Pirjo Mattila

Effects of dietary phytase and cholecalciferol on phosphorus bioavailability in rainbow trout /Oncorhynchus mykiss

A study was conducted to examine the influence of dietary inclusion of phytase and high levels of cholecalciferol on phytate phosphorus (P) utilization of rainbow trout. Triplicate groups of rainbow trout, initial weight 51.6 g, were fed in excess diets containing 0 or 1500 units phytase kg−1 and 2500, 250,000 or 2,500,000 IU cholecalciferol kg−1 for 12 weeks. The basal diet provided 5.8 and 3.2 g total and phytate P kg−1 dry matter, of which most was derived from soy protein concentrate. Weight gain of fish was increased by phytase supplementation but decreased by higher cholecalciferol concentrations. Inclusion of phytase improved P availability as indicated by significantly higher apparent availability of P and bone ash and plasma and body P concentrations. Dietary cholecalciferol content did not influence P utilization. An increase in dietary cholecalciferol concentration caused higher deposition of calcium, magnesium and zinc in the kidney. Both cholecalciferol and phytase supplementation significantly (P<0.05) increased the hepatic cholecalciferol concentration. In summary, dietary phytase supplementation was effective in reducing P load of rainbow trout fed soybean protein concentrate-based diet. High levels of cholecalciferol (250,000 and 2,500,000) had no beneficial effect on P utilization.

Proanthocyanidins in Common Food Products of Plant Origin

The contents of extractable and unextractable proanthocyanidins were determined in a large number of commercial food products of plant origin available in Finland. Proanthocyanidins were extracted with aqueous acetone-methanol and quantified by normal phase high-performance liquid chromatography (HPLC) according to their degree of polymerization. Unextractable proanthocyanidins were analyzed from the extraction residue by reversed phase HPLC after acid-catalyzed depolymerization as free flavan-3-ols (terminal units) and benzylthioethers (extension units). Proanthocyanidins were detected in 49 of 99 selected food items. The highest contents per fresh weight were determined in chokeberries, rose hips, and cocoa products. Berries and fruits were generally the best sources of proanthocyanidins, whereas most of the vegetables, roots, and cereals lacked them completely. Many of the samples contained a significant proportion of insoluble proanthocyanidins, which need to be quantified as well if total proanthocyanidins are studied. Considerable variation was observed in proanthocyanidin contents in berries, which requires further research.

Sterol and vitamin D2 contents in some wild and cultivated mushrooms

Abstract The contents of vitamin D 2 and sterols in some wild and cultivated mushrooms were determined, and the distribution of these compounds in different parts of the wild mushrooms was evaluated. In addition, the variation in vitamin D 2 contents between individual fruiting bodies of wild mushrooms was studied. Vitamin D 2 was determined using an HPLC method, including saponification and semipreparative normal-phase HPLC purification before analytical reversed-phase quantification with an internal standard. Sterol contents were analysed with gas chromatography using an internal standard method, including saponification before derivatizing sterols to trimethylsilyl ethers. Mass-spectral analyses were used to further confirm the identification of sterols. Vitamin D 2 was almost totally absent in cultivated mushrooms, while some wild mushrooms contained high concentrations of this vitamin (4.7–194 μg/100 g dry weight). Ergosterol was the most abundant sterol found in mushrooms, and its contents were higher in cultivated mushrooms (602.1–678.6) than in wild mushrooms (296–489 mg/100 g dry weight). The contents of vitamin D 2 and ergosterol varied greatly and moderately, respectively, between different parts of the mushrooms and were lowest in stipes. In addition, high variation in vitamin D 2 contents between individual fruiting bodies was found.

Simultaneous HPLC analysis of fat-soluble vitamins in selected animal products after small-scale extraction.

Abstract A method for simultaneous determination of fat-soluble vitamins in animal products is presented. Milk, and two fish products containing many ingredients, were used as test materials. The vitamins determined were tocopherols, β-carotene, all- trans -retinol and in the case of fish, cholecalciferol. The sample preparation procedure, consisting of saponification and extraction by n -hexane–ethyl acetate was carried out in small-scale. To cope with the highly different composition of the test materials, some modifications were needed. Normal-phase HPLC separation using diisopropyl ether- n -hexane gradient elution with UV and fluorescence detections for tocopherols, β-carotene and all- trans -retinol was used. Cholecalciferol was determined from the same extract by reverse-phase HPLC (with UV detection) after semi-preparative HPLC purification. Recoveries of spiked samples varied from 80 to 111% for all determined fat-soluble vitamins. The day-to-day repeatability was in most cases under 7%, and the within-day variation of this method was small, under 5.5% for all vitamins. The described analytical method is effective and fast, enabling the processing of a large number of samples.

Determination of phylloquinone in oils, margarines and butter by high-performance liquid chromatography with electrochemical detection

Abstract A high-performance liquid chromatographic (HPLC) method for the determination of phylloquinone in oils and margarines following purification of hexane solutions of oils or hexane extracts of margarines by straight-phase semipreparative HPLC is described. Phylloquinone was quantified by reverse-phase HPLC with a dual-electrode electrochemical detector operating in the redox mode. Menaquinone-4 (MK-4) was used as an internal standard. By this method the phylloquinone present in the main oils and margarines available on the Finnish market was determined.The same method, but without the internal standard, was applied to butter. The detection limit of phylloquinone was 50 pg per injection, and its recovery when added to oil and margarine samples and quantified by the internal standard method was 98% and 102%, respectively. The mean phylloquinone content of oils ranged from 1·5 μg (refined rapeseed oil) to 0·10 μg (sunflower oil). In the soft margarines with 80% fat, the phylloquinone levels were 0·89 – 1·1 μg g −1 . The phylloquinone content of margarines with 40% and 60% fat correlated with their fat content. Blended and hard margarines contained less phylloquinone than the soft margarines with corresponding fat contents. The contribution of oils and margarines to the average daily dietary intake of phylloquinone in Finland was estimated to be approximate 40 μg.

Bioavailability of various polyphenols from a diet containing moderate amounts of berries.

Berries are a rich source of various polyphenols. The objective of this study was to investigate the bioavailability of polyphenols from berries. Middle-aged subjects (n = 72) consumed moderate amounts of berry or control products for 8 weeks in a randomized, placebo-controlled dietary intervention trial. Average intake of berries was 160 g/day (bilberries, lingonberries, black currants, and chokeberries). Plasma and urine polyphenols were analyzed by GC-MS and HPLC and berry polyphenols by HPLC. The total intake of polyphenols was 837 mg/day. Plasma quercetin, p-coumaric acid, 3-hydroxyphenylacetic acid, caffeic acid, protocatechuic acid, vanillic acid, homovanillic acid, and 3-(3-hydroxyphenyl)propionic acid increased significantly from the baseline in the berry group compared to the control group (p < 0.05). The urinary excretion of quercetin, p-coumaric acid, and 3-hydroxyphenylacetic acid increased significantly in the berry group compared to the control group (p < 0.05). In conclusion, a number of polyphenols are bioavailable from a diet containing moderate amounts of blue and red berries.

Polyphenol and vitamin C contents in European commercial blackcurrant juice products

Vitamin C and polyphenol contents (anthocyanins, proanthocyanidins, phenolic acids and flavonols) were analysed in commercial blackcurrant juice products purchased from various European countries (Finland, Poland, Germany, United Kingdom) using HPLC methods. The aim was to study variation between countries, as well as evaluate the intake of polyphenols from commercial juices. There was significant variation in the contents of polyphenols and vitamin C between countries. Expressed as the ready-to-drink beverages, German, Polish, Finnish and British products averaged anthocyanin contents of 38, 32, 12 and 7.5mg/2.5dl, proanthocyanidin contents of 27, 24, 10 and 1.2mg/2.5dl, flavonol contents of 16, 15, 5.2 & 1.9mg/2.5dl and phenolic acid contents of 12, 8.9, 3.7 and 1.5mg/2.5dl, respectively. The mean vitamin C content was highest in British (70mg/2.5dl) and lowest in Finnish products (15mg/2.5dl). The intake of polyphenols from German and Polish ready-to-drink beverages was clearly higher than that from Finnish, and especially, British beverages.

Determination of vitamin D3 in egg yolk by high-performance liquid chromatography with diode array detection

Abstract A high-performance liquid chromatographic (HPLC) method for the determination of cholecalciferol (vitamin D 3 ) in egg yolk is described. The egg yolk samples were purified by saponification and extraction as well as efficient solid-phase extraction and semipreparative straight-phase HPLC. Vitamin D 3 was quantitated with reverse-phase HPLC using an internal standard (vitamin D 2 ) method and diode array detection. The suitability of the method was tested by analyzing eggs on sale through retail outlets as well as samples obtained straight from farms. The detection and determination limits of the method were 2 and 8 ng per injection, respectively. Overall mean recovery for both vitamins was 70%. The recovery of vitamin D 3 calculated on the basis of the internal standard was 94%. The day to day repeatability of the method expressed by the coefficient of variation was 3.6%. The method introduced here is well suited for the determination of vitamin D 3 contents in egg yolks; quantification was reliable and accurate.

INFLUENCE OF LOW DIETARY CHOLECALCIFEROL INTAKE ON PHOSPHORUS AND TRACE ELEMENT METABOLISM BY RAINBOW TROUT (ONCORHYNCHUS MYKISS, WALBAUM)

Abstract A study was conducted to investigate the relationship between dietary cholecalciferol and phosphorus (P) metabolism in rainbow trout ( Oncorhynchus mykiss ). Triplicate groups of rainbow trout fingerlings, initial weight 12.4±0.7 g, were fed to satiation one of four semipurified diets containing either 3.2 or 5.6 g kg −1 P and either 100 or 2600 IU kg −1 cholecalciferol in a 2×2 factorial design. The fish were reared in freshwater for 20 weeks on a 12 h photoperiod regime without exposure to UV-radiation. Growth and feed efficiency of fish were not influenced by dietary cholecalciferol and P levels. Fish fed the diet containing 3.2 g P kg −1 exhibited poor bone mineralization, low plasma phosphate level and reduced carcass P concentration. The low level of dietary vitamin D 3 (100 IU kg −1 ) significantly improved bone mineralization. Liver cholecalciferol concentration was higher (4.9 vs 9 ng g −1 ) at 2600 IU kg −1 cholecalciferol but dietary P had no effect on the liver content of this vitamin. Higher dietary P significantly increased urine P concentration whereas dietary cholecalciferol level had no effect. Dietary P supplementation increased body ash, Ca and P but Mg and Mn levels decreased significantly. Although cholecalciferol supplementation increased Mn deposition, carcass Zn content decreased. While tripling in weight during the trial, rainbow trout reared in freshwater effectively utilized phosphorus from a low-cholecalciferol diet.

New analytical aspects of vitamin D in foods

Abstract A comprehensive study of vitamin D in foods was undertaken during 1991–1994 in Finland to develop high-performance liquid chromatographic (HPLC) methods for analysing cholecalciferol, ergocalciferol and their 25-hydroxylated metabolites in fish, egg yolk, meat, milk, edible mushrooms and fortified foods, and to evaluate which vitamin D compounds should be analysed to estimate the total vitamin D contents in these foods. The procedures included saponification, extraction and purification using one or two chromatographic steps prior to quantification by HPLC using internal standard methods. Ergocalciferol was used as an internal standard for cholecalciferol and vice versa, and, as first presented in the present study, 25-hydroxyergocalciferol was used as an internal standard for 25-hydroxycholecalciferol and vice versa. The methods developed were well suited to determining the contents of ergocalciferol, cholecalciferol and their 25-hydroxylated metabolites in food. Good estimates of the vitamin D content of fish or edible mushrooms are obtained if only cholecalciferol or ergocalciferol, respectively, are determined. On the other hand, in addition to cholecalciferol 25-hydroxycholecalciferol should also be determined from egg yolk, meat and milk.