Carnitine Precursors and Short-Chain Acylcarnitines in Water

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Food and Beverage Chemistry/Biochemistry

Carnitine Precursors and Short-Chain Acylcarnitines in Water Buffalo Milk Luigi Servillo, Nunzia D'Onofrio, Gianluca Neglia, Rosario Casale, Domenico Cautela, Massimo Marrelli, Antonio Limone, Giuseppe Campanile, and Maria Luisa Balestrieri J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02963 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Journal of Agricultural and Food Chemistry

Carnitine Precursors and Short-Chain Acylcarnitines in Water Buffalo Milk

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Luigi Servillo§, Nunzia D’Onofrio§, Gianluca Neglia#, Rosario Casale§, Domenico Cautela†, Massimo Marrelli¶, Antonio Limone‡, Giuseppe Campanile#, Maria Luisa Balestrieri§*

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§

Department of Precision Medicine, University of Campania “L. Vanvitelli”, Naples, Italy

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#

Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.

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†

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¶

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‡

Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi, Azienda Speciale della Camera di Commercio di Reggio Calabria, Italy Marrelli Health, Maxillofacial Surgery Section, Crotone, Italy Istituto Zooprofilattico Sperimentale del Mezzogiorno, Naples, Italy

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*Correspondence: Maria Luisa Balestrieri, Department of Precision Medicine, University of

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Campania “L. Vanvitelli”, via L. De Crecchio 7, 80138, Naples, Italy. Tel.: +39 081 5667635; Fax:

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+39 081 5665863. Email: [email protected]

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ORCID: 0000-0001-6001-1789

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Key words: Nε-Trimethyllysine, γ-Butyrobetaine, δ-Valerobetaine, Carnitine, Short-chain

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Acylcarnitines, Buffalo, Milk.

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ACS Paragon Plus Environment


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Abstract

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dietary Nε-trimethyllysine. Among ruminant’s milk, the occurrence of δ-valerobetaine, along with

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carnitine precursors and metabolites, has not been investigate in buffalo milk, the second most

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worldwide consumed milk, well known for its nutritional value. HPLC-ESI-MS/MS analyses of

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bulk milk revealed that the Italian Mediterranean buffalo milk contains δ-valerobetaine at levels

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higher than bovine milk. Importantly, we detected also γ-butyrobetaine, the L-carnitine precursor,

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never described so far in any milk. Of interest, buffalo milk shows higher levels of acetylcarnitine,

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propionylcarnitine, butyrylcarnitine, isobutyrylcarnitine, and 3-methylbutyrylcarnitine

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(isovalerylcarnitine) than cow milk. Moreover, buffalo milk shows isobutyrylcarnitine and

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butyrylcarnitine at a 1 to 1 molar ratio, while in cow's milk this ratio is 5 to 1. Results indicate a

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peculiar short-chain acylcarnitine profile characterizing buffalo milk widening the current

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knowledge about its composition and nutritional value.

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Ruminants’ milk contains δ-valerobetaine originating from rumen through the transformation of

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1. Introduction L-Carnitine

(Cnt),

a

water-soluble

quaternary

amine

(3-hydroxy-4-N,N,N-

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trimethylaminobutyric acid) ubiquitous in plant, animal, and microbial kingdoms, plays a key role

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in energy metabolism as it facilitates the long-chain fatty acid shuttling from the cytosol into the

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mitochondrial matrix, regulates the mitochondrial acyl-CoA/CoA ratio and prevents acylation of

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free CoA buffering the free CoA pool through the formation of acylcarnitines.1 The active transport

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of carnitine from plasma into tissues occurs via a family of carnitine/organic cation transporters

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(OCTN) with the plasmalemmal OCTN22-5 showing a broad specificity also for other cationic

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metabolites in animal tissues, including carnitine esters and γ-butyrobetaine (γ-BB), the ultimate

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precursor of carnitine.6-8 Mammals are able to synthesize carnitine endogenously from Nε-

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trimethyllysine (TML) released in the course of protein breakdown or from free TML occurring in

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plants.9,10

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Free TML, present at consistent levels in animal plant feedstuff, is particularly abundant in

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alfalfa, an important forage for ruminants in which, differently from non-ruminants, carnitine

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degradation and synthesis are specifically regulated by rumen microbes.10,11 TML of dietary source

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is also transformed by rumen microbiota into δ-valerobetaine (δ-VB), a recently identified

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constitutive metabolite of ruminant milk and meat.

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differs between ruminants and non-ruminants milk and meat with higher levels in ruminant (cattle,

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goat, sheep)

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Indeed, the distribution of δ-VB consistently

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Among ruminants, buffalo milk (Bubalus bubalis), the second most consumed milk

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worldwide, is one of the richest milks from a compositional point of view having fat, mineral, and

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protein content higher than cow milk.13-18 These peculiar features make it highly suitable for the

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production of dairy products, including yogurt, superior cream, butter, soft and hard cheeses, with

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particular regard to mozzarella cheese, produced in Italy under the European Union’s protected

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designation of origin scheme. In this regard, Italian Mediterranean buffalo has reached high 3



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productivity and reproductive standards thanks to the remarkable advancements of the Italian

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buffalo-breeding program.18,19 The fatty acid fraction of Italian Mediterranean buffalo milk, from

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the qualitative point of view, includes also those fatty acids present in small concentrations that may

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have important effects on human health.

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Short-chain fatty acids are produced in the rumen and represent a primary energy source for

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ruminants. In fact, during anaerobic fermentation in the rumen, carbohydrates ingested with the diet

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are converted mainly into acetic acid, propionic acid and butyric acids, generally referred to as

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volatile fatty acids (VFA), which are the most important source of energy for these animals.21 VFA,

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which provide about 75% of the metabolizable energy, can have major effects on production and

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product composition as they may indirectly influence animal cholesterol synthesis, insulin or

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glucagon secretion, and affect functions of the large intestine, cecum, and rumen. 21 As for branched

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short-chain fatty acids, isobutyric, 2-methylbutyric, and 3-methylbutyric acids, collectively called

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isoacids, they are efficiently produced in the rumen by deamination and subsequent decarboxylation

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of valine, isoleucine and leucine, respectively, which come largely from dietary protein digestion.22

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Then VFA and isoacids, produced in the rumen, are rapidly absorbed in the blood by action of

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specific carriers of the family of the monocarboxylate transporters present on the epithelium of

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various tracts of the animal's gastrointestinal tract, and used by body tissues for lipid biosynthesis,

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gluconeogesis or milk formation.23-25 Given the richness of buffalo milk composition, showing

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health properties different from those of cow milk, as it possesses a low allergenic potential and

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provides benefits for obesity, hypertension and osteoporosis13, an increase of knowledge regarding

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the natural substance content could be useful to drive the dairy industry toward the production of

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buffalo milk and buffalo milk-based products with improved nutritional properties. The presence of

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δ-VB, recently described in cattle, goat, and sheep milk, opened a new scenario about the ruminant

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metabolite with important roles in animal physiology and consumers health.12 To our knowledge,

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the content of carnitine precursors (TML and γ-BB) in buffalo milk is unexplored. In the present 4


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study, with the aim at enhancing our understanding on the buffalo milk components with potential

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health benefits, we investigated the possible presence of carnitine precursors and carnitine esters.

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2. Materials and Methods

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2.1 Reagents

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Nε-trimethyllysine

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carboxypropyl)trimethylammonium chloride (γ-butyrobetaine chloride), L-carnitine (Cnt) inner salt,

(TML),

(C2Cnt),

5-aminovaleric

propionyl-L-carnitine

acid,

chloride,

acetyl-L-carnitine

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isobutyryl-L-carnitine

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methylbutyryl-L-carnitine) (3-MeC4Cnt) were from Sigma-Aldrich (Milan, Italy). 2-Methylbutyryl-

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L-carnitine (mixture of diastereomers) was from Toronto Chemical Research (North York, Canada).

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Milli-Q water was used for all the preparations of solutions and standards. The solutions of 0.1%

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formic acid in water and 0.1% formic acid in acetonitrile used for the HPLC-ESI-MS/MS analyses

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were from Sigma-Aldrich (Milan, Italy).

valeryl-L-carnitine

butyryl-L-carnitine

(3-

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(i-C4Cnt),

(C3Cnt),

pivaloyl

(n-C5Cnt),

(n-C4Cnt),

isovaleryl-L-carnitine

(3-

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2.2 Animals management

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The study was conducted in Southern Italy (between 40.5° N and 41.5° N). A total of 250 Italian

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Mediterranean buffaloes (130.7±4.4 days in milk) and 190 lactating Holstein cows (228.7±5.4 days

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in milk) of 129 kg metabolic weight. Buffalo and Holstein cows were maintained in open yards and

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fed with different diets: 50% forage, 0.93 MFU/kg of dry matter (DM), 14% crude protein/DM,

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42% NDF/DM and 26% ADF/DM in buffalo; 40% forage, 0.96 MFU/kg of dry matter (DM), 16%

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crude protein/DM, 34% NDF/DM and 20% ADF/DM in cattle. The average milk yield in buffalo

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and cattle were respectively 14.6 and 25.2 equivalent corrected milk (ECM = 730 Kcal).

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2.3 Milk sampling and preparation

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Bulk milk samples were collected every two weeks for 3 months. Aliquots of bulk were centrifuged

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at 3000xg for 15 min a 4°C to remove the fat globules, filtered through a 5 µm Millipore filters, and

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then stored frozen in aliquots until used. Before mass spectrometric analysis, aliquots were filtered

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through Amicon Ultra 0.5 mL centrifugal filters (3kDa molecular weight cutoff).

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2.4 Synthesis and purification δ-VB

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δ-VB was prepared as previously described.

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dissolved in 20 mL of methanol, added with 1 g of KHCO3, 10 mL of iodomethane, and stirred 12 h

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at room temperature. The addition of KHCO3 (1 g) and iodomethane (10 mL) was repeated twice

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more. At the end of the incubation, the mixture was centrifuged, the supernatant was evaporated to

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dryness at 40 °C, and the residue was dissolved in 10 mL of Milli Q grade water. Sample was

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applied on 10 cm column filled with a mixed-bed resin of Dowex-1-OH- and Biorex-70-H+ (1:1

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v/v). The aqueous wash from the Dowex-1-OH- and Biorex-70-H+ column was then applied to a 10

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x 2 cm column with AG50WX8-H+ resin. After a wash with 20 mL of water, the product was eluted

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with 30 mL of 6M NH4OH and evaporated to dryness under a stream of air.

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Briefly, about 100 mg of 5-aminovaleric acid was

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2.5 Synthesis of pivaloylcarnitine

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Pivaloylcarnitine (pivalCnt) was synthesized by reaction of pivaloyl chloride with carnitine. Briefly,

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5 mg of carnitine inner salt was suspended in 2 mL of anhydrous acetonitrile. The reaction started

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with the addition of 40 µL of triethylamine followed by 20 µL of pivaloyl chloride. The product

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formation was monitored over time by HPLC-ESI-MS/MS analyses of the reaction mixture after

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due dilution of the reaction mixture in 0.1% formic acid in water.

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2.6 Analysis by HPLC-ESI-MS/MS 6


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HPLC-ESI-MS/MS were performed with an Agilent LC-MSD SL quadrupole ion trap and a 1100

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series liquid chromatograph using two different chromatographic conditions. More in details, the

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milk content of TML, γ-BB, δ-VB, Cnt, C2Cnt and C3Cnt (Table 1) was determined by employing

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a Supelco Discovery C8 column, 250 x 3.0 mm, particle size 5 µm. The chromatography was

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conducted isocratically with 0.1% formic acid in water at flow rate of 100 µL/min. Instead, the

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content of n-C4Cnt, i-C4Cnt, 2-MeC4Cnt, 3-MeC4Cnt, and n-C5Cnt (Table 1) was determined by

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employing a Supelco Discovery C18 column, 12.5 x 3.0 mm, particle size 5 µm under isocratic

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conditions with a mixture (92:8 v:v) of 0.1% formic acid in water and 0.1% formic acid in

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acetonitrile, at flow rate of 100 µL/min. Volumes of 10 µL of standard solution or sample were

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injected.12 The HPLC-ESI-MS/MS analyses, performed in positive multiple reaction monitoring

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(MRM), allowed the compound identification on the basis of their retention times and MS2

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fragmentation patters. Quantification of each substance was obtained by comparison of the peak

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area of its most intense MS2 fragment with the respective calibration curve built with standard

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solutions. The standard addition method was used to assess the matrix effect in quantitative

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determinations. The instrumental conditions of the mass spectrometer, operating utilizing nitrogen

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as the nebulizing and drying gas, were as follows: nebulizer pressure, 30 psi; drying temperature,

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350°C; drying gas 7 l/min. The ion charge control (ICC) was applied with target set at 30000 and

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maximum accumulation time at 20 ms. The concentration of each compound was determined by

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comparison with the relative calibration curve built using standard solutions (0.2, 0.1, 0.05, 0.02,

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0.002 and 0.001 mg/L) prepared by serial dilution of standard stock solutions (2 mg/L) with water

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containing 0.1% formic acid. The linear regression analysis was carried out by plotting the peak

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areas of the monitored fragment ions versus the concentrations of the analyte standard solutions.

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The linearity of the instrumental response was assessed by correlation coefficients (r2) > 0.99 for all

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analytes. The stability of the compounds of interest was monitored by analysis after keeping them at

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room temperature for three days. No one of them showed any appreciable variation of its 7



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concentration over the time. The intra-day variability was measured by analysing spiked milk

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samples at four different concentrations in three replicates for each level for all the compounds

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analysed. Precision and accuracy for all the compounds in milk ranged from 95% to 105%. Carry-

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over was minimized by washing the injector with pure solvent before and after the injection.

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Each sample was analysed in triplicate and the mean concentration value of each compound was

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expressed as µmoles/L of milk.

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2.7 Statistical analysis

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Data are expressed as mean±SD. Differences were assessed by Student’s t-test, and P

Carnitine Precursors and Short-Chain Acylcarnitines in Water

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