Vitamin D content and variability in fluid milks from a US Department of Agriculture nationwide sampling to update values in the National Nutrient Database for Standard Reference.

This study determined the vitamin D(3) content and variability of retail milk in the United States having a declared fortification level of 400 IU (10 µg) per quart (qt; 1 qt=946.4 mL), which is 25% daily value per 8 fluid ounce (236.6 mL) serving. In 2007, vitamin D(3) fortified milk (skim, 1%, 2%, whole, and 1% fat chocolate milk) was collected from 24 statistically selected supermarkets in the United States. Additionally, 2% milk samples from an earlier 2001 USDA nationwide collection were reanalyzed. Vitamin D(3) was determined using a specifically validated method involving HPLC with UV spectroscopic detection and vitamin D(2) as an internal standard. Quality control materials were analyzed with the samples. Of the 120 milk samples procured in 2007, 49% had vitamin D(3) within 100 to 125% of 400 IU (10 µg)/qt (label value), 28% had 501 to 600 IU (12.5-15 µg)/qt, 16% had a level below the label amount, and 7% had greater than 600 IU (15 µg)/qt (>150% of label). Even though the mean vitamin D(3) content did not differ statistically between milk types, a wide range in values was found among individual samples, from nondetectable [<20 IU (0.5 µg)/qt] for one sample to almost 800 IU (20 µg)/qt, with a trend toward more samples of whole milk having greater than 150% of the labeled content. On average, vitamin D(3) in 2% milk was higher in 2007 compared with in 2001 [473 vs. 426 IU (11.8 vs. 10.6 µg)/qt].

Atmospheric pressure chemical ionization mass spectrometry for analysis of lipids.

Atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) has proven to be a very valuable technique for analysis of lipids from a variety of classes. This instrumental method readily produces useful ions with gentle fragmentation from large neutral molecules such as triacylglycerols and carotenoids, which are often difficult to analyze using other techniques. Molecules that are easily ionized, such as phospholipids, produce molecular ions and diagnostically useful fragment ions that are complementary to those produced by methods such as electrospray ionization MS with collision-induced dissociation. The simplicity and versatility of APCI-MS make it an ideal tool for use in solving hitherto very difficult analytical problems.

Analysis of triacylglycerol positional isomers in food products as brominated derivatives by high-performance liquid chromatography coupled with a flame ionization detection.

Reversed-phase HPLC resolution and HPLC-flame ionization detection quantitation of model triacylglycerol positional isomer pairs (important in the study of food formulation lipids) after facile conversion to brominated derivatives is reported. The positional isomers in the triacylglycerol pairs were at least 98% resolved from each other during reversed-phase HPLC. Triacylglycerol quantitation obtained by HPLC-flame ionization detector was checked against standard positional isomer pairs known by mass. The flame ionization detection area percent gave absolute error range of 0.3-1.6% per triacylglycerol.

Effect of oleic and linoleic acids on the production of deep-fried odor in heated triolein and trilinolein.

To determine sources of desirable deep-fried flavor in frying oils, degradation products from heated triolein and trilinolein with 5-31% polar compounds representing low to high deterioration were evaluated by purge-trap gas chromatography-mass spectrometry-olfactometry. (E,E)-2,4-Decadienal, 2-heptenal, 2-octenal, 2,4-nonadienal, and 2,4-octadienal produced deep-fried odor at moderate-strong intensities in heated trilinolein. However, unexpected aldehydes-2,4-decadienal, 2,4-undecadienal, 2,4-nonadienal, and 2-octenal (all <15 ppm)-were produced in triolein heated for 6 h. These dienals possibly were produced by hydroperoxidation and/or hydroxylation followed by dehydration of 2-alkenals. The 2-alkenals were produced from thermal decomposition of hydroperoxides, epoxides, and keto and dimeric compounds produced during the heating of triolein. These aldehydes produced low intensities of deep-fried odor in triolein. This information helps to explain sources of the deep-fried flavor that is characteristic of high linoleic frying oils but which is only at low intensity levels in high oleic frying oils.

Autoxidation products of normal and genetically modified canola oil varieties determined using liquid chromatography with mass spectrometric detection.

Normal, high stearic acid and high lauric acid canola oil varieties were heated in the presence of air to allow autoxidation to occur. After the reaction, the oils were analyzed using a non-aqueous reversed-phase high-performance liquid chromatographic separation followed by detection using atmospheric pressure chemical ionization mass spectrometry. Oxidized products were separated and identified. The major autoxidation products which remained intact were epoxides and hydroperoxides. Two classes of epoxy triacylglycerols (TAGs) were formed. One class with the epoxy group replacing a site of unsaturation and one class adjacent to a site of unsaturation, as was previously reported for model TAGs. Intact oxidation products resulted mostly from oxidation of oleic acid, while oxidation products of linoleic and linolenic acid chains decomposed to yield chain-shortened species. Both neutral and polar chain-shortened products were observed. Polar chain-shortened decomposition products eluted at very short retention times and required a different chromatographic gradient to separate the molecules. This class of molecules was tentatively identified as core aldehydes. The high stearic acid canola oil yielded more intact oxidation products containing stearic acid, as expected. The high lauric acid oil produced intact oxidation products which contained lauric acid.

Triacylglycerol analysis of potential margarine base stocks by high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry and flame ionization detection.

Several margarine base stock candidates have previously been prepared for the purpose of finding better, more oxidatively stable food components: high-saturate vegetable oils, randomized vegetable oils, vegetable oil-hard stock blends, and interesterified vegetable oil-hard stock blends. Here are reported the triacylglycerol compositions of these products, determined using reverse-phase high-performance liquid chromatography (HPLC) coupled with a flame ionization detector or a quadrupole mass spectrometer with an atmospheric pressure chemical ionization source. Triacylglycerol percent composition results for samples of known composition (randomized and interesterified samples) exhibited less average error by HPLC coupled with a quadrupole mass spectrometer with an atmospheric pressure chemical ionization source, after application of response factors, than the results by HPLC coupled with a flame ionization detector. The fatty acid compositions calculated from the mass spectrometric data exhibited less average error than the fatty acid compositions resulting from the flame ionization detector data. The average error of the fatty acid compositions by the mass spectrometer was lowest for interesterified blend samples, next lowest for randomized samples, then followed by high-saturated fatty acid oils, normal oils, and blends. Analysis of the vegetable oil-hard stock blends by mass spectrometer required special treatment for calculation of response factors.

Non-volatile products of triolein produced at frying temperatures characterized using liquid chromatography with online mass spectrometric detection.

Oxidation products from triolein under model heated frying conditions have been analyzed using liquid chromatography with an evaporative light scattering detector and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Triolein was heated at 190 degrees C with 2% water added each hour, to simulate the moisture of a frozen product, until polar components reached approximately 30%. The samples were separated using reversed-phase high-performance liquid chromatography with APCI-MS detection. Triolein oxidation products included hydroperoxides, epoxides and a ketone. Other products were formed by shortening of an acyl chain on the intact triolein. Normal and oxygen-containing products formed by the dimerization of triolein were also observed. Other products included chain addition products formed by addition of acyl chain subunits to intact triolein to form higher molecular weight products.

Dual parallel mass spectrometers for analysis of sphingolipid, glycerophospholipid and plasmalogen molecular species.

Analysis of phospholipids was performed using a liquid chromatographic separation with two mass spectrometers in parallel providing electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) data simultaneously from a triple quadrupole instrument and a single quadrupole instrument, respectively. The output from UV-Vis and evaporative light scattering detectors were also acquired by the two mass spectrometers, respectively, for four detectors overall. This arrangement was used to identify and calculate area percents for molecular species of dihydrosphingomyelin (DHS) and sphingomyelin (SPM) in commercially available bovine brain SPM, in human plasma extract and in porcine lens extract. Molecular species of phosphatidylethanolamine and its plasmalogen, and phosphatidylcholine and its plasmalogen were identified and semi-quantitative analysis performed. Commercially available bovine brain SPM was found to contain 11.5% DHS and 88.5% SPM. The only DHS molecular species identified in human plasma was 16:0-DHS, at or below 1% of the sphingolipid content. Porcine lens membranes were found to contain 14.4% DHS and 85.6% SPM. Other findings reported here include: (1) phospholipids were found to undergo dimerization in the electrospray source, giving masses representing combinations of species present. (2) Triacylglycerols gave usable mass spectra under electrospray ionization conditions, as well as under APCI-MS conditions. (3) Triacylglycerols gave ammonium adducts as base peaks in their APCI mass spectra, which reduced fragmentation and increased the proportions of molecular ions. (4) Mass spectra were obtained for phospholipids which underwent both protonation and sodium adduct formation in different chromatographic runs.

Liquid chromatography/mass-spectrometric characterization of sphingomyelin and dihydrosphingomyelin of human lens membranes.

The identity of the major phospholipid component of human lens membrane extracts, previously referred to as the 'unknown phospholipid', was recently proposed to be 4,5-dihydrosphingomyelin (DHS). Using high-performance liquid-chromatographic separation with atmospheric pressure chemical ionization and electrospray ionization mass-spectrometric detection, we report here the first identification of the molecular species of DHSs and sphingomyelins (SPMs) of the human eye lens. The most abundant molecular species were palmitic and tetracosenoic (24:1) DHSs, representing 57.8 and 23.3% of human lens DHSs, respectively. Lesser amounts of hexacosanoic, hexacosenoic, tetracosanoic, docosanoic, docosenoic, stearic, palmitoleic, myristic and other DHSs were found. The most abundant normal SPM molecular species in the human lens were similar to those of DHS. Palmitic SPM represented 53.9% of the uncorrected SPM peak areas, while tetracosenoic SPM represented 17.6% of the SPM in the human lens. The sphingolipids of the human lens were determined to be composed of 76.9% DHS species and 23.1% SPM species. Commercially available SPM was also found to contain significant amounts of DHS species.

Quantitative analysis of triglycerides using atmospheric pressure chemical ionization-mass spectrometry.

Atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) was used for quantitative analysis of triglycerides (TG) separated by reverse-phase high-performance liquid chromatography. APCI-MS was used for analysis of mono-acid TG standards containing deuterated internal standard, of a synthetic mixture of heterogeneous TG, of randomized and normal soybean oils and of randomized and normal lard samples. Quantitation of the TG by four approaches based on APCI-MS were compared, and these were compared to quantitation obtained using liquid chromatography (LC) with flame-ionization detection (FID). The APCI-MS methods were based on (i) calibration curves from data for mono-acid TG standards, (ii) response factors obtained from a synthetic mixture of TG, (iii) response factors calculated from comparison of randomized samples to their statistically expected compositions, and (iv) response factors calculated from comparison of fatty acid (FA) compositions calculated from TG compositions to FA compositions obtained by calibrated gas chromatography (GC) with FID. Response factors derived from a synthetic mixture were not widely applicable to samples of disparate composition. The TG compositions obtained using APCI-MS data without application of response factors had average relative errors very similar to those obtained using LC-FID. Numerous TG species were identified using LC/APCI-MS which were undetected using LC-FID. Two quantitation methods, based on response factors calculated from randomized samples or on response factors calculated from FA compositions, both gave similar results for all samples. The TG compositions obtained using response factors calculated from FA compositions showed less average relative error than was obtained from LC-FID data, and were in good agreement with predicted compositions for the synthetic mixture and for randomized soybean oil and lard samples.

Lipid-protein interactions in human and bovine lens membranes by Fourier transform Raman and infrared spectroscopies.

In other systems, proteins have been shown to alter the molecular structures of lipids in the cell membrane bilayer. We wished to determine if proteins altered the structure of lens lipids. The structure of lipid hydrocarbon chains in urea purified human lens membrane vesicles containing intrinsic, hydrophobically bound proteins was compared to the structure of lipids in vesicles without protein. Fourier transform Raman spectroscopy was used to characterize lipid and protein structure. To study lipid interactions with extrinsic, surface bound proteins, the lipid structure was compared in bovine lipid vesicles with and without alpha-crystallin bound to the surface of the membrane. Lipid structure was studied using Fourier transform infrared spectroscopy. No change in lipid structure was detected even at protein/lipid weight ratios of two to one. Human lens intrinsic proteins contained a high amount of a helical structure (60%), but did not alter hydrocarbon chain interactions.

Thermodynamic phase transition parameters of human lens dihydrosphingomyelin.

Dihydrosphingomyelin (DHS) is the major phospholipid in the human lens. The influence of this phospholipid on membrane structure and function is not known. In this study we used infrared spectroscopy to determine the thermodynamic and molecular structural properties of the hydrocarbon chains of DHS membranes isolated from human lenses. The phase transition temperature of human lens DHS was 9 degrees C higher than for bovine brain sphingomyelin membranes and 14 and 7 degrees C higher than human lens cortical and nuclear membranes, respectively. This increase in the phase transition temperature results in 20% increase in lipid order at 36 degrees C in comparison to that of native membranes and bovine brain sphingomyelin. DHS is likely to provide structural order to the hydrocarbon chain region and upholds the integrity of native membranes under oxidative conditions.

Structural characterization of clear human lens lipid membranes by near-infrared Fourier transform Raman spectroscopy.

Regional differences in human lens membrane lipid composition have been documented and could be responsible for alterations in the function of lens membranes. The phospholipid composition of epithelial membranes of human lenses has been shown to be different from that of fiber membranes. To establish lipid composition-membrane structure relationships, we have examined spectroscopically the structure of lipid membranes from human lens epithelium, cortex and nucleus. Near-infrared Fourier transform Raman spectroscopy was used to obtain the lipid structure of membranes in which the lipid composition was determined previously by 31P-NMR. The disorder (fluidity measured structurally) of the epithelium was evaluated to be 80%, whereas that of the lipids from the cortical and nuclear regions was 55%. The large size of the band at 1650 cm-1 arising from sphingolipids supported the compositional studies which indicate that the major component of human lens membranes is a sphingolipid. Sphingolipids probably account for the high degree of lipid order found in lens membranes. Epithelial membranes were found to contain more glycerolipids and less sphingolipids than fiber cell membranes. This compositional difference would be expected to disorder the epithelial membrane.

Analysis of triglycerides using atmospheric pressure chemical ionization mass spectrometry.

Atmospheric pressure chemical ionization (APCI) mass spectrometry was investigated as a new method for analysis of a mixture of triglycerides separated by reverse-phase high-performance liquid chromatography. A mixture of homogeneous (monoacid) triglyceride standards containing fatty acids with zero to three double bonds was analyzed to demonstrate the quality of mass spectra obtained by using the APCI interface. The mass spectra showed that minimal fragmentation occurs, resulting primarily in diglyceride [M-RCOO]+ ions and [M + 1]+ protonated molecular ions. The degree of unsaturation within the acyl chains had a marked effect on the proportion of diglyceride ions vs. the [M + 1]+ ions formed in the APCI source. The mass spectra of triglycerides containing fatty acids with two or three double bonds showed predominantly protonated triglyceride ions, with diglyceride peaks representing 13 to 25% of the base peak. The triglycerides containing singly unsaturated fatty acids gave diglyceride ions as the base peak, and [M + 1]+ ions with an intensity 20 to 28% that of the base peak. Only diglyceride ions were observable in the spectra of triglycerides containing saturated fatty acids.

Separation and characterization of the unknown phospholipid in human lens membranes.

PURPOSE: The major component of human lens membranes was thought to be sphingomyelin until 1991, when a study by phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy revealed the presence of an unknown phospholipid that constituted approximately half the human lens phospholipids. The objective of this work was to isolate this phospholipid and to elucidate its identity. METHODS: The separation of sphingomyelin from the unknown was accomplished using high-performance liquid chromatography (HPLC) and an amino-bound column. Sphingomyelin standard and the membranes from human lenses were chromatographed. Chromatographic fractions were collected and spectrally characterized by proton (1H) NMR and 31P NMR spectroscopy. RESULTS: The chromatographic method did not affect the integrity of the sphingomyelin. Besides the bands corresponding to the unknown components, the chromatogram of the human lens membranes showed three large peaks, the central one with a shoulder, with elution times similar to that for sphingomyelin. The 1H NMR spectra for the fractions collected during the elution of these peaks showed differences. The study by 31P NMR indicated that the first peak contained the unknown phospholipid. The subsequent fractions showed the presence, in different relative levels, of both the unknown and sphingomyelin. By comparison and interpretation of the two-dimensional 1H NMR spectra for sphingomyelin and for the fraction containing the unknown, the unknown phospholipid is proposed to be 4,5 dihydrosphingomyelin, in which the site of unsaturation present in the sphingosine moiety is no longer present. CONCLUSIONS: The ability to separate the unknown from sphingomyelin and the power of 1H NMR spectroscopy allowed the proposition of the identity of the major component of human lens membranes as 4,5-dihydrosphingomyelin. Although the synthetic compound is known to be involved in the formation of extended hydrogen-bonding networks, its biologic and physicochemical properties need further study.

Regional and age-dependent differences in the phospholipid composition of human lens membranes.

PURPOSE: The long-term purpose of this research was to establish the relationships between composition, structure, and function that affect human lens membranes. The authors hypothesized that the functional differences of epithelial, cortical, and nuclear lens membranes are related to compositional differences. Furthermore, age-dependent alterations in membrane function and structure can also be related to variations in the phospholipid composition. To explore these possibilities, the authors determined the phospholipid composition of epithelial, cortical, and nuclear membranes from pools of human lenses of different ages. METHODS: Membranes were extracted from pools of clear human lenses of different ages using a monophasic methanolic extraction that minimizes the interfacial fluff produced with biphasic extractions. The phospholipid composition was determined by 31P NMR: RESULTS: Only minor differences were detected between cortical and nuclear fractions. All phospholipids, except sphingomyelin, phosphatidylethanolamine, and the phospholipid with a shift of 0.12 parts per million (ppm) in the 31P NMR spectrum, showed significant differences in the epithelial fractions of all age groups compared to the fiber fractions; the percentage of phosphatidylcholine was considerably higher than that in the cortical and nuclear membranes of the same age. Conversely, the percentage of phosphatidylglycerol and lysophosphatidylglycerol was significantly smaller in the epithelial membranes than in the fiber membranes. The age-related changes in the composition of cortical and nuclear membranes were identical. These membranes showed a steady increase with age in the percentage of sphingomyelin and of an unidentified component with a shift of 1.2 ppm. The percentage of phosphatidylcholine decreased with age in both epithelial and fiber membranes. The rate of decrease was greater in the epithelial membranes than in the fiber membranes. Epithelial membranes contained approximately five times more phosphatidylcholine than fiber membranes of corresponding age. CONCLUSION: Regardless of age, the composition of epithelial cell membranes was different than that of cortical and nuclear membranes, which showed similar phospholipid content. This suggests that significant compositional changes occur when epithelial cells become elongated to form fiber cells.

Comparison of specific blue and green fluorescence in cataractous versus normal human lens fractions.

PURPOSE: The authors compared the specific green and blue fluorescence levels in soluble and insoluble fractions (cortical and nuclear) extracted from cataractous lenses with those corresponding to clear lenses and tried to establish the nature of the role of extrinsic fluorophores in cataractogenesis. METHODS: Laser-induced fluorescence was measured with an optical-fiber sensor. The specific fluorescence was evaluated as the ratio of the fluorescence intensity to the protein concentration. Four brunescent, three pure nuclear, and four mixed nuclear-subcapsular cataractous lenses were investigated. RESULTS: Specific blue fluorescence levels in cataractous fractions were similar to those in clear lens fractions, except for the insoluble nuclear fractions in which the levels were slightly lower. The specific green fluorescence in the soluble cataractous fractions showed marked increases compared with the clear lens fractions of similar ages. The insoluble cataractous fractions had similar (cortical fractions) or slightly lower (nuclear fractions) specific green fluorescence levels compared with the normal lens fractions. CONCLUSIONS: The similarity in blue fluorescence levels suggests that the blue fluorophore(s), although increasing in concentration with age, are not indicators of cataractogenesis and may not play an active role in opacification. The levels of specific green fluorescence indicate either a dramatic increase in the fluorescence quantum efficiency and/or an increase in the number of fluorescent sites per protein unit. The green-to-blue ratios were greater by a factor of as much as six in all cataractous soluble fractions versus clear ones. This suggests an accelerated formation of the green fluorescent species in cataractous tissues.