Choline In Milk Research Articles

Enzymatic determination of choline in milk using a FIA system with potentiometric detection.

The development of a FIA system for the determination of total choline content in several types of milk is described. The samples were submitted to hydrochloric acid digestion before injection into the system and passed through an enzymatic reactor containing choline oxidase immobilised on glass beads. This enzymatic reaction releases hydrogen peroxide which then reacts with a solution of iodide. The decrease in the concentration of iodide ion is quantified using an iodide ion selective tubular electrode based on a homogeneous crystalline membrane. Validation of the results obtained with this system was performed by comparison with results from a method described in the literature and applied to the determination of total choline in milks. The relative deviation was always < 5%. The repeatability of the method developed was assessed by calculation of the relative standard deviation (RSD) for 12 consecutive injections of one sample. The RSD obtained was < 0.6%.

Determination of Choline in Milk and Infant Formulas by Enzymatic Analysis: Collaborative Study

A collaborative study was conducted on a coupled enzymatic-spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45-175 micrograms/100 g sample. After the combined acid hydrolysis-phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26-6.34%) with a HORRAT value of 0.91 (range, 0.76-1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42-0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.

Milk Choline Secretion as an Indirect Indicator of Postruminal Choline Supply

The objectives of this study were to evaluate milk choline as an indicator of choline absorption and to use milk choline to evaluate the efficacy of a rumen-protected choline supplement. In a preliminary 4-wk experiment, two Holstein cows in early lactation were abomasally infused with either 0 or 60 g/d of choline chloride in 2L of water, which was used as a carrier. Choline infusion increased milk choline secretion from 1.95 to 3.95 g/d during the 2-wk choline infusion period. In Experiment 2, four Holstein cows in early lactation were abomasally infused with 0, 25, 50, and 75 g/d of choline chloride in 2L of water using a 4 × 4 Latin square design with 1-wk experimental periods. Milk choline secretion was 2.56, 3.62, 3.72, and 3.82 g/d for the respective choline treatments. In Experiment 3, 10 Holstein cows in midlactation were fed either 0 or 50 g/d of choline using an experimental rumen-protected choline supplement during a 2-wk experiment. Milk choline secretion was increased from 2.12 to 2.99 g/d with the supplemental choline. Results of these experiments demonstrated that milk choline is responsive to postruminal choline supply and can be used as a qualitative indicator of choline absorption.

Determination of Choline in Milk and Dairy Products by an Enzymatic Method

An enzymatic method for determination of choline in milk and dairy products was developed. Samples were hydrolyzed for 45min at 100°C with 6N hydrochloric acid, and neutralized with 6N sodium hydroxide. For decomposition of residual H2O2 produced during the hydrolysis reaction, 0.8ml of 0.8% catalase solution was added, and decolorized with active carbon. Then 2.5ml of the filtrate was added to an equal volume of color reagent (Phospholipids C-Test Wako), and allowed to react for 5min at 37°C, and the absorbance was read at 600nm by spectrophotometer.A linear calibration plot for choline chloride was obtained between 1 and 20μg/ml. The recovery of choline (8-13mg/100ml) added to milk was more than 96%.This method is simple, rapid and accurate, and may be useful for the determination of choline in food.

Uptake of choline by rat mammary-gland epithelial cells.

The neonatal mammal requires especially large amounts of choline to sustain growth. Much of this choline is derived from the newborn's only source of food, milk. The concentration of choline in rat milk [182 +/- 24 microM (S.E.M.)] was much higher than that in maternal serum (11.6 +/- 0.9 microM), suggesting that a mechanism capable of concentrating choline into milk must exist. We characterized choline uptake by mammary epithelial cells (the site of milk production) of the lactating rat. We observed two uptake processes, one saturable and obeying Michaelis-Menten kinetics, and the other non-saturable and linear. At physiological blood choline concentrations, the saturable component of choline uptake predominated. The saturable component had Kapp. = 35 +/- 16 microM, and Vmax. = 1.24 +/- 0.19 nmol/h per mg of protein. Saturable uptake of choline was inhibited by hemicholinium-3. Ca2+ was required for uptake, but Mg2+ was not. Replacement Na+ with K+, Li+ or sucrose inhibited transport. Ouabain did not inhibit choline uptake. Choline concentration in epithelial cells was 67.7 +/- 1.9 nmol/g wet wt. at the start of incubation at 37 degrees C and rose to 80.9 +/- 6.5 nmol/g wet wt. over 30 min. Much of the choline accumulated by the mammary gland (in the presence of endogenous concentrations of choline) remained in the form of choline (50 +/- 1.2%), phosphatidylcholine (12 +/- 2.3%), lysophosphatidylcholine (0.1 +/- 0.03%), betaine (7 +/- 0.3% and phosphocholine (6 +/- 0.5%). In addition, we isolated 25 +/- 1.2% of choline-derived radiolabel in an unidentified compound.

Choline, Phosphatidylcholine and Sphingomyelin in Human and Bovine Milk and Infant Formulas

Choline is a precursor for the biosynthesis of phosphatidylcholine (lecithin), sphingomyelin, and choline plasmalogens—all essential constituents of membranes. Choline is also needed to make acetylcholine, a major neurotransmitter. The major choline-containing compounds of human milk (unesterified choline, phosphatidylcholine, sphingomyelin) were measured in samples obtained from mothers of full-term infants. Unesterified choline concentrations were highest (> 600 nmol/ml) during the first week of lactation, but thereafter remained relatively constant at 70–200 nmol/ml. There was no difference among foremilk, middle milk and hind milk, nor was there a diurnal pattern of variation in unesterified choline concentrations. Milk phosphatidylcholine and sphingomyelin concentrations remained relatively constant throughout lactation (100–200 nmol/ml). Hind milk always contained more of these phospholipids than did foremilk or middle milk. There was no consistent diurnal pattern of variation in milk concentrations of phosphatidylcholine or sphingomyelin. Milk contained no phospholipase activity capable of forming free choline from phosphatidylcholine or sphingomyelin. Bovine milk contained approximately the same concentrations of choline, phosphatidylcholine and sphingomyelin as did human milk from mothers more than 15 d postpartum. The same was true of “humanized” infant formulas made from cow’s milk. Soy protein-based formulas had much more unesterified choline (up to 650 nmol/ml) and much less sphingomyelin than did mature human milk.

Evidence for Lack of Phosphatidic Acid and Phospholipase D Activity in Milk

Phosphatidic acid could not be demonstrated when milk was assayed for phospholipase D activity even when lecithin was added. The concentration of “free” choline in milk remained the same before and after addition of lecithin. However, phosphatidic acid and an increased concentration of “free” choline were demonstrated when cabbage phospholipase D was added to raw milk. The concentration of phosphatidic acid and “free” choline in milk increased appreciably when both lecithin and cabbage phospholipase D were added to raw milk.Phospholipase D activity in milk was assayed at 23.5C, pH 5.6, in the presence of 10% vol/vol of diethyl ether and .015M calcium chloride. “Free” choline was determined by adjusting the acid whey from milk to pH 11 and adding a freshly prepared 3% methanolic solution of ammonium reineckate. The precipitated choline reineckate was washed, dissolved in acetone, and its absorbance measured at 520nm. The absence or presence of phosphatidic acid was shown by thin-layer chromatography.These results refute a previous report which proposed that both phosphatidic acid and phospholipase D were in milk.