Validated Method for the Quantification of Free and Total Carnitine, Butyrobetaine, and Acylcarnitines in Biological Samples

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)­HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes

Plasma and tissue BCAA and BCKA concentrations in lean and obese Zucker rats.

<p>Concentrations of BCAAs (Leu, Val and Ile: A, C, E) and BCKAs (KIC, KIV, and KMV: B, D, F) are shown for plasma (A, B), gastrocnemius muscle (C, D) and liver (E,F). Bars are mean ± SE; *P<0.05, n = 9 and 8 determinations for lean and obese rats, respectively.</p

Branched chain keto acid dehydrogenase activity in lean and obese Zucker rats^*.

*<p>Data from cohort C. Enzyme activities were measured at 30°C. Values are means ± SEM; n = 10 rats per group. NS indicates not significant, P>0.05.</p>a<p>Muscle and adipose tissue BCKDC activities were measured using the ¹³C method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone.0059443-Matsumoto1" target="_blank">[57]</a>, which does not use U indicating µmol of NADH formed. BCKDC total activities were unavailable for muscle; neither total nor actual BCKDC activities were available for adipose tissue. Unfortunately, activation of BCKDH using lambda phosphatase did not work in these experiments. Additionally, the adipose tissue values were very low and too close to the level of detection and contamination from room air and are not reported.</p

Whole-body proteolysis and muscle proteolysis in lean and obese Zucker rats.

<p>Whole-body proteolysis was expressed either per kg of body weight (BW) (A) or fat free mass (FFM) (B). Values are mean ± SE; * P<0.05, n = 9 and 8 for lean and obese rats, respectively, in A and B. The ratio of urinary muscle derived 3-MeHis to creatinine (C) was calculated from 24-h urinary amounts of 3-MeHis and creatinine to normalize 3-MeHis excretion by creatinine (D). Values are mean ± SE; * P<0.05, n = 10 for both groups in C and D.</p

24 h Urine amino acids in lean and obese Zucker rats (µmol/g creatinine).

<p>Values are means ± SEM; n = 10 rats per group. NS indicates a P>0.05 or that the comparison was flagged as a potential false discovery when the Q was set to 0.01 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone.0059443-Benjamini1" target="_blank">[58]</a>.</p

Plasma concentrations (µM) of amino acids and related metabolites in lean and obese rats.

<p>Values are means ± SEM; n = 10 rats per group. NS indicates a P>0.05 or that the comparison was flagged as a potential false discovery when the Q was set to 0.01 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone.0059443-Benjamini1" target="_blank">[58]</a>.</p

Plasma ¹⁴C KIC and Leu specific activity (SA) and turnover in lean and obese Zucker rats.

<p>Plasma ¹⁴C-SA for KIC (A) and Leu (B) were calculated dividing DPM radioactivity of KIC and Leu with their amounts in each sample. Leu turnover (Leu flux or rate of appearance) was calculated by dividing Leu infusion rate with the plasma ¹⁴C KIC SA and expressed per kg of body wt (BW) (C) or fat free mass (FFM) (D). Values are mean ± SE; * P<0.05, n = 9 and 8 for lean and obese rats, respectively.</p

Illustration of experimental set up for measuring Leu flux in rats.

<p>After being filtered through mist (Fuller’s Earth) and CO₂ (Soda lime) absorbent, a constant stream of air flowed through one tube to a closed metabolic cage with a quiet convection fan (Columbus Instruments, Columbus, OH) and out through two outlet tubes which bubbled into a 50 ml conical tube containing Hyamine 10X hydroxide (Perkin Elmer, Waltham, MA)-ethanol (1∶1, vol/vol) for CO₂ fixation. ¹⁴CO₂ samples were collected every 10 min throughout the infusions. The rat was implanted with a jugular vein catheter and infused with [¹⁴C]-NaHCO₃ (Moravek Biochemical, Brea, CA) and then [1-¹⁴C]-Leu (Moravek Biochemical, Brea, CA). [¹⁴C]-NaHCO₃ and [1-¹⁴C]-Leu were each sequentially infused for 2 h. Three blood samples were collected at the 90 and 105 min (0.5 ml) and 120 min (∼3 ml) after start of [1-¹⁴C]-Leu infusion. A carotid catheter (not shown) came through the same sealed cage port as the jugular cannula pair and was used for blood sampling.</p

Food, fluid, protein and Leu intakes with urine outputs in lean and obese Zucker rats^*.

*<p>Data from cohort C (the cohort in which no radioactivity was used for metabolomics/enzyme activity assays). Values are means ± SE, n = 9–10 per group. NS indicates not significant (P>0.05). Leucine and protein intake are determined from the values provided by the manufacturer of the diet. Small differences in percent changes (+98 vs +97%) are due to removal of significant figures and rounding.</p

Body weight, organ weights and plasma glucose and insulin concentrations of lean and obese rats^*.

*<p>Weights in g were obtained from cohort B (the cohort used for protein synthesis using the [³H]-Phe) and represent the sum of the weight of the left and right side where applicable. Plasma values are from cohort A (the cohort used for [¹⁴C]-Leu metabolism studies). Values are means ± SE, n = 9–10 per group. Percent differences were determined before rounding. NS indicates not significant (P>0.05). Data on amount of protein per g wet weight for some tissues is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059443#pone-0059443-t007" target="_blank">Table 7</a>.</p