Serum depletion and bioavailability of lutein in Type I diabetic patients

Summary Background Lutein, a non-provitamin A carotenoid, is frequently consumed in the human diet. It is distributed preferentially in certain human tissues (i. e., retina) and shows a high antioxidant activity. Type 1 diabetic patients have been considered to be at risk of increased oxidative stress that may contribute to accelerated atherogenesis and to the microangiopathic complications of the disease. Aim of the study To assess the influence of type 1 diabetes mellitus on the serum depletion rate and bioavailability of lutein. Subjects and Methods Ten type 1 diabetics and eight controls consumed a low carotenoid diet for 21 days and the bioavailability study was performed in 7 diabetics and 5 controls on day 15 with the administration of a capsule of lutein esters from marigold extract. Samples were collected at baseline and on days 1, 2, 3, 6, 11, 15 (eight times during 9 h), 16, 17 and 21. Lutein and other carotenoids were determined by HPLC in triglyceride-rich lipoprotein (TRL) fractions and plasma or serum. Results Serum depletion curve, area of concentrations under time curve (AUC) and final concentration percentages were similar in diabetics and controls. In the bioavailability study, all-trans-lutein increased in both groups and AUC, maxima concentrations in TRL and serum and time required for maxima concentration in serum were similar in diabetics and controls. Conclusions These data suggest that in the group of patients assessed, type 1 diabetes mellitus does not apparently influence the absorption and depletion rate of lutein in serum. Received: 24 July 2001, Accepted: 18 December 2001     

Evaluation of polyphenol bioavailability in rat small intestine

Summary Background Dietary polyphenols, which are contained in several foods of plant origin, have been reported to be effective protective agents against cardiovascular diseases and cancer. However, data on their absorption from the gastrointestinal tract are still scarce and, often, contradictory. Aim of the study In this report, evaluation of polyphenol bioavailability was carried out by using segments of the small intestine from rat. The extent of absorption throughout the small intestine of rat was evaluated with two model compounds, tannic acid and catechin, as representatives of high and low molecular weight polyphenols, respectively. The consequence of the binding of tannic acid to BSA on both tannic acid absorption and in vivo protein digestibility was also examined. Methods Polyphenol solutions of different concentrations were injected into the lumen of ligated segments (6 cm) of the small intestine and the segments incubated in buffer for 5 min. The residual amount of polyphenol in the lumen of each segment was assayed by maximum absorption in the UV/VIS optical spectrum as was the amount of compound that had crossed the gut wall into the incubation buffer. Digestibility of BSA and of a BSA-tannic acid complex was assayed with rats. Results The results indicated a significant, concentration-dependent, disappearance of both polyphenols from the small intestine of the rat, with higher uptake levels being evident for tannic acid (50 %) than catechin (30 %). However, complete transfer through the gut wall was not observed with tannic acid whilst low but significant amounts (10 %) were detected in incubation buffers with catechin. Partial binding of polyphenols by endogenous proteins in the intestinal lumen was also demonstrated. Complexing tannic acid with BSA (1:10 mol/mol) was not found to affect either the extent of interaction of tannic acid with the small intestine or the in vivo digestibility of the protein. Conclusions Our experiments show that tannic acid and catechin both interact with the gut but only catechin appears able to traverse the gut. In addition, they provide evidence for binding of tannic acid and catechin by endogenous proteins in the intestinal lumen. This may limit their absorption from the small intestine. BSA complexed with tannic acid was as readily digested as BSA alone. This may suggest that tannic acid exerts anti-nutritional effects by binding to proteins of the gut wall and interfering with gut function rather than by inhibition of dietary protein digestion. Received: 1 February 2001 / Accepted: 14 May 2001     

The lack of effect of added dibasic calcium phosphate on the bioavailability of electrolytic iron in infant cereals

Infant cereals are generally fortified with about 1.0% Ca, 0.8% P and 0.03% Fe. The effect of such high levels of added Ca and P on the bioavailability of iron is not known. The purpose of this investigation was, therefore, to determine the effect of adding 0.25, 0.50 and 1.00% Ca as CaHPO₄.2H₂O to iron-fortified (0.03% added as electrolytic iron) rice cereal on the 2-week hemoglobin regeneration in iron-depleted male Sprague-Dawley rats. The mixed cereal with banana was also tested but only at the normal level of Ca and P. The cereals provided 30 or 60 mg Fe/kg diet. From the initial and final body weights, 2-week food intake and hemoglobin levels it was concluded that there was no significant effect of the Ca-P addition on the bioavailability of iron.     

Malvidin-3-glucoside bioavailability in humans after ingestion of red wine, dealcoholized red wine and red grape juice

Summary Background & Aims Dietary polyphenols, including anthocyanins, are suggested to be involved in the protective effects of red wine against cardiovascular diseases. Very little data are available concerning the bioavailability of anthocyanins, major sources of red pigmentation in red wine. The aim of this study was to compare changes in plasma malvidin-3-glucoside (M-3-G), a red wine anthocyanin, and its urinary excretion after ingestion of red wine, dealcoholized red wine and red grape juice. Design Six healthy male subjects were studied in a randomized cross over setting in a human nutrition research unit under controlled conditions. All subject consumed 500 mL of each beverage on separate days providing the following M-3-G quantities: red wine 68 mg, dealcoholized red wine 58 mg, and red grape juice 117 mg. M-3-G was measured by HPLC and photodiode detection. Results M-3-G was found in plasma and urine after ingestion of all the beverages studied. The aglycon, sulfate or glucuronate conjugates of M-3-G were not detected in plasma and urine. Increases in plasma M-3-G concentrations were not significantly different after the consumption of either red wine or dealcoholized red wine and were about two times less than those measured after consumption of red grape juice. This difference may be caused by the about two times higher M-3-G concentration determined in red grape juice. Area under the plasma concentration curves were as follows: 288±127nmol × h/L (red wine), 214±124nmol × h/L (dealcoholized red wine) and 662±210 nmol × h/L (red grape juice) and showed a linear relationship with the amount of anthocyanin consumed (mean±SD). Conclusions M-3-G is poorly absorbed after a single ingestion of red wine, dealcoholized red wine, or red grape juice and seems to be differentially metabolized as compared to other red grape polyphenols. Our results suggest that not anthocyanins such as M-3-G themselves but rather not yet identified anthocyanin metabolites and/or other polyphenols in red wine might be responsible for the observed antioxidant and health effects in vivo in subjects consuming red wine. Received: 28 May 2001, Accepted: 17 July 2001     

Enzymatic treatment of pork protein for the enhancement of iron bioavailability

The typical intervention for iron-deficiency anaemia is through oral supplementation with iron salts, which have unpleasant side effects. Therefore, there is a need for the development of supplements which will be absorbed more effectively and may have fewer side effects. This study investigated the effects of partially hydrolysed pork proteins on the bioavailability of non-haem iron. The peptides were derived using either pepsin or a combination of bacterial and fungal proteases, and their ability to deliver iron was evaluated in a rat intestine epithelial tissue model. The greatest iron absorption was achieved with peptides hydrolysed by pepsin of low molecular weight (<6–8?kDa). The peptides hydrolysed with bacterial and fungal enzymes may have bound to the iron too strongly, affecting bioavailability. Finally, hydrolysing proteins using pepsin in the presence of iron produces a complex that resulted in more ferritin expression than mixing the peptides with iron after hydrolysis.     

Functionality of endogenous folates from rye and orange juice using human <Emphasis Type="Italic">in vivo</Emphasis> model

Summary. Background: Cereals contribute about a quarter of the daily folate intake from a typical diet in several European countries. However, studies on bioavailability of endogenous folates, in particular of cereal sources, are scarce. Aim of the study: We aimed to study how well natural folates from rye (different rye breads and muesli made of malted rye) and orange juice function in improving folate status of human volunteers compared to a diet containing folic acid fortified wheat bread. Methods: Healthy human volunteers aged 20–66 y took part in a four-week intervention trial in which bread, breakfast cereal and juice were provided. The study had a parallel design with two groups, 1) rye and orange juice group (33 volunteers) and 2) fortified wheat bread and apple juice group (31 volunteers). The test foods provided on average 184 μg and 188 μg folate per day in rye and wheat groups, respectively. Test foods were consumed as part of the subjects' normal diet. Results: In both groups statistically significant increases in serum and red cell folates were observed after the intervention period. The serum folate increased 26 % and 31 %, and red cell folate levels increased 17 % and 15 % in rye and orange juice and wheat and apple juice groups, respectively. The effects did not differ between the rye and wheat groups. Increases in serum and red cell folate were more profound among subjects with low starting folate levels. Decrease in the plasma homocysteine concentrations was observed only in the highest tertile of both groups but not in the group means. Conclusions: Endogenous folates incorporated into a healthy diet, even in moderate amounts, is an efficient way to improve folate status among healthy adults. Folates from different rye products and orange juice showed good bioavailability that was similar to folic acid from fortified white bread. Received: 19 July 2002, Accepted: 6 October 2002 Correspondence to: Dr. Liisa Vahteristo     

Antioxidant and radical scavenging properties in vitro of polyphenolic extracts from red wine

Summary Background & Aims Red wine polyphenols inhibit chemically-induced oxidative DNA damage in vivo in experimental animals through a mechanism which is still unclear. On this basis, we tried to clarify the mechanisms of inhibition of DNA oxidation in vitro by wine extracts containing monomeric and polymeric phenols (WE) and monomer-free complex polyphenols and tannins (WCPT) from red wine. Methods Oxidative DNA damage was induced by incubating DNA with GSH/Fe³⁺ or cumene hydroperoxide (CumOOH) in vitro and using 8-OH–2-deoxyguanosine (8-OHdG) levels as a measure of DNA oxidation. Levels of 8-OHdG were determined by HPLC coupled with electrochemical detector (ESA). Results and conclusions WCPT and WE, at μM concentrations, reduced concentration-dependently oxidative DNA damage induced by GSH/Fe³⁺. WCPT and WE also reduced DNA oxidation by CumOOH. In conclusion, complex polyphenols and tannin extracts from red wine, with or without small molecular phenols, prevent oxidative DNA damage through a dual mechanism, iron binding and direct free radical scavenging. Received: 27 November 2000 / Accepted: 11 April 2001     

Plasma concentration response to drinks containing beta-carotene as carrot juice or formulated as a water dispersible powder

Summary. Background: Bioavailability of β-carotene is highly variable and depends on the source, the formulation and other nutritional factors. Objective: It was the aim of the study to compare β-carotene plasma response to b-carotene dosing with two commercially available drinks, containing β-carotene from carrot juice or as water dispersible β-carotene powder. Design In a randomized, parallel group study design, 4 volunteers per group received daily β-carotene doses of 6–7 or 18–22 mg of either drink over 6 weeks. Blood samples for determination of carotenoid and vitamin A plasma concentrations were collected before supplementation and over the dosing period. Results: Apparent steady-state β-carotene concentrations were attained after 40 days of supplementation. Consumption of the beverage containing β-carotene as a water dispersible powder resulted in a higher response of β-carotene plasma concentrations with increments of 3.84 ± 0.60 μmol/L (p < 0.05, dose: 7.2 mg/d) and 5.04 ± 0.72 μmol/L (p < 0.05, dose: 21.6 mg/d), respectively, in comparison to the carrot juice-based drink with increments of 0.42 ± 0.33 μmol/L (dose: 6 mg/d) and 1.71 ± 0.55 μmol/L (dose: 18 mg/d), respectively. β-carotene was cleared from the plasma with an apparent half-life of 6–11 days. Plasma concentrations of α-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene remained almost unchanged, whereas retinol plasma concentrations increased slightly. By contrast, with the exception of elevated 13-cis-retinoic acid in one group (21.6 mg/d, water dispersible powder), the concentrations of all-trans-retinoic acid, and the oxo-derivatives or retinoic acid were not significantly affected by b-carotene supplementation. Conclusions: The results confirm that the relative bioavailability of β-carotene depends largely on the source of b-carotene and demonstrate the superior bioavailability of β-carotene powder in comparison to that in carrot juice. Received: 7 May 2002, Accepted: 22 August 2002 This work was supported by a grant from F. Hoffmann-La Roche Ltd., Vitamins and Nutrition Research, Basle, Switzerland. Correspondence to: Prof. Dr. med. Petra A. Thürmann     

The acid-base hypothesis: diet and bone in the Framingham Osteoporosis Study

Summary Background There continues to be considerable debate about the role of acid vs. basic components of the diet on the long-term status of bone mineral density. Aim In a set of two analyses, we examined the effect of components in the diet thought to have basic effects (magnesium, potassium, fruit, vegetables) and acid effects (protein) on bone mineral density in an elderly cohort. Methods Bone mineral density of participants in the Framingham Osteoporosis Study was measured at three hip sites and one forearm site at two points in time, four years apart. At the time of baseline measurement, participants ranged in age from 69–97 years. Dietary intake was assessed at baseline by food frequency questionnaire. Results As hypothesized, magnesium, potassium, fruit and vegetable intakes were significantly associated with bone mineral density at baseline and among men, with lower bone loss over four years. In contrast to the hypothesis, higher rather than lower protein intakes were associated with lower bone loss. Conclusion Together these results support the role of base forming foods and nutrients in bone maintenance. The role of protein appears to be complex and is probably dependent on the presence of other nutrients available in a mixed diet. A balanced diet with ample fruit and vegetables and adequate protein appears to be important to bone mineral density. Received: 4 August 2001, Accepted: 14 August 2001     

The carbohydrate crystalean and colonic microflora modulate expression of glutathione S-transferase subunits in colon of rats

Summary Background Glutathione S-transferases (GSTs)* are an important class of phase II, predominantly detoxifying, enzymes. The supergene family is composed of several isoenzymes, hetero- and homodimers, with tissue specific distribution and levels of expression. The hypothesis is that a higher expression of individual proteins within a specific tissue may be associated with a decreased burden of exposure to reactive carcinogens and ultimately with a decreased cancer risk in this tissue. Aims of the study Since nutrition is expected to contribute to the gene expression, it was the aim of this study to investigate the impact of dietary factors, especially resistant starch, and of the gut microflora, which may be influenced by diet, on the GSTs in colon cells of rats. Methods For this, a technique using high pressure liquid chromatography was established with which for the first time GST isoenzymes were analysed in colon cells and compared to the levels of the corresponding proteins in the liver of the same rat. Results It was found that colon cells contain mainly GST π and low amounts of μ but not GST α. In contrast, the predominant form of GSTs in the liver was α, then μ and hardly any π. Altogether, liver cells had approximately tenfold more total GSTs than colon cells. The feeding of “Crystalean”, a retrograded, high amylose starch which alters the fermentation profile and the composition of the microflora, led to higher levels of GST π in the colon. Furthermore, the comparison of GSTs in colon cells of germ-free rats revealed they were much lower than those observed in rats with conventional microflora. Conclusions These findings clearly demonstrate that the gut bacteria, or their metabolic products, enhance GST expression. The studies support the hypothesis that nutrition – by affecting the gut flora – may induce this potentially protective and important class of phase II enzymes in important tumor target cells.     

The status of plasma homocysteine and related B-vitamins in healthy young vegetarians and nonvegetarians

Summary. Background: Exclusion of animal products and having only plant protein in vegetarian diets may affect the status of certain B-vitamins, and further cause the elevation of plasma homocysteine concentration. Aim: The purpose of this study was to assess the status of homocysteine and related B-vitamins in vegetarians and nonvegetarians. The effects of biochemical parameters of B-vitamins and dietary protein on plasma homocysteine were also examined. Methods: The study was performed at the Chung Shan Medical University, Taichung, in the central part of Taiwan. Thirty-seven vegetarians (28.9 ± 5.5 y) and 32 nonvegetarians (22.9 ± 1.6 y) were recruited. Nutrient intake was recorded using 3-day dietary records. Fasting venous blood samples were obtained. Plasma homocysteine, folate and vitamin B-12 were measured. Vitamin B-6 status was assessed by direct measures [plasma pyridoxal 5'-phosphate (PLP) and urinary 4-pyridoxic acid (4-PA)] and indirect measures [erythrocyte alanine (EALT-AC) and aspartate (EAST-AC) aminotransaminase activity coefficient]. Results: There was no significant difference in vitamin B-6 intake between the two groups, although the vegetarian group had a significantly lower vitamin B-12 intake than the nonvegetarian group. Vegetarian subjects had significantly lower mean plasma PLP and vitamin B-12 concentrations than did nonvegetarian subjects (p < 0.05); however, a significantly higher mean plasma folate concentration was found in the vegetarian group. Vegetarian subjects had a significantly higher mean plasma homocysteine concentration than nonvegetarian subjects (13.2 ± 7.9 vs. 9.8 ± 2.2 μmol/L). Negative correlations were seen between plasma homocysteine and vitamin B-12 concentrations in the vegetarian (p = 0.004), nonvegetarian (p = 0.026), and pooled (p < 0.001) groups. From best subsets regression analyses, the plasma homocysteine concentration could be significantly predicted by total protein intake (p = 0.027) and plasma vitamin B-12 concentration (p = 0.005) in the pooled group. When the intake of protein is not considered, vitamin B-12 concentration is still a strong predictor of plasma homocysteine concentration (p = 0.012). Conclusions: Vitamin B-12 intake and mean plasma vitamin B-12 concentration were lower for vegetarian subjects than for nonvegetarian subjects, leading to an increase in plasma homocysteine concentration. Vitamin B-6 and folate had little effect on plasma homocysteine concentration when individuals had adequate vitamin B-6 and folate status. Received: 15 July 2002, Accepted: 24 October 2002 The study was supported by National Science Council (NSC 89–2320-B-040–046), Taiwan. Correspondence to: Y. C. Huang     

Bioavailability and potency of natural-source and all-racemicα-tocopherol in the human: a dispute

Summary Alpha-tocopherol occurs in nature as a single stereoisomer (RRR) while synthetic vitamin E is a mixture of eight stereoisomers (all-racemic, all-rac). The presently accepted ratio of biopotency (RRR: all-rac) is 1.36, based on the fetal resorption test in rats. This ratio has been disputed for humans. Clinical endpoint studies in humans are lacking, but plasma responses to RRR-and all-rac were measured in bioavailability studies. In nine studies comparing unlabeled forms, the ratio of plasma parameters (AUC, C_max or steady-state concentration) concurred with the accepted ratio of biopotency within accepted bounds of equivalence. Four recent studies with simultaneous application of trideutero-RRR and hexadeutero-all-rac resulted in ratios of up to 2 for plasma, and of ≈ 2.7 and ≈ 3.4 for α-CEHC (a urinary metabolite) and umbilical cord plasma, respectively. Because these results have been widely assumed to reflect the difference in biopotency, this has prompted a proposal to the Food and Nutrition Board, National Academy of Sciences, USA to change the biopotency factor to 2:1. We challenge the validity of bioavailability data in lieu of clinical endpoints. Because RRR and all-rac are not chemically identical and differ in plasma and tissue kinetics and metabolism, the ratio of bioavailability parameters does not reflect the ratio of biopotency. This needs to be determined in adequately designed studies using clinical and biochemical endpoints. Until such studies have been performed it does not appear prudent to exchange the presently accepted ratio based on valid bioassays, albeit in a model animal, for another that is based on erroneous conclusions from human studies. Received: 17 April 2000, Accepted: 24 July 2000     

Effects of non-esterified stanols in a liquid emulsion on cholesterol absorption and synthesis in hypercholesterolemic men

Summary Background Background Numerous studies have shown that dietary plant sterols (phytosterols and phytostanols) and their esters can decrease cholesterol absorption. However, few researchers have examined the effects of plant sterols on cholesterol absorption and synthesis using stable isotope tracers, instead of relying on endogenous pathway precursors. Further, we have worked with non-esterified lecithin-solubilized stanols as opposed to the more frequently studied esterified sterols and stanols. The vehicle was an oil-in-water liquid emulsion rather than the more common spread vehicle typically employed. Aim of the study To determine the effects of relatively low doses of lecithin-solubilized non-esterified stanols in liquid emulsions on cholesterol absorption and synthesis in mildly hypercholesterolemic subjects. Methods In a randomized, double blind crossover design, 12 mildly hypercholesterolemic men received either a free phytostanol supplement (3 g/d in 3 servings) or a control treatment for 3 days. Cholesterol endogenous synthesis rate was determined using the rate of incorporation of deuterium from body water into newly formed cholesterol molecules. Cholesterol absorption at the intestinal level was determined using the dual isotope method using ¹³C cholesterol injected intravenously and ¹⁸O cholesterol given orally. Results Cholesterol absorption was 55.7 ± 6.5 % for the control and 33.5 ± 5.3 % for the phytostanol treatment. This massive reduction of the cholesterol absorption did not induce, on average, a difference in cholesterol endogenous synthesis which was measured at 0.074 ± 0.0015 pool/d for plant sterols and 0.0736 ± 0.0015 pool/d for controls (p > 0.05). Conclusions The results demonstrated that lecithin-solubilized stanols administrated during a short period of time (3 days) in an oil-in-water emulsion can dramatically decrease cholesterol absorption, without a consistent, concomitant increase in synthesis, which is highly suggestive of effective LDL cholesterol lowering. The effects of synthesis should be verified in a longer study with more subjects. Received: 17 September 2001, Accepted: 17 December 2001     

The fatty acid composition of human colostrum

Summary We reviewed 15 studies reporting on the fatty acid composition of colostrum lipids from 16 geographic regions: 11 European studies and one study each from Central America, the Caribbean, Australia and Asia. The contents of essential fatty acids, saturates and polyunsaturates were similar in the southern European countries Spain, Slovenia and France. Colostrum of St. Lucian women was high in saturates and low in oleic acid, reflecting a high-carbohydrate, low-fat diet. Abundant fish intake was reflected in high contents of docosahexaenoic acid and total n-3 long-chain polyunsaturated fatty acids in St. Lucia. Two French studies published with an interval of two years showed a very similar colostrum fatty acid composition, whereas two German studies obtained with an interval of 14 years showed higher docosahexaenoic acid and arachidonic acid contents in the later study, with an unchanged n-6/n-3 long-chain polyunsaturated fatty acid ratio. Studies from Spain reported a decline of α-linolenic acid in colostrum over a time period of 13 years. Colostrum of Australian women contained the lowest polyunsaturated/saturated and n-6/n-3 long-chain polyunsaturated fatty acids ratios (0.28 and 1.58) and the lowest contents of linoleic and α-linolenic acids (7.8 and 0.4 wt. %). In contrast, the contents of docosahexaenoic acid, eicosapentaenoic acid and total n-3 long-chain polyunsaturated fatty acids (0.6, 0.4 and 1.4 wt. %) were higher in Australian than in European samples. Fatty acid composition of human colostrum appears to be markedly influenced by geographic differences in maternal dietary composition. Received: 3 January 2000, Accepted: 25 February 2000     

The concept of bioavailability as it relates to iron nutrition

Bioavailability is discussed in conceptual terms; the definition proposed for all nutrients is that it is a measure of the proportion of the total in a food or diet that is digested, absorbed and metabolised by normal pathways. The availability of Fe is affected by a number of dietary and physiological variables, and various techniques used to quantify these effects and thereby predict availability are reviewed. Emphasis is placed on the modifying influence of physiological factors on Fe absorption and hence measurements of availability. Methods of estimating Fe status as a means of incorporating the utilisation element of Fe availability are described.     

The hypocholesterolemic effect of lemon peels,lemon pectin,and the waste stream material of lemon peels in hybrid F<Subscript>1</Subscript>B hamsters

Summary Background We found in preliminary studies with hamsters that citrus peels have a cholesterol lowering effect comparable to that of pectin extracted from these peels. Aim of the study We wanted to examine whether the cholesterol lowering effect of the peels could be completely accounted for by the pectin in the peels. Methods We fed cholesterol enriched (0.1 %,w/w) semipurified diets containing 3 % (w/w) of cellulose, lemon peels, lemon pectin, and the waste stream material of the lemon peels to hybrid F₁B hamsters for a period of 8 weeks. The waste stream of the lemon peels is the left over after extraction of the lemon pectin. Results Feeding the semipurified diets resulted in an increase of plasma cholesterol levels in all the dietary groups after 2 and 4 weeks on the diets. Cholesterol concentrations in the cellulose fed hamsters continued to increase after 4 weeks on the diet, whereas cholesterol levels in the other groups had reached a plateau. As a consequence, the plasma cholesterol levels in the hamsters fed the peels (5.59 ± 0.74 mmol/L, mean ± SD, n = 14), pectin (5.19 ± 0.48 mmol/L), or waste stream (5.53 ± 0.94 mmol/L) were lower than those in the hamsters fed cellulose (6.71 ± 1.52 mmol/L) after 8 weeks on the diets. Differences in total plasma cholesterol were reflected in differences in both VLDL and LDL cholesterol concentration, but this effect was more distinct for the VLDL. There was no effect of the type of fiber on HDL cholesterol levels. Liver cholesterol concentrations paralleled the concentrations of plasma cholesterol and the liver cholesterol concentrations in the hamsters fed the peels (3.57 ± 1.01 μmol/g liver, mean ± SD, n = 14), pectin (4.86 ± 1.42), and the waste stream (4.96 ± 1.89) were lower than those in the cellulose group (7.19 ± 2.32). The hamsters fed the peels, pectin, or waste stream tended to have a higher excretion of fecal bile acids and neutral sterols then the cellulose fed hamsters. Conclusion The results of this study suggest that lemon peels and the waste stream of the lemon peels are as effective in lowering plasma and liver cholesterol in hamsters as the pectin extracted from the peels and that also compounds other than pectin are probably responsible for the cholesterol lowering effect of the citrus peels. Received: 20 September 2001, Accepted: 17 December 2001     

Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen

Summary Background: Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacitiy for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples. Objective: To investigate the occurence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1. Methods: The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. “Hot-spots” for IgE-reactivity were identified by site-directed mutagenesis. Results: Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of DG26. Moreover, the allergenicity was similar to another rMal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen. Conclusion: We conclude that divergent allergenicity of apple strains mainly depends on differnet expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects. Received: 7 June 1999, Accepted: 30 July 1999     

The effects of calcium supplementation to patients with primary hyperparathyroidism and a low calcium intake

Summary. Background: In patients with primary hyperparathyroidism (PHPT) a low calcium intake might cause increased bone loss and thus aggravate osteoporosis, and a high intake might increase serum calcium level and the risk of nephrolithiasis. Aim of the study: Generally, guidelines recommend a normal calcium intake, and accordingly, those with a low intake might benefit from a modest calcium supplementation. This hypothesis was tested in the present study. Methods: Thirty-one patients with asymptomatic PHPT were recruited from an epidemiological study (The Troms? study 1994/95). Those with a daily calcium intake below 450 mg were given calcium supplementation (500 mg Ca²⁺), and those with an intake above 450 mg were followed without supplementation. The study was open and lasted 1 year. Serum levels of calcium, PTH, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D, urinary calcium excretion, blood pressure, and bone mineral density (BMD) were measured. Results: Three subjects dropped out without reason, 1 developed abdominal discomfort from the calcium supplementation, and 3 had an increase in serum calcium of more than 0.2 mmol/L and were therefore excluded. The latter three did not differ from the rest of the group at baseline. Of the remaining 24 that completed the study, 17 were given calcium. In this group there was a non-significant increase in serum calcium and urinary calcium excretion, a significant decrease in PTH after 4 weeks (13.2 (6.0) vs 9.4 (3.0) pmol/L, P < 0.05), and a significant increase in BMD at the femoral neck at the end of the study (0.849 (0.139) vs 0.870 (0.153) g/cm², P < 0.05). The blood pressure was not significantly affected. Conclusions: Most patients with mild PHPT and a low calcium intake tolerate a moderate calcium supplement. This may have beneficial effects on the bones, but the patients must be followed carefully. Received: 30 April 2002, Accepted: 17 September 2002 Correspondence to: R. Jorde     

Improved general health status in an unselected infant population following an allergen reduced dietary intervention programme The ZUFF-STUDY-PROGRAMME Part I: study design and 6-month nutritional behaviour

Summary Background: The best nutritional option for newborn infants is mother's milk. However, some newborn babies may not be exlusively breastfed during the first months of life, potentially leading to reduced overall health status and the early onset of allergic diseases in some infants. Considerable research has been devoted to the development and assessment of infant nutrition programmes, particularly to the prevention of allergies in high-risk infants. However, equal numbers of infants with and without an elevated familial risk of allergies will eventually develop allergic diseases. Therefore, optimizing nutritional programmes for the early infant population as a whole is an important – but as yet insufficiently studied – area of investigation. Moreover, although safe and effective nutrition must primarily support healthy development of the infant, few studies have evaluated the overall health benefits of nutritional interventions, but have focussed on specific allergic manifestations. In animal models, an allergen-reduced moderate whey hydrolysate formula (pHF, Nestlé Beba HA) induces the development of oral tolerance towards cow's milk proteins, without inducing sensitization. In infants with a high risk for allergies, pHF formulae reduce the early onset of allergic disease during the first 5 years of life by approximately 50% compared with a dietary regimen of unaltered proteins. At present, very little is known about the overall health benefits of such a dietary intervention on the unselected infant population as a whole. Aim of the study: The aim of our prospective, controlled study was to investigate the overall health benefits of an allergen-reduced nutritional programme in a newborn infant population unselected for atopic risk factors. The population in our study was a comparable as possible to the general population of healthy newborn infants. Our study included exclusive breastfeeding, use of a moderate whey hydrolysate formula (pHF, Nestlé Beba HA) if infant formula was needed, and delayed introduction of low-allergenic weaning foods. The study included assessments of compliance with the dietary programme, and evaluated nutritional habits, growth, and overall health status for 24 months. The health evaluation included allergic manifestations but die – by purpose – not define or evaluate them specifically. Part I of this paper gives results for nutritional habits during the first 6 months of life, Part II gives results for growth and general health status for the same time period, Part III will present feeding habits during the second half of the first year of life, and Part IV will present results to 24 months of age. The complete study report is published as a supplement to this journal. Methods: Nuritional assignment was to demographically comparable intervention (Z) or control (FF) cohorts according to the infant's place of birth. In the intervention cohort (Z, n=564), the recommended dietary regimen was breastfeeding and/or the pHF formula, with no weaning food before 4 months of age. In the control cohort (FF, n=566), there was no intervention. Longitudinal diet groups, defined for 4 months, excluding dropouts and noncompliants, were exclusive breastfeeding (eBF, Z, n=201, FF, n=162), partial breastfeeding (pBF, Z, n=222, FF, n=311), or non-breastfeeding (nBF, Z, n=42, FF, n=62). Imbalances between groups and cohorts in confounding factors that could influence health-related symptoms were integrated as covariates into the main analyses using logistic regression. Nutritional surveillance was carried out using continuous prospective monitoring. Results: The overall rate of breastfeeding, irrespective of partial or excluive breastfeeding or the additional use of weaning foods, was similar in both cohorts at 4 and 6 months. However, from ages 3 to 6 month, significantly more Z than FF infants were excluively breastfed (p<0.05), and weaning foods were introduced at a significantly later age in Z than FF (22 versus 18 weeks; p<0.0001). Infants in the Z cohort received fewer different kinds of weaning foods compared with FF (2 versus 10; p<0.0001). Six-month rates of dropout were very low in both groups (Z=4.3%, FF=1.8%) and compliance with the programme was excellent over the strict intervention period of 4 months (Z=86.9%) until the sixth month. Conclusions: A dietary recommendation that promotes breastfeeding, includes a moderate whey hydrolysate formula (Nestlé Beba HA), and delays the introduction of highly allergenic weaning foods (an allergen-reduced dietary regimen) significantly increases breastfeeding and delays weaning in a normal infant population without any compliance problem to the moderate hydroysate formula that is quite often seen in the eHF. The regimen was followed closely, with a high degree of compliance and a low dropout rate compared with previous allergy-prevention studies. Compliance with the dietary regimen was excellent over the 4 months of intervention and throughout the following 2 months. Received: 14 December 1999, Accepted: 2 May 2000     

In vitro evaluation of iron solubility and dialyzability of various iron fortificants and of iron-fortified milk products targeted for infants and toddlers

The objectives of the present study were: to compare the solubility and dialyzability of various iron fortificants (iron pyrophosphate, ferrous bis-glycinate, ferrous gluconate, ferrous lactate, ferrous sulfate) added, in the presence of ascorbic acid, to pasteurized milk samples produced under laboratory conditions; and to compare the solubility and dialyzability of iron in commercial pasteurized, UHT and condensed milk products available in the Greek market fortified with various vitamins and minerals including iron and targeted towards infants (6–12 months old) and toddlers. Iron solubility and dialyzability were determined using a simulated gastrointestinal digestive system. Ferrous dialyzable iron (molecular weight lower than 8000) was used as an index for prediction of iron bioavailability. Ferrous dialyzable iron in pasteurized milk samples fortified with iron pyrophosphate, ferrous lactate and ferrous bis-glycinate was higher (P?<?0.05) than that in milk samples fortified with ferrous sulfate and ferrous gluconate. In commercial liquid pasteurized or UHT milk products, formation of ferrous dialyzable iron in products fortified with ferrous lactate was not different (P?>?0.05) from those fortified with ferrous sulfate. Ferrous dialyzable iron in four condensed commercial milk products was higher (P?<?0.05) than the corresponding values of the liquid UHT milk samples fortified with the same fortificant (ferrous sulfate). Ferrous dialyzable iron was higher (P?<?0.05) in products targeted for infants compared with those targeted for toddlers. In conclusion, the type of iron source, milk processing and the overall product composition affect formation of ferrous dialyzable iron and may determine the success and effectiveness of iron fortification of milk.