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ORIGINAL ARTICLE

Changes in circulating concentrations of vitaminsand trace elements after cessation of nocturnalenteral tube feeding

Christine Baldwina, Odile Dewitb, Marinos Eliac,*

aDepartment of Medicine and Therapeutics, Imperial College, Chelsea & Westminster Hospital, FulhamRoad, London SW10 9NH, UKbClinical Pharmacology Department, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park(South), Third Avenue, Harlow CM19 5AW, UKcInstitute of Human Nutrition (University of Southampton), Southampton General Hospital, Mailpoint 113,Tremona Road, Southampton SO16 6YD, UK

Received 19 May 2003; accepted 10 July 2003

Summary Background: This study aimed to examine whether circulating concentra-tions of a range of vitamins and trace elements in patients receiving long-term cyclicenteral tube feeding vary during the day, and whether standardised time points forblood sampling are required for assessment of nutrient status.Methods: Circulating concentrations or activities of water-soluble vitamins

(thiamine, riboflavin, and vitamins B6, B12, folate and C), fat-soluble vitamins (A,D, E) and trace elements (iron, zinc, copper and selenium (assessed by glutathioneperoxidase activity), were measured at 0,3,6 and 9–12 h after cessation of nocturnalfeeding (fasting), in eight clinically stable patients receiving cyclic nocturnal enteralnutrition.Results: The circulating concentrations of the nutrients did not change between the

fed and fasted state (repeated-measures-ANOVA) except the following: plasma folateincreased progressively from 10.9 (SD 4.6) nmol/l in the fed state to 14.0 (SD4.4) nmol/l at 9–12 h after cessation of feeding (Po0.05); plasma zinc increasedprogressively throughout the fasting period by 33.5% (8.57, SD 0.68 vs. 11.44, SD1.85 mmol/l, in fed state vs. 9–12 h fast respectively, Po0.05); and total tocopherol/cholesterol ratio decreased by 9.6% during the study period (Po0.02), while g-tocopherol increased by 59.2% (Po0.05). For all analytes, the concentrations in bloodsamples taken at 3 and 6 h after cessation of feeding were not significantly differentfrom those at 9–12 h.Conclusions: Although cessation of nocturnal tube feeding had no significant effect

on the circulating concentrations of most micronutrients, it increased plasma folateand zinc concentrations, and decreased the tocopherol/cholesterol ratio. The timingfor blood sampling should be standardised when the status of these nutrients isassessed in patients receiving cyclic tube feeding.& 2003 Elsevier Ltd. All rights reserved.

KEYWORDS

Fasting;

Tube feeding;

Nutritional status;

Zinc;

Folic acid;

Tocopherol

*Corresponding author.E-mail address: elia@soton.ac.uk (M. Elia).

0261-5614/$ - see front matter & 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.clnu.2003.07.003

Clinical Nutrition (2004) 23, 249–255

Introduction

The description of a range of trace element andvitamin deficiencies in patients receiving long-termenteral tube-feeding1–6 has led to the recommen-dation that micronutrient status should be mea-sured more frequently. This is particularlyimportant in patients who have little or no oralfood intake. However, attempts to assess micro-nutrient status have often been made using bloodsamples taken at variable time points in relation tothe feeding. In some studies, samples have beentaken in the fed state.1 In others they have beentaken after an overnight fast,3,4,7 or 4–6 h aftercessation of feeding,2,6 whilst others have notspecified the timing.5,8 Since reference ranges forcirculating concentrations of nutrients have usuallybeen established after a period of fasting (typically8–14 h), adequate interpretation of randomly ob-tained samples may be difficult or impossible.

Diurnal variations in the circulating concentra-tions of some micronutrients in normal subjectsingesting normal food intermittently has beendocumented.9–12 This may be due to the effectsof variable physical activity, or variable foodingestion.9 However, there is a notable lack ofinformation on the effects of food ingestion aloneon the circulating concentrations of micronutri-ents, and even less in relation to enteral tubefeeding. The aims of this study were two-fold: toassess the extent to which circulating concentra-tions of a range of vitamins and trace elementschange during a 9–12 h period of fasting aftercessation of nocturnal enteral feeding; and todetermine the need for practical time points forblood sampling for assessment of micronutrientstatus in patients receiving enteral tube feeding.

Patients and methods

Patients

The study group consisted of eight clinically stablepatients, six males and two females, aged 24–89years (mean 64, SD 24), with swallowing difficultiesresulting from cerebrovascular accidents (n¼ 3),persistent vegetative state (n¼ 2), multiple sclerosis(n¼ 1), cerebellar astrocytoma (n¼ 1) and supranuclear palsy (n¼ 1). Subjects had been receivingnocturnal feeding for a duration ranging from 2months to 7 years (median 4.5 months). Feed wasinfused for a mean of 12.5h (range 10–15) overnight,typically 19:00 to 07:00h. Three patients had alsobeen receiving boluses of 100–125ml given twice to

four times daily. Oral intake was nil in seven of thesubjects and minimal in one (less than 50kcal/day,210kJ/day). Patients were receiving three differentfeeds (Nutrison Standard, Nutrison Fibre and NutrisonEnergy, Nutricia, Zoetermeer, The Netherlands)providing 1000–1500kcal/l (4.2–6.3MJ) with 49%energy from carbohydrate, 35% from fat, and 16%from protein. The micronutrient content of thesethree formulas (per litre) was identical except for theslightly higher mineral and trace element content ofthe fibre-containing product (indicated below inparentheses) received by five patients: vitamin A667mg, vitamin D 5.0mg, vitamin E 11.0mg, thiamin1.0mg, riboflavin 1.1mg, vitamin B6 1.3mg, folicacid 133.0mg, vitamin B12 2.0mg, vitamin C 50.0mg,iron 10.0mg (11.0), zinc 10.0mg (11.0), copper1500mg (1700), selenium 430mg (460), and othermicronutrients, such as chromium, iodine and cho-line, which were not measured.

The protocol received ethical approval from theLocal Ethics Research Committee and informedconsent was obtained from the patients, or relativeswhen the patients were incapable of giving consent.

Methods

Blood was withdrawn from a venous cannula in theantecubital fossa at the end of the feeding period,with the enteral feeding still being infused (fedstate), and at 3,6 and 9–12 h of fasting followingcessation of feeding. The initial blood sample wasusually taken while the patients were recumbent inbed, but subsequent samples were usually takenwhilst they were sitting on a chair. Patients did notreceive feed, food, drink or bolus feeding duringthe study period other than water. An equal volumeof water was given in place of daytime bolus feeds.The volume given was not greater than 250 ml.

Sample preparation

Blood was collected in lithium/heparin Vacutainertubes (LH-Metall-Analytik, Sarstedt, Numbrecht,Germany). From this, 0.1ml whole blood was addedto 0.9ml of 1% ascorbic acid, a stabilising agent, forthe red cell folate assay. A further 0.5ml of wholeblood was portioned off and frozen. The remainingwhole blood was centrifuged, and the plasmaseparated. Five hundred microlitres of plasma wasadded to an equal volume of 10% metaphosphoricacid to stabilise the ascorbic acid for the vitamin Cassay. The buffy coat was discarded and the packederythrocytes washed three times with 0.9% normalsaline.

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250 C. Baldwin et al.

All sample portions were stored at �801C untilrequired for analyses.

Assay methods

Roche diagnostic kits (Roche Diagnostica Instru-ments, Basle, Switzerland) were used to measurecholesterol (UNIMATE 5 CHOL), triglycerides (UN-IMATE 5 TRIG), iron (UNIMATE 5 IRON), and totaliron-binding capacity (IBC test and UNIMATE 5IRON). Glutathione peroxidase activity, an indexof selenium status, was measured using the RANSELkit (Randox Laboratories Ltd., Co. Antrim, NorthernIreland, UK). Zinc was measured using the Wako Zncolorimetric kit (Art.No.435-14909, Wako Chemi-cals GmbH, Neuss, Germany). Copper was mea-sured using the Cu MPR1 colorimetric kit(Boehringer Mannheim, Mannheim, Germany). Aturbidimetric assay was used to measure albumin(DAKO A/S, Glostrup, Denmark). Vitamin B1, B2,and B6 were determined by red cell enzymestimulation tests (erythrocyte transketolase, ery-throcyte glutathione reductase and erythrocyteaspartate amino-transferase respectively.13 Vita-min C was measured by the automated fluorimetricmethod of Vuilleumier.14 All the above assays wereperformed on the Roche ‘Cobas-Fara’, an auto-mated centrifugal spectrophotometer. Radioimmu-noassays (RIA) were employed to measure thefollowing: plasma vitamin B12, plasma folate andred cell folate, using the Quantaphase II B12/Folateand Quantaphase II Folate Radioassay kits, with RedCell Folate Reagent Pack (Bio-Rad DiagnosticsGroup, Hercules, CA, USA); vitamin D, using the25-hydroxy-vitamin D 125I RIA kit (INCSTAR Corpora-tion, Stillwater, MN, USA); ferritin using Bio-Rad

Quantimune Ferritin Immunoradiometric assay kit(Bio-Rad, CA, USA). These assays were counted on aWallac 1470 Wizard automated gamma counter(Wallac UK, EG&G Instruments Ltd, Milton Keynes,UK). Fat-soluble vitamins and carotenoids weremeasured by liquid chromatography.15 C-reactiveprotein was measured using a DAKO protocol,employing double antibody sandwich ELISA. Alpha-1-antichymotrypsin was measured by immunoturbi-dimetry, using a DAKO protocol.

The autoanalyser analysed all samples from thesame patient together. The intra-assay coefficientsof variation for the analytes were as follows:vitamin A (retinol), 4%; thiamin, 3.8%; riboflavin,1.7%; vitamin B6, 3.0%; vitamin B12,11%; folic acid11%; vitamin C, 1.8%; vitamin E, 3.2%; ferritin, 10%;iron, 7%; total iron binding capacity 3.7%; copper,3.8%; zinc 2.5%, cholesterol, 3.2%; and albumin2.1%.

Statistics

Repeated measures ANOVA was undertaken usingSPSS version 7.5. Post-hoc paired t-tests were usedwhen significant differences were obtained.

Results

Tables 1–3 present the circulating concentrations ofvitamins and trace elements at the end ofnocturnal feeding (time 0), and at 3, 6, and 9–12 h after cessation of feeding. Of the vitamins,significant changes were observed only for folate,g-tocopherol and total tocopherol to cholesterol

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Table 1 Circulating concentrations and activation coefficients of water-soluble vitamins following cessation ofnocturnal enteral feeding.

Fed Fasting Reference for

9–12 h

0 h3 h 6 h 9–12 h

Thiamin (activation coefficient) 1.13 (0.15) 1.17 (0.13) 1.23 (0.13) 1.16 (0.16) o1.220

Riboflavin (activation coefficient) 1.13 (0.08) 1.24 (0.33) 1.14 (0.06) 1.10 (0.07) o1.317

Vitamin B6 (activation coefficient) 1.65 (0.15) 1.65 (0.14) 1.64 (0.16) 1.63 (0.15) o1.821

Vitamin B12 (pmol/l) 599.18 (392.05) 571.39 (355.42) 609.55 (387.05) 587.19 (337.80) 414717

Red cell folate (nmol/gHb) 1.23 (0.18) 1.24 (0.24) 1.17 (0.24) 1.17 (0.21) 42.2618

Plasma folate (nmol/l)n 10.93 (4.57) 12.53 (3.29) 13.55 (3.14) 14.04n (4.42) 413.417

Vitamin C (mmol/l) 41.75 (16.51) 44.65 (20.92) 46.88 (20.79) 47.86 (24.09) 41717

Vitamin 25 (OH)D (nmol/l) 43.29 (18.87) 43.72 (20.33) 46.82 (25.45) 47.10 (23.23) 42517

Values are mean (SD). References which suggest acceptable status are given for fasting samples (superscript numbers indicatethe corresponding literature references; NA not available).nValue significantly different from 0h, at Po0.05.

Changes in circulating concentrations of vitamins 251

ratio. Plasma folate concentration increased pro-gressively throughout the fasting period by anaverage of 3.11 nmol/l. The value at 9–12 h was28.5% higher than in the fed state (Po0.05). Thecirculating concentration of a-tocopherol did notchange during the study period, but that of g-tocopherol decreased as fasting progressed(Po0.05). The circulating concentration of g-tocopherol at 9–12 h was 59.2% lower than that inthe fed state. Total tocopherol concentration wasdefined as the sum of a-tocopherol and g-tocopher-ol concentrations, and their proportions were 92–97% and 3–8% of total tocopherol, respectively.Total circulating concentrations of tocopherol didnot change significantly, but the molar ratio of totaltocopherol to cholesterol16 decreased (Po0.02), a

change that was mainly attributable to a rise incholesterol concentration from 2.93mmol/l (SD0.64) at time 0 to 3.25mmol/l (SD 0.78) at 6 h(Po0.01) and to 3.22mmol/l (SD 0.65) at 9–12 h(an increase of 9.9%, Po0.02). Concentrations ofHDL-cholesterol increased from 0.89mmol/l (SD0.32) on cessation of feeding to 1.03 (SD 0.36) at 6 hof fasting (an increase of 15.7%, Po0.01). Incontrast, circulating concentrations of triglyceridesdecreased significantly from 1.20mmol/l (SD 0.53)on cessation of feeding to 0.90 (SD 0.31) after 6 h offasting (a decrease of 25%, Po0.05), whilst thetotal tocopherol to triglycerides ratio did notchange significantly during this time.

Circulating zinc concentrations rose progressivelyas fasting progressed. The value at 9–12 h of fasting

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Table 2 Circulating concentrations of fat-soluble vitamins and glutathione peroxidase (GPX) activity followingcessation of nocturnal enteral feeding.

Fed Fasting Reference for9–12 h

0 h3 h 6 h 9–12 h

Retinol (mmol/l) 1.62 (0.43) 1.58 (0.39) 1.59 (0.48) 1.55 (0.40) 40.717

a-Carotene (mmol/l) 0.011 (0.007) 0.009 (0.006) 0.009 (0.005) 0.007 (0.006) 0.02–0.2218

b-Carotene (mmol/l) 0.036 (0.026) 0.033 (0.024) 0.034 (0.026) 0.038 (0.026) 0.09–0.7516

a-Cryptoxanthin (mmol/l) 0.008 (0.005) 0.007 (0.006) 0.008 (0.005) 0.008 (0.005) NAb-Cryptoxanthin (mmol/l) 0.009 (0.005) 0.009 (0.005) 0.009 (0.005) 0.009 (0.005) 0.05–0.5218

a-Tocopherol (mmol/l) 21.11 (5.61) 21.13 (5.33) 21.02 (5.19) 21.43 (4.80) 411.617

g-Tocopherol (mmol/l)n 1.74 (0.99) 0.97 (1.00) 0.95n (0.28) 0.71n (0.92) NAðaþ gÞ-Tocopherol:cholesterol 7.96 (2.03) 7.82 (1.71) 6.74n (1.62) 7.19n (1.82) w

ratio (mmol/mmol)n

GPX activity (IU/g Hb) 36.00 (6.19) 32.17 (12.10) 37.16 (10.63) 33.90 (9.57) NA

Values are mean (SD). References which suggest acceptable status are given for fasting samples (superscript numbers indicatethe corresponding literature references; NA not available).nValue significantly different from 0h, at Po0.05.wReference value17 available for a-tocopherol:cholesterol ratio 42.22mmol/mmol; mean (SD) a-tocopherol:cholesterol ratio at9–12 h in the present study¼ 6.73 (1.69).

Table 3 Iron status indicators and plasma concentrations of trace elements following cessation of nocturnalenteral feeding.

Fed Fasting Reference for9–12 h

0 h3 h 6 h 9–12 h

Iron (mmol/l) 7.68 (4.75) 6.13 (4.00) 8.78 (5.61) 9.19 (5.67) 410.719

TIBC (mmol/l)n 44.90 (8.24) 44.17 (7.15) 51.47n (6.70) 46.40 (11.15) o7019

Transferrin saturation (%) 16.6 (8.2) 13.5 (7.0) 16.5 (9.6) 19.4 (9.5) 415%19

Ferritin (ng/ml) 125.71 (108.69) 122.33 (94.60) 128.50 (110.62) 136.63 (130.24) 12–20020

Zinc (mmol/l)n 8.57 (0.68) 10.06 (2.75) 11.75n (2.09) 11.44n (1.85) 11–2320

Copper (mmol/l) 18.65 (1.52) 19.33 (1.60) 19.42 (1.68) 18.99 (2.09) 11–2420

Values are mean (SD). References which suggest acceptable status are given for fasting samples (superscript numbers indicatethe corresponding literature references; NA not available).nValue significantly different from 0h, at Po0.05.

252 C. Baldwin et al.

was 33.5% (þ 2.9 mmol/l on average) higher than inthe fed state (Po0.05). Plasma albumin increasedfrom a mean of 24.8 g/l (SD 5.7) in the fed state to27.9 g/l (SD 6.4) after 9–12 h of fasting (an increaseof 12.5%, Po0.01) and was associated with anacute phase protein response (six out of the eightpatients had elevated C-reactive protein values,ranging from 9 to 66mg/l). Total iron bindingcapacity increased by 6.6 mmol/l (14.6% increase)in the first 6 h of fast (P¼ 0.001), but the plasmaconcentrations of iron and transferrin saturationdid not change significantly during the 9–12 h fast.

The circulating concentrations of all vitamins andtrace elements measured in blood samples taken at3 and 6 h after cessation of feeding were notsignificantly different from those at 9–12 h.

Tables 1–3 show the ‘reference’ ranges sugges-tive of an ‘acceptable status’17 against whichcirculating concentrations at 9–12 h following ces-sation of enteral feeding were compared. Thestatus of most analytes was judged to be accep-table but this was not the case for a- and b-carotene (all subjects had plasma concentrationsbelow the 5th centile values for a-carotene18 andfor b-carotene15), folic acid (red cell folatecirculating concentrations ranged from 0.93 to1.42 nmol/g Hb, while concentrations below1.53 nmol/g Hb are suggestive of increased risk ofvitamin deficiency17). Indicators of iron status wereabnormal in some cases: low plasma iron19 in fivesubjects for whom values ranged from 1.59 to7.62 mmol/l; low transferrin percentage satura-tion19 in two subjects (3.0% and 14.5%); normalferritin plasma concentrations20 in seven subjects,and elevated20 in one (424.8 mg/l); haemoglobinconcentrations low in three subjects (9.6–11.3 g/dl, with normal mean red cell volume). One subjecthad low plasma concentration of vitamin 25(OH)D(17.6 nmol/l).17 In another subject, vitamin B6activation coefficient was 1.86, while a valueabove 1.8 is suggestive of deficiency.21

There were significant between-subject differ-ences in the circulating concentrations of all analytes.

The patients had an acute phase protein response(as judged by elevated (46mg/l) CRP in sixsubjects, ranging from 9.1 to 76.6mg/l; andelevated (40.32 g/l) a1-antichymotrypsin in eightsubjects, ranging from 0.41 to 0.86 g/l). In somecases this was considered to be due to a chronic lowgrade chest infection.

Discussion

In normal subjects blood sampling for assessingmicronutrient status usually takes place after an

overnight fast. In patients receiving nocturnalenteral tube feeding, an early morning sample isobtained in the ‘fed’ state. Therefore applyingfasting references ranges of nutrient status tosamples obtained during tube feeding may beinappropriate. To our knowledge, this is the firststudy to assess the effect of cessation of enteralfeeding on the circulating concentrations of a widerange of trace elements, water-soluble and fat-soluble vitamins for a period of up to 12 h whilst thesubjects remained fasted. Apart from the plasmaconcentration of folate, tocopherol, and zinc, thecirculating concentration of the other measuredtrace elements and vitamins did not changesignificantly during 9–12 h of fasting after cessationof nocturnal enteral tube feeding.

In healthy subjects circulating concentrations oftrace elements and minerals, such as zinc, calcium,phosphate,9 iron,10 and vitamins, such as 25-hydroxy vitamin D,11 riboflavin,22 vitamin C,23 havebeen reported to follow a circadian rhythm, whichcould be due to intermittent dietary intake and/orphysical activity. In patients receiving tube feed-ing, the lack of change for most micronutrientsbetween the fed and fasted states may be due tothe slow administration and hence slow absorptionof nutrients, or to the high flux of nutrients throughthe plasma relative to the absorption rate. Red cellfolate is an example of a nutrient that essentiallydoes not exchange with plasma folate, since folateis sequestered in red cells at the time of theirformation. No change in red cell or whole bloodfolate would therefore be expected on a short-termbasis (see Table 1). However, plasma folate roseprogressively and significantly on cessation offeeding and throughout the 9–12 h fasting period.A two-fold increase has been observed previouslyduring 36 h of fasting in normal subjects.24 As folateis excreted in urine and bile, with the enterohepa-tic circulation accounting for a flux of about 60–90 mg per day,24 it is possible that reduced or absentgall bladder contraction during fasting may lead toa marked reduction in passage of folate into bile,and hence a rise in plasma folate level.24 Vitamin Econcentration (a- and g-tocopherol) is usuallyexpressed as a molar ratio of total tocopherol tocholesterol16 because the vitamin is carried by thelipid fraction of plasma. In the present study, totaltocopherol to cholesterol ratio decreased withfasting but the ratio of tocopherol to triglyceridesdid not. Meal ingestion in normal subjects has beenreported to produce variable effects on plasmacholesterol25 which result from complex relation-ships in the proportions of different lipoproteins.These were not measured in the present study,which limits the interpretation of the observed rise

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Changes in circulating concentrations of vitamins 253

in cholesterol during the period of fasting followingcessation of nocturnal tube feeding. Plasma zincconcentration increased by approximately 33% inthe 9–12 h following cessation of nocturnal enteralfeeding in the present study. This is consistent withprevious reports in healthy subjects of a significantrise (33%) in plasma zinc concentration between anovernight fast and 2 days of starvation,26 which isassociated with a 2- to 3-fold increase in urine zincexcretion.26 It is suggested that the net catabolismof tissue which occur after cessation of feedingreleases zinc into the circulation and causes a risein its concentration. Zinc is a type II nutrient27

which participates in the accretion and loss ofprotein during the feeding–fasting cycle. Thisexplanation is consistent with the effect of recentfood ingestion in healthy subjects, which causes adecrease in plasma zinc concentrations,9 whilstlean tissues are accreted. The significant rise inalbumin concentrations, which may have resultedfrom a change in posture from lying to sitting,28

may also have contributed to the rise in zincconcentration, as about 97% of plasma zinc is boundto albumin and a2 macroglobulin.29 Dehydration isalso a theoretical possibility for the rise in plasmaalbumin concentration, but precautions were takento prevent this during the fasting period byproviding water in place of bolus feeds.

Reference ranges are usually established insubjects after an overnight fast (typically 8–14 hfast). As there was no significant difference in thecirculating concentrations of any of the nutrientsmeasured between blood samples taken at 6 and 9–12 h of fast from cessation of feeding, it islegitimate to compare results obtained at 6 h withreference ranges. This has a practical implication:for patients receiving nocturnal enteral feeding,who therefore stop feeding early morning, taking ablood sample 6 h after cessation of enteral feedingrepresents a more practical time point than a 9–12 h fast. A 3–6 h fast also represents a minimaldisruption to the feeding regimen of the majority ofpatients and still allows daytime bolus feeds to begiven. For all nutrients, blood sampling at 3 h wouldgive a result comparable with reference rangesestablished after 9–12 h fast. However, for plasmafolate, vitamin E and zinc, there is a differencebetween the concentrations in the fed and fastedstate, and therefore the fed state may not berecommended for comparison with referencesranges, while sampling after a 6–9 h fast may berecommended.

Finally, a preliminary comment can be made onthe vitamin and trace element status of thesepatients receiving long-term enteral feeding. Thelow circulating concentrations of carotenes can be

explained by the absence of carotenes in theenteral formulas. The low concentration of zincand iron could be due to the low concentrations ofbinding proteins (e.g. albumin for zinc, andtransferrin for iron) which are reduced during anacute phase protein response. Although deficien-cies of other vitamins or trace elements may havebeen present, this requires confirmation in a largercohort of patients.

Acknowledgements

The study was supported by a grant from NumicoResearch BV, The Netherlands. We wish to acknowl-edge Graham Jennings and Justin Shaw for helpingwith the laboratory analyses and David I. Thurnhamfor analysing some of the fat-soluble vitamins.

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