Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Abstract

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.

Fig. 1.

CDP-choline (A) and phosphatidylethanolamine-N-methyltransferase (PEMT) (B) pathways for synthesis of liver phosphatidylcholine. Enzymes indentified are CK, choline kinase; CT, CTP-choline phosphate cytidylyltransferase; CPT, CDP-choline phosphotransferase; CDH, choline dehydrogenase; BADH, betaine aldehyde dehydrogenase; and BHMT, betaine-homocysteine methyltransferase.

Fig. 2.

Incorporation of methyl-d₉-labeled choline into mouse liver_. Methyl-d₉-choline (1 mg) was injected ip, and the mouse was euthanized 1.5 h later. A total lipid extract of liver was analyzed by diagnostic ESI-MS/MS scans. A: precursors of m/z 184 (P184) for endogenous PC; (B) precursors of m/z 193 (P193) for PC species synthesized by the CDP-choline pathway (methyl-d₉-labeled) (C) precursors of m/z 187 (P187) for PC species synthesized by the PEMT pathway (methyl-d₃-labeled); and (D) neutral loss of m/z 141 (NL141) for diacyl PE. Spectra are aligned on the x-axis to enable direct comparisons of phospholipid species with equivalent combinations of fatty acids. The molecular species nomenclature is A:a/B:b, where A and B represent the total number of carbon atoms in each fatty acid at the sn-1 and sn-2 positions of the glycerophosphate backbone, and a and b represent the number of unsaturated double bonds in each fatty acid.

Fig. 3.

Fractional enrichment of methyl-d₉ and methyl-d₃ labels in liver, lung, or plasma total PC in vivo. A: Direct incorporation of methyl-d₉ label by the CDP-choline pathway into mouse liver (circles) and lung (squares) total PC over a 24 h period after ip injection of 1 mg of methyl-d₉-choline chloride (n = 5 mice/group). B: Incorporation of methyl-d₃ label in SAMe mice, demonstrating the absence of the PEMT pathway in mouse lung. C and D: Time courses of incorporation of the methyl-d₉ (squares) and methyl-d₃ (circles) labels into, respectively, mouse an human plasma. Results were calculated as a percentage of total PC, given by the sum of P184, P187, P190, and P193 for all selected species (means ± SD).

Fig. 4.

Molecular specificity of incorporation of methyl-d₉ (CDP-choline pathways, panels A, C, and E) and methyl-d₃ (PEMT pathway, panels B, D, and F) into PC from mouse liver (A, B), mouse plasma (C, D), and human plasma (E, F). Incorporations into four individual PC species were chosen as representative of PC synthesis by both pathways. Results are percentages of incorporations into each PC species at each time point, calculated relative to total incorporation. Solid bars represent the fractional concentration of the SAMe species in endogenous PC.

Fig. 5.

Incorporation of methyl-d₃ label into selected PC species in mouse liver (A), mouse plasma (B) and human plasma (C). Fractional incorporation of methyl-d₃ into PC16:0/22:6, the major product of the PEMT pathway, and PC16:0/18:2, the major product of the CDP-choline pathway.

Fig. 6.

Enrichment of stable isotope labels in liver choline phosphate and S-adenosylmethionine (SAMe). A: Methyl-d₉ enrichment in mouse liver choline phosphate was calculated from the ratio of the MRM transitions from 183.8 → 85.8 and 192.8 → 94.8. Methyl-d₃ enrichment of liver SAMe was calculated by MIDA from the P187 and P190 scans from mouse liver and plasma (B) and human plasma (C).

Fig. 7.

Absolute rates of liver PC synthesis by the PEMT pathway. Results were derived from MIDA-corrected methyl-d₃ incorporations into mouse liver and plasma (A) and human plasma (B) and are presented as fractional synthetic rates of the pool of total liver PC.

Fig. 8.

Absolute rates of synthesis by the PEMT pathway of selected molecular species of liver PC. Rates of PC synthesis were calculated from MIDA-corrected methyl-d₃ incorporations into five individual PC molecular species for mouse liver (A), mouse plasma (B), and human plasma (C). Results are presented as fractional synthetic rates for each individual PC species.