Different choline supplement metabolism in adults using deuterium labelling

Choline has been defined as an essential nutrient, with daily adequate intake (AI) values for healthy adult men of 550 mg according to NAM [15] and 400 mg according to EFSA [24]. Whilst choline is a critical nutrient throughout the population [25, 26], its adequate supply is particularly important in preterm infants with higher needs because of exponential growth, and in patients in whom choline deficiency is pathognomonic. The latter includes cystic fibrosis (CF) patients suffering from exocrine pancreas insufficiency and those with short bowl disease, both suffering from a disturbed enterohepatic cycle of bile PC, and the latter being additionally dependent on long-term total parenteral nutrition, still mostly devoid of free choline [4, 9]. For all these patients, optimal ways of choline supplementation need to be investigated. In this study, we administered four different deuterium-labelled choline (D9-choline) supplements, to follow their assimilation and the plasma kinetics of D9-choline and its deuterium-labelled metabolites. We compared free D9-choline in the form of its chloride salt, the water-soluble organic esters D9-glycerophosphocholine (D9-GPC) and D9-phosphocholine, whose native correlates predominate in milk [27], and 1-palmitoyl-2-oleoyl-D9-PC (D9-POPC) in analogy to the predominant PC compound in egg yolk.

All water-soluble supplements rapidly and similarly increased D9-choline and D9-betaine plasma levels, with maximum concentrations achieved at 0.5–1 h. This is consistent with the results on single administration of an AI dosage of unlabeled choline supplements in adults [28]. Consistently, (D9-)choline in the form of salts or water-soluble organic esters results in rapid absorption and via portal circulation and incomplete hepatic absorption increase in plasma D9-choline. In line with this process and the rapid plasma turnover of choline (~ 1 h), all these water-soluble choline compounds exhibited only a transient elevation of plasma choline concentrations after single oral intake. Consequently, a continuous elevation of plasma concentration to physiological values requires either a retarded choline formulation or a more frequent intake, i.e. during every meal, rather than once or twice per day [3, 28].

The increase in plasma D9-choline concentration after D9-POPC administration, however, was different in two ways. First, plasma concentrations increased later, peak concentrations were delayed from 0.5–1 h to ~ 1.5 h and the peaks were broader, indicating different mechanisms of D9-choline assimilation from lipidic D9-POPC. Such more retarded and longer lasting increase may be of benefit for achieving a balanced increase in plasma choline concentrations. After ingesting the AI value of choline (550 mg) in the form of egg-PC (4012 mg), maximum values of plasma choline were delayed to 3 h, but maximum concentrations were similar to water-soluble choline components. This discrepancy may be due to the higher dosage of ~ 7 mg/kg choline in our previous study, compared to 2.7 mg/kg D9-choline equivalent used here as a tracer, and the higher sensitivity of metabolic follow-up using deuterium-labelled compared to native compounds [3, 7, 28]. Such differences compared to water-soluble compounds are explained by the complex mechanisms involved: after formation of gastric chymus and participation of (D9-)PC in triglyceride (butter pretzel, Table s1) emulsification, all PC must first be cleaved, mainly by pancreatic phospholipase A2 (sPLA2IB), as only lyso-PC is absorbed by enterocytes. These further degrade a part of lyso-PC to release choline, but partly resynthesize PC from lyso-PC for chylomicron formation [29] (see below). Hence, D9-POPC was degraded to D9-lyso-PC within the duodenal lumen at the surface of lipid micelles, absorbed and further processed within enterocytes, finally resulting in a delayed and lower increase in plasma (D9-)choline. We used a standardised butter pretzel comprising ~ 12 g fat, content together with the ingestion of D9-choline components. Similar to higher choline dosages in the form of PC, higher triglyceride amounts may further delay the increase of free (D9-) choline in response to (D9-PO)PC.

Second, the maximum value and AUC of plasma D9-choline were lower after D9-POPC compared to water-soluble D9-choline components. It was suggested that lyso-PC is only partly degraded to free choline within the enterocytes, whereas ~ 50% are re-synthesised to PC, using available coenzyme A-activated fatty acids (acyl-CoA) and lyso-PC acyltransferases (LPCAT) [30]. From plasma analyses in humans, we cannot calculate D9-choline and D9-PC pools as is feasible in animal experiments [20]. Nevertheless, the lower AUC of D9-choline (-50%) and of its water-soluble downstream metabolites D9-betaine (-65%) and D6-DMG (-82%) together with a higher AUC of D9-PC (+ 50%) in response to D9-POPC compared to the water-soluble D9-choline compounds suggest that (D9-PO)PC administration partly circumvents full hydrolysis to D9-choline for PC synthesis de novo and the synthesis of downstream metabolites for methyl donation [31, 32]. Nevertheless, endogenous (D3-)PC and (D3-)choline synthesis via the PEMT pathway is not significantly inhibited in response to (D9-PO)PC as a choline supplement (see below).

Plasma D9-PC and D3-PC metabolism in response to D9-choline supplements

PC is the major carrier for the plasma transport of long-chain polyunsaturated fatty acids (LC-PUFA), mainly arachidonic (ARA) and docosahexaenoic acid (DHA) to organs, via lipoproteins [33]. ARA and DHA deficiencies in preterm infants are associated with neonatal morbidity [34] and the clinical status of CF patients [10]. LC-PUFA are integrated in structural and functional membrane phospholipids of organs, like PC, phosphatidylethanolamine (PE) and others. Plasma transport of LC-PUFA via PC may, therefore, be essential for adequate development of the organs’ ‘lipidome’, especially that of the cerebrum, cerebellum and retina. Whereas hepatic very low-density lipoproteins (VLDL) comprise ~ 20% PC, chylomicrons contain ~ 8% PC as a coating so that their assembly within enterocytes requires PC formation. Such enterocytic PC synthesis partly originates from absorbed lyso-PC, using LPCAT3 that prefers polyunsaturated acyl-CoA as a substrate, and is, therefore, essential for plasma lipid transport of LC-PUFA [30]. Our results clearly show that LA, ARA, DHA and EPA are preferentially incorporated into (D9-)PC by the intestine, when (D9-PO)PC is used as a (D9-)choline supplement. In contrast, however, in the liver, DHA-PC is primarily and ARA-PC by 50% formed by methylation of PE via the PEMT pathway, requiring the formation of (D9-)betaine from (D9-)choline that is demethylated to (D6-) dimethylglycine for (D3-)methionine synthesis from homocysteine (Figs. 1B, 2A) [7].

In general, the formation of betaine, and its D9-enrichment correlated with the plasma concentrations of D3-PC (see supplementary Figure s5A). In this context, the lower increase in plasma D9-betaine and, subsequently, in D3-PC in response to D9-POPC, compared to its water-soluble D9-choline analogues, may be important for the homeostasis of methyl groups. In response to the ingestion of D9-POPC, compared to that of D9-choline chloride, D9-GPC and D9-phosphocholine, the generation of free D9-choline, D9-betaine and D6-dimethylglycine was decreased and, instead, (D9-PO)PC was used by the enterocytes via (D9-) lyso-PC re-acylation to PC for chylomicron formation. In line with this, D9-PC concentrations in plasma rapidly increase and maximal concentrations are highest after (D9-PO)PC as a choline supplement. Whilst D9-POPC resulted in lower plasma concentrations of free D9-choline, D9-betaine and D6-dimethylglycine (D6-DMG), it should be noticed that the administration of any water-soluble components not only normalises plasma choline concentrations, but may increase plasma betaine above physiological values [9, 35]. On the other hand, whereas the plasma concentrations of free (D9-)choline and (D9-)betaine were lower following (D9-PO)PC compared to water-soluble choline supplements, the synthesis of D3-PC via PE methylation by PEMT was not significantly decreased (Supplemental Fig. 5), suggesting that (D9-PO)PC as a choline source did not impair hepatic methyl group homeostasis.

Differential kinetics of plasma D9-PC in response to D9-choline supplements

Administration of water-soluble D9-choline compounds resulted in an increase of plasma D9-PC that showed uniform peak concentrations at 24-33 h, and a plasma half-life of 2-3d as previously demonstrated after both enteral and parenteral D9-choline chloride administration [3, 7]. They all result in the preferential hepatic synthesis and secretion of D9-PC species that are dominated by linoleic (LA) (D9-C18:2-PC) and oleic acid (OA) (D9-C18:1-PC) PC species, followed by those containing an ARA (D9-C20:4-PC), DHA (D9-C22:6-PC) or eicosapentaenoic acid (EPA) residue (D9-C20:5-PC) as described before [3, 7, 10].

Such fatty acid specificity is consistent with the PC synthesis de novo by adult liver, and contrasts to the preferential synthesis of C22:6-PC and C20:4-PC synthesis by the PEMT pathway using betaine as a methyl donor [7]. However, such fatty acid specificity does not apply to other organs, foetuses and newborns, where large amounts of C22:6-PC and C20:4-PC are derived from de novo PC synthesis, depending on fatty acid availability [1, 20, 35].

Importantly, assimilation of D9-POPC resulted in an earlier (9 h) and twofold higher peak concentration of D9-PC without plateau and an increased AUC. This indicates that (D9-)choline assimilation from (D9-PO)PC is faster with respect to plasma (D9-)PC increase, suggesting preferential use of ingested (D9-PO)PC for the (D9-)PC moiety of chylomicrons (see above). The similarly rapid decrease of D9-PC after D9-POPC and other compounds, reaching similarly low levels at 72 h (see Fig. 3a, insert), suggests that (D9-PO)PC results in higher (D9-) PC disposal in other organs. This is consistent with animal experiments showing high accretion of plasma PC by the brain and lung [20]. Finally, our data suggest that the intestine does not have a reservoir function for choline or PC like the liver, but only a ‘permissive’ function for peripheral supply derived from actually administered choline sources.

Impact of different choline supplements on endogenous PC synthesis by PEMT

Our data show that, in response to (D9-PO)PC, the plasma concentrations of free (D9-)choline and (D9-)betaine are decreased but their hepatic use for the synthesis of (D3-)methionine as a methyl donor and (D3-)PC synthesis from PE via the PEMT pathway is not decreased. Hence, hepatic secretion of VLDL, where both PC synthesis de novo and via the PEMT pathway are essential [23], should not be impaired by POPC supplementation compared to supplementation with water-soluble choline compounds. The contribution of the PEMT pathway to PC synthesis is low in many humans, just like in adult males studied here, e.g. in postmenopausal women, women with characteristic single nucleotide polymorphisms (SNPs) and, particularly, preterm infants. Notably, such individuals with low PEMT (e.g. SNP rs12325817) activity only suffer from decreased VLDL formation and hepatosteatosis in severe choline deficiency [36]. Moreover, the selectivity of ARA- and DHA-PC synthesis via the PEMT pathway only exists in the liver, whereas foetal (and preterm infant) requirements and plasma concentrations of ARA- and DHA-PC are high in spite low PEMT activity, and extrahepatic synthesis of these PC components is high, although they do not express PEMT (lung, intestine) [20, 29]. Our data are consistent with this, as after D9-POPC, there was a rapid and increased formation of polyunsaturated D9-PC, with increased contents not only of LA, but also of ARA, DHA and EPA.

Potential consequences of choline supplementation via PC instead of water-soluble components

The assimilation of D9-POPC, via cleavage to D9-lyso-PC, preferential small intestinal re-acylation to D9-PC and chylomicron assembly (see above), contrasted the hepatic D9-choline, -betaine, -PC and -VLDL metabolism as represented by the other D9-choline supplements. Although D9-POPC belongs to the D9-C18:1-PC subgroup, D9-C18:1-PC concentration was not increased here, but contrary to the other D9-choline supplements, remained a minor fraction of plasma D9-PC (see Figs. 4A and 5A). Instead, D9-POPC resulted in twofold to –threefold increases in D9-PC comprising LA, ARA and EPA. As the butter pretzels’ fat was dominated by OA (24%) over LA (22%), ARA (0.1%) and DHA (0%), the domination of polyunsaturated D9-PC is explained by the molecular specificity of LPCAT3 [31]. Such specificity of LPCAT3 suggests that, simultaneous supplementation of POPC as well as ARA and DHA at the expense of LA, might increase the ARA and DHA supply of organs. Further investigation is required to assess the quantitative impact of such combinations of increased LC-PUFA and reduced LA in combination with PC as a choline supplement.

Formation of (D9-)TMAO in response to (D9-)choline supplements

In addition to the efficacy of a choline supplement, safety is paramount. Therefore, we also investigated the concentration of D9-TMAO, the hepatic oxidation product of D9-TMA that is a bacterial D9-choline degradation product. TMAO has been described as a cardiovascular risk factor [17]. Notably, neither D9-POPC nor D9-phosphocholine resulted in detectable D9-TMAO concentrations. Whereas this was previously shown for (egg-)PC [28], absence of TMAO formation from phosphocholine is a new finding. Notably, D9-phosphocholine showed the earliest peak concentration of plasma D9-choline (0.75 h), compared to any other choline compound. Further research is required here to address phosphocholine assimilation as a choline supplement, possibly occurring earlier and more proximally in the small intestine than other compounds.

Differences in absorption may also apply to (D9-) choline chloride and (D9-)GPC, where the latter showed a later increase of TMAO (> 6 h) that was not detected in a previous study at 0–6 h [28]. Unfortunately, phosphocholine as a rapidly absorbed choline compound is not commercially available in nutritional bulk quantities, but taking the effects on (D9-)choline, (D9-)betaine and (D9-)PC levels as well as those on D9-PC fatty acid kinetics and (D9-) TMAO formation into account, we conclude that (PO)PC might be the best suitable supplement for adults, possibly in combination with phosphocholine. A water-soluble choline supplement in combination with PC appears important, as adults with choline deficiency also have low betaine concentrations in plasma and free choline is an important source of betaine as the major methyl donor [1, 3, 6, 20].

Limitations

The main limitation of the study is the small number of participants which limited the power to detect smaller differences in plasma kinetics, particularly between the different water-soluble supplements. Additionally, we only assessed healthy male adults, excluding the oestrogen impact on choline metabolism via higher PEMT expression in pre-menopausal women [36]. Moreover, adult men’s metabolic rate does not represent that of the fast growing preterm infant, nor is the impact of exocrine pancreas dysfunction, small intestinal pathologies and dysbiosis represented by these volunteers [9]. Nevertheless, a significant number of healthy pre-menopausal women have deleterious single nucleotide polymorphisms resulting in low PEMT expression like males [36]. Finally, in spite of exocrine pancreas insufficiency in CF patients, and massive fecal choline losses due to pancreatic phospholipase A2 deficiency, these patients profit from choline administration in the form of PC as intestinal phospholipase activity is not fully absent [9, 37, 38]. Hence, whilst further studies must follow with specific clinical cohorts, our data provide a frame work to optimise choline supplementation (Table 3).

Table 3 Time to peak (h) of concentrations of D9-choline and D9-choline metabolites in response to different D9-choline tracers

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