% and 115%).
In order to assess intra- and inter-day precision and accuracy, five replicates of five different quality control (QC) samples with concentrations ranging from 0.002 μM to 2 μM were extracted and quantified using freshly prepared calibrators in charcoal treated rat serum. Replicates of each QC sample were analyzed in a given day in order to determine intra-day accuracy and precision as well as over a period of three days (inter-day) using freshly prepared calibration curves.
Recovery experiments were performed using untreated and charcoal-treated serum samples in order to mimic extraction conditions similar to those of real samples and to assess the impact of matrix components on extraction recoveries. In order to assess extraction recovery, twelve untreated and charcoal-treated serum samples were taken for each of the concentration levels (2000 nM, 200 nM and 20 nM). From these twelve samples, six were spiked with the appropriate amount of standard stock solution and IS prior to extraction, and the remaining six samples were extracted as blanks and reconstituted with the same amount of standard stock solution and IS after extraction. Six additional unspiked serum samples were extracted in order to determine endogenous concentrations of BAs. Thereafter, samples were evaporated, reconstituted and injected. Correction of the spiked serum samples was performed by subtracting the endogenous amounts of the respective BAs. Recovery results were obtained by expressing the average of the mean peak area of samples spiked prior to extraction as a percentage of that of samples spiked after extraction.
Matrix effects were assessed by using untreated pooled rat serum in order to mimic chemical conditions of those of real samples. For that purpose, six samples were spiked with defined amounts of standard stock solutions (2000 nM, 200 nM and 20 nM) and IS after extraction. Six additional unspiked serum samples were extracted in order to determine the endogenous concentration of BAs. Thereafter, all samples were evaporated and reconstituted in the mobile phase. Correction of the spiked serum samples was performed by subtracting the endogenous amounts of the respective BAs, and matrix effects were calculated by expressing the peak area of spiked serum samples after extraction as a percentage of the peak area of that of net solutions containing only the pure standard in methanol.
Fig. 1 Structure of BAs. |
Fig. 2 Representative chromatogram of bile acids extracted from a plasma sample and charcoal-treated rat serum spiked with 24 bile acids standard. |
QC 2 nM | QC 4 nM | QC 100 nM | QC 1000 nM | QC 2000 nM | ||||||
---|---|---|---|---|---|---|---|---|---|---|
CV (%) | Accuracy (%) | CV (%) | Accuracy (%) | CV (%) | Accuracy (%) | CV (%) | Accuracy (%) | CV (%) | Accuracy (%) | |
a CV: coefficient of variation. N.D.: not determined owing to concentration below lower limit of quantification. | ||||||||||
Intra-day | ||||||||||
CA | N.D. | N.D. | 6.4 | 100.0 | 2.5 | 111.1 | 1.5 | 93.0 | ||
CDCA | N.D. | N.D. | 2.1 | 88.0 | 2.2 | 99.3 | 1.9 | 102.7 | ||
DCA | N.D. | N.D. | 3.1 | 90.6 | 2.6 | 101.0 | 4.8 | 103.3 | ||
7-oxoDCA | N.D. | N.D. | 8.3 | 104.6 | 3.2 | 101.0 | 2.7 | 98.3 | ||
HDCA | N.D. | N.D. | 10.2 | 88.7 | 9.6 | 110.4 | 11.1 | 103.4 | ||
LCA | N.D. | N.D. | 6.2 | 94.2 | 3.8 | 95.0 | 6.4 | 98.5 | ||
7-oxoLCA | N.D. | N.D. | 5.1 | 109.9 | 3.2 | 102.4 | 6.3 | 102.7 | ||
α-MCA | N.D. | N.D. | 2.4 | 106.8 | 1.4 | 98.6 | 5.1 | 105.9 | ||
β-MCA | N.D. | N.D. | 6.5 | 103.7 | 4.3 | 108.0 | 2.4 | 101.7 | ||
ω-MCA | N.D. | N.D. | 5.9 | 95.8 | 2.2 | 108.7 | 9.5 | 98.6 | ||
UDCA | N.D. | N.D. | 8.6 | 96.3 | 3.5 | 106.0 | 3.9 | 102.3 | ||
G-CA | N.D. | 12.9 | 95.2 | 2.9 | 87.5 | 4.2 | 105.8 | 4.5 | 96.5 | |
G-CDCA | N.D. | N.D. | 2.4 | 91.2 | 2.3 | 104.2 | 1.8 | 90.6 | ||
G-DCA | 8.9 | 102.0 | 8.3 | 92.5 | 2.0 | 90.1 | 3.6 | 104.8 | 1.9 | 103.1 |
G-LCA | 10.4 | 111.1 | 7.4 | 102.6 | 2.7 | 90.1 | 6.4 | 100.2 | 1.9 | 99.5 |
G-7-oxo-LCA | 5.4 | 113.8 | 3.3 | 88.5 | 1.5 | 86.8 | 3.2 | 108.0 | 3.6 | 94.6 |
G-UDCA | 7.7 | 106.8 | 11.7 | 102.6 | 3.2 | 95.4 | 6.8 | 112.5 | 1.7 | 94.7 |
T-CA | 9.3 | 96.2 | 8.8 | 105.3 | 4.7 | 94.3 | 1.9 | 101.9 | 3.2 | 96.3 |
T-CDCA | 10.6 | 88.4 | 8.2 | 91.7 | 3.8 | 93.8 | 2.6 | 105.0 | 3.2 | 104.5 |
T-DCA | 9.4 | 103.3 | 3.0 | 87.7 | 4.8 | 98.0 | 2.4 | 96.6 | 3.3 | 99.4 |
T-LCA | 8.6 | 108.2 | 12.6 | 101.0 | 4.2 | 99.4 | 4.6 | 100.0 | 4.0 | 96.7 |
T-7-oxo-LCA | 14.8 | 92.6 | 6.4 | 97.6 | 3.4 | 92.9 | 4.2 | 93.5 | 3.2 | 98.6 |
T-β-MCA | 7.2 | 114.9 | 12.6 | 101.0 | 4.1 | 100.6 | 6.9 | 97.7 | 4.2 | 99.4 |
T-UDCA | 12.2 | 92.3 | 5.2 | 97.1 | 3.9 | 96.5 | 3.0 | 104.7 | 4.5 | 97.6 |
Inter-day | ||||||||||
CA | N.D. | 5.5 | 96.8 | 5.2 | 99.4 | 4.4 | 97.0 | 3.8 | 93.8 | |
CDCA | N.D. | 6.0 | 93.4 | 2.2 | 97.4 | 1.9 | 95.1 | 3.8 | 97.1 | |
DCA | N.D. | 3.5 | 93.8 | 4.0 | 93.0 | 1.7 | 96.4 | 2.0 | 100.0 | |
7-oxoDCA | N.D. | N.D. | 5.1 | 106.6 | 1.9 | 100.2 | 1.2 | 104.0 | ||
HDCA | N.D. | N.D. | 4.6 | 92.0 | 3.0 | 94.8 | 3.7 | 96.1 | ||
LCA | N.D. | N.D. | 4.3 | 97.8 | 1.8 | 93.3 | 2.9 | 99.8 | ||
7-oxoLCA | N.D. | N.D. | 4.4 | 104.0 | 5.9 | 92.3 | 4.4 | 99.1 | ||
α-MCA | N.D. | N.D. | 3.8 | 99.4 | 3.5 | 92.4 | 5.4 | 98.1 | ||
β-MCA | N.D. | N.D. | 5.5 | 96.5 | 4.3 | 94.9 | 5.3 | 99.2 | ||
ω-MCA | N.D. | N.D. | 3.7 | 95.0 | 2.1 | 97.5 | 6.7 | 95.6 | ||
UDCA | N.D. | N.D. | 7.1 | 97.2 | 4.8 | 93.7 | 1.8 | 98.3 | ||
G-CA | N.D. | 4.8 | 102.9 | 1.8 | 91.8 | 2.4 | 99.5 | 3.4 | 101.7 | |
G-CDCA | N.D. | N.D. | 2.3 | 92.9 | 1.8 | 97.9 | 1.9 | 93.2 | ||
G-DCA | 12.3 | 90.1 | 7.2 | 96.0 | 2.4 | 93.3 | 2.3 | 98.7 | 4.9 | 101.4 |
G-LCA | 9.2 | 105.6 | 4.8 | 94.9 | 1.3 | 88.9 | 1.3 | 98.0 | 1.4 | 99.8 |
G-7-oxo-LCA | 9.3 | 109.5 | 2.1 | 90.6 | 2.2 | 90.7 | 1.7 | 99.0 | 1.8 | 98.3 |
G-UDCA | 9.5 | 102.7 | 7.8 | 100.8 | 2.9 | 96.1 | 2.4 | 98.2 | 1.5 | 94.7 |
T-CA | 8.5 | 96.8 | 4.7 | 90.9 | 2.8 | 95.1 | 1.8 | 99.3 | 1.4 | 101.4 |
T-CDCA | 12.0 | 90.6 | 4.6 | 91.5 | 3.3 | 96.4 | 2.9 | 104.4 | 4.0 | 96.8 |
T-DCA | 14.2 | 102.1 | 2.5 | 86.8 | 2.1 | 96.2 | 5.0 | 99.9 | 1.1 | 104.2 |
T-LCA | 8.2 | 111.4 | 4.2 | 98.0 | 2.2 | 98.1 | 3.2 | 101.4 | 1.7 | 102.7 |
T-7-oxo-LCA | 15.0 | 95.2 | 2.2 | 93.9 | 3.2 | 94.9 | 1.3 | 95.6 | 1.4 | 102.1 |
T-β-MCA | 11.7 | 109.5 | 5.5 | 92.4 | 2.1 | 95.1 | 4.3 | 99.9 | 2.8 | 104.3 |
T-UDCA | 12.3 | 95.4 | 2.5 | 87.2 | 3.1 | 94.3 | 3.7 | 104.7 | 3.1 | 105.5 |
Bile acids | LLOQ (nM) | SNR | RT (min) | Linearity (R2) | Calibration range (nM) | Extraction recoveriesa (%) | ||
---|---|---|---|---|---|---|---|---|
20 nM | 200 nM | 2 μM | ||||||
a Data are presented as the average ± %R.S.D of three levels of QC concentrations (20 nM, 200 nM and 2 μM), six samples per concentration. b N.A. not applicable: LLOQs were determined as the lowest concentration in which these analytes were quantified with sufficient precision (CV of 15%) and accuracy (between 85% and 115%). | ||||||||
CA | 3 | 11 ± 4 | 9.3 | 0.9921 | 4000–0.98 | 78 ± 30 | 97 ± 14 | 70 ± 11 |
CDCA | 3 | 13 ± 4 | 12.3 | 0.9914 | 4000–0.98 | 76 ± 3 | 94 ± 12 | 83 ± 8 |
DCA | 3 | 10 ± 1 | 12.6 | 0.9985 | 4000–0.98 | 75 ± 12 | 86 ± 26 | 63 ± 7 |
7-oxoDCA | 25 | 11 ± 1 | 6.6 | 0.9955 | 4000–7.8 | 78 ± 9 | 71 ± 16 | 73 ± 5 |
HDCA | 8 | 11 ± 2 | 9.6 | 0.9957 | 4000–7.8 | 43 ± 8 | 33 ± 6 | 39 ± 9 |
LCA | 13 | 10 ± 2 | 15.5 | 0.9963 | 4000–7.8 | 95 ± 13 | 60 ± 4 | 68 ± 4 |
7-oxoLCA | 8 | 10 ± 2 | 10.5 | 0.9902 | 4000–7.8 | 76 ± 22 | 68 ± 5 | 79 ± 3 |
α-MCA | 13 | 17 ± 3 | 6.3 | 0.9952 | 4000–7.8 | 77 ± 6 | 64 ± 12 | 60 ± 9 |
β-MCA | 13 | 11 ± 2 | 6.7 | 0.9922 | 4000–7.8 | 77 ± 11 | 66 ± 13 | 61 ± 8 |
ω-MCA | 13 | 16 ± 4 | 5.9 | 0.9954 | 4000–7.8 | 76 ± 7 | 59 ± 7 | 54 ± 2 |
UDCA | 13 | 11 ± 2 | 9.3 | 0.9962 | 4000–7.8 | 62 ± 15 | 68 ± 6 | 74 ± 4 |
G-CA | 3 | N.A.b | 6.6 | 0.9926 | 4000–0.98 | 79 ± 13 | 71 ± 15 | 52 ± 12 |
G-CDCA | 6 | N.A.b | 9.8 | 0.9970 | 4000–0.98 | 61 ± 6 | 58 ± 12 | 61 ± 8 |
G-DCA | 1 | N.A.b | 10.4 | 0.9978 | 4000–0.98 | 57 ± 24 | 62 ± 3 | 58 ± 5 |
G-LCA | 1 | 17 ± 4 | 13.1 | 0.9936 | 4000–0.98 | 53 ± 1 | 56 ± 5 | 69 ± 4 |
G-7-oxoLCA | 1 | 18 ± 4 | 7.5 | 0.9910 | 4000–0.98 | 55 ± 1 | 64 ± 3 | 77 ± 6 |
G-UDCA | 1 | 12 ± 3 | 6.1 | 0.9951 | 4000–0.98 | 60 ± 12 | 60 ± 6 | 73 ± 5 |
T-CA | 1 | 14 ± 3 | 4.5 | 0.9955 | 4000–0.98 | 67 ± 18 | 92 ± 10 | 80 ± 4 |
T-CDCA | 0.2 | 10 ± 4 | 7.7 | 0.9900 | 4000–0.1221 | 65 ± 17 | 87 ± 9 | 89 ± 6 |
T-DCA | 0.2 | 10 ± 3 | 8.3 | 0.9947 | 4000–0.1221 | 78 ± 12 | 73 ± 6 | 83 ± 5 |
T-LCA | 0.4 | 12 ± 1 | 11.4 | 0.9910 | 4000–0.1221 | 62 ± 2 | 79 ± 5 | 93 ± 4 |
T-7-oxoLCA | 1 | 14 ± 3 | 5.2 | 0.9963 | 4000–0.98 | 60 ± 4 | 81 ± 3 | 96 ± 4 |
T-β-MCA | 0.2 | 10 ± 3 | 1.9 | 0.9945 | 4000–0.1221 | 71 ± 10 | 110 ± 3 | 80 ± 6 |
T-UDCA | 0.2 | 11 ± 1.5 | 4.1 | 0.9967 | 4000–0.1221 | 73 ± 3 | 78 ± 4 | 93 ± 3 |
Low extraction recoveries were observed for a few BAs (HDCA, G-DCA, G-LCA and G-UDCA), in contrast to a previous study using a similar extraction procedure.14 A possible explanation for these discrepancies may be the presence of matrix components interfering with the extraction of the aforementioned BAs, because identical experiments using charcoal-treated instead of untreated rat serum provided superior extraction recoveries (Table S1†). Overall, extraction recoveries using acetonitrile were reproducible across the concentration studied and ranged from 33% to 110% in untreated serum and from 70% to 88% in charcoal-treated serum. According to the FDA guidelines for validation of analytical methods, the recovery of a given analyte does not need to be 100%, but the amount of recovery must be consistent, precise and reproducible.34 Due to the fact that HDCA extraction recovery was discrepant between untreated and charcoal-treated samples, its value may not reflect the absolute concentration. Regarding matrix effects, the ionization of BAs studied was not affected by the matrix components at the three concentrations studied (Table 4). Overall, our findings are in line with that of other investigators.18
Relative response (%) | |||
---|---|---|---|
20 nM | 200 nM | 2000 nM | |
a Data is presented as the average ± CV (%). | |||
CA | 108 ± 6 | 99 ± 14 | 114 ± 5 |
CDCA | 91 ± 3 | 110 ± 6 | 115 ± 1 |
DCA | 90 ± 15 | 114 ± 1 | 114 ± 3 |
7-oxoDCA | 87 ± 10 | 104 ± 10 | 110 ± 2 |
HDCA | 100 ± 10 | 111 ± 5 | 111 ± 1 |
LCA | 77 ± 9 | 105 ± 1 | 106 ± 3 |
7-oxoLCA | 87 ± 10 | 115 ± 7 | 114 ± 5 |
α-MCA | 103 ± 15 | 103 ± 8 | 109 ± 1 |
β-MCA | 95 ± 13 | 104 ± 10 | 115 ± 3 |
ω-MCA | 92 ± 13 | 99 ± 6 | 112 ± 2 |
UDCA | 97 ± 7 | 105 ± 9 | 113 ± 2 |
G-CA | 89 ± 15 | 102 ± 8 | 113 ± 2 |
G-CDCA | 92 ± 12 | 102 ± 7 | 114 ± 1 |
G-DCA | 96 ± 15 | 114 ± 2 | 111 ± 4 |
G-LCA | 107 ± 2 | 113 ± 2 | 115 ± 2 |
G-7-oxo-LCA | 112 ± 3 | 113 ± 1 | 111 ± 2 |
G-UDCA | 105 ± 4 | 120 ± 1 | 115 ± 3 |
T-CA | 98 ± 3 | 96 ± 6 | 115 ± 2 |
T-CDCA | 114 ± 6 | 107 ± 3 | 114 ± 2 |
T-DCA | 111 ± 10 | 113 ± 1 | 118 ± 1 |
T-LCA | 106 ± 7 | 114 ± 1 | 112 ± 3 |
T-7-oxo-LCA | 99 ± 2 | 112 ± 2 | 111 ± 2 |
T-β-MCA | 84 ± 12 | 99 ± 6 | 97 ± 4 |
T-UDCA | 98 ± 4 | 115 ± 4 | 115 ± 2 |
1 Week sham | 1 Week UNX | Fold increase | 1 Week sham | 2 Weeks UNX | Fold increase | 4 Weeks sham | 4 Weeks UNX | Fold increase | |
---|---|---|---|---|---|---|---|---|---|
a The results are expressed in nM as mean ± standard deviation (n = 8). N.D.: not detected. Underlined values represent below lower limit of quantification. Sham, sham-operated control rats; UNX, uninephrectomized rats. Statistics: * for p ≤ 0.05. | |||||||||
Primary BAs | |||||||||
α-MCA | 381 ± 332 | 642 ± 326 | 1.7 | 616 ± 486 | 481 ± 391 | 0.8 | 256 ± 187 | 328 ± 2000 | 1.3 |
β-MCA | 357 ± 335 | 428 ± 166 | 1.2 | 346 ± 256 | 429 ± 384 | 1.2 | 252 ± 201 | 338 ± 235 | 1.3 |
CDCA | 483 ± 420 | 1052 ± 436* | 2.2 | 801 ± 576 | 794 ± 577 | 1 | 644 ± 602 | 380 ± 310 | 0.6 |
CA | 795 ± 653 | 1860 ± 1515 | 2.3 | 1037 ± 881 | 1192 ± 1094 | 1.1 | 884 ± 576 | 830 ± 669 | 0.9 |
Total | 2016 ± 202 | 3983 ± 632* | 2 | 2800 ± 292 | 2896 ± 351 | 1 | 2035 ± 310 | 1878 ± 242 | 0.9 |
Secondary BAs | |||||||||
LCA | 14 ± 7 | 17 ± 9 | 1.3 | 9 ± 6 | 14 ± 4 | 1.6 | 5 ± 1 | 6 ± 2 | 1.2 |
DCA | 22 ± 15 | 46 ± 34 | 2.1 | 38 ± 27 | 58 ± 48 | 1.5 | 40 ± 25 | 55 ± 24 | 1.4 |
UDCA | 176 ± 161 | 300 ± 137 | 1.7 | 243 ± 162 | 230 ± 168 | 0.9 | 131 ± 62 | 160 ± 93 | 1.2 |
HDCA | 199 ± 119 | 214 ± 158 | 1.1 | 372 ± 371 | 398 ± 419 | 1.1 | 209 ± 247 | 213 ± 248 | 1 |
7-oxoLCA | 23 ± 25 | 38 ± 23 | 1.6 | 35 ± 30 | 55 ± 52 | 1.6 | 22 ± 16 | 23 ± 14 | 1.1 |
7-oxoDCA | 482 ± 590 | 479 ± 222 | 1 | 716 ± 779 | 645 ± 745 | 0.9 | 249 ± 254 | 323 ± 184 | 1.3 |
ω-MCA | 69 ± 36 | 111 ± 61 | 1.6 | 107 ± 79 | 163 ± 155 | 1.5 | 90 ± 64 | 121 ± 67 | 1.4 |
Total | 986 ± 169 | 1204 ± 170* | 1.2 | 1519 ± 256 | 1563 ± 228 | 1.0 | 746 ± 94 | 901 ± 114 | 1.2 |
Taurine-conjugated BAs | |||||||||
T-UDCA | 3 ± 1 | 4 ± 1* | 1.2 | 3 ± 1 | 5 ± 4* | 1.9 | 2 ± 1 | 4 ± 3 | 1.5 |
T-CDCA | 52 ± 22 | 59 ± 16 | 1.1 | 33 ± 19 | 31 ± 13 | 0.9 | 25 ± 10 | 34 ± 21 | 1.3 |
T-CA | 150 ± 38 | 189 ± 79 | 1.3 | 134 ± 73 | 213 ± 85* | 1.6 | 123 ± 60 | 190 ± 124 | 1.5 |
T-DCA | 6 ± 4 | 8 ± 6 | 1.3 | 7 ± 3 | 12 ± 9 | 1.8 | 7 ± 5 | 12 ± 9 | 1.8 |
T-LCA | N.D. | 1 ± 0.7* | N.D. | 2 ± 4 | N.D. | 1 ± 0.6* | |||
T-α-MCA | 320 ± 100 | 300 ± 88 | 0.9 | 279 ± 99 | 348 ± 159 | 1.2 | 269 ± 92 | 286 ± 168 | 1.1 |
T-β-MCA | 56 ± 31 | 47 ± 7 | 0.8 | 48 ± 29 | 85 ± 64 | 1.8 | 62 ± 26 | 67 ± 48 | 1.1 |
T-7oxo-LCA | 3 ± 1 | 3 ± 1 | 1.3 | 2 ± 1 | 5 ± 3* | 2 | 3 ± 1 | 3 ± 2 | 1.2 |
Total | 591 ± 112 | 612 ± 110 | 1 | 506 ± 98 | 700 ± 127 | 1.4 | 490 ± 94 | 597 ± 107 | 1.2 |
Glycine-conjugated BAs | |||||||||
G-CA | 29 ± 13 | 27 ± 15 | 0.9 | 51 ± 58 | 93 ± 95 | 1.8 | 79 ± 84 | 84 ± 76 | 1.1 |
G-UDCA | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |||
G-CDCA | 3 ± 1 | 3 ± 2 | 0.9 | 5 ± 2 | 8 ± 8 | 1.6 | 7 ± 6 | 7 ± 6 | 1 |
G-DCA | 0.4 ± 0.4 | 1 ± 0.9 | 1.2 | 1.5 ± 1.6 | 4 ± 6 | 2.9 | 3 ± 4 | 4 ± 3 | 1.2 |
G-LCA | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |||
G-7oxo-LCA | N.D. | N.D. | N.D. | N.D. | N.D. | N.D. | |||
Total | 33 ± 14 | 31 ± 13 | 0.9 | 57 ± 24 | 106 ± 44 | 1.8 | 90 ± 38 | 95 ± 40 | 1.1 |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ay26520j |
This journal is © The Royal Society of Chemistry 2013 |