Digestibility of resistant starch containing preparations using two in vitro models Background Resistant starch -LRB- RS -RRB- is known for potential health benefits in the human colon . To investigate these positive effects it is important to be able to predict the amount , and the structure of starch reaching the large intestine . Introduction Resistant Starch -LRB- RS -RRB- is defined as starch that is not absorbed in the small intestine of humans . Three different types of RS were defined by Englyst et al. -LSB- 1 -RSB- . Type 1 is defined as physically inaccessible starch , type 2 -LRB- RS2 -RRB- as native starch granules and type 3 -LRB- RS3 -RRB- as retrograded starch . More recently , a fourth type of RS has been classified , comprising chemically modified starches -LSB- 2 , 3 -RSB- . RS is claimed to be a good substrate for colonic fermentation and to be beneficial because of its high ratio of butyrate production , which may play a major role in the prevention of colon cancer , as shown in several studies on animal models -LSB- 4 , 5 -RSB- . The fermentation products from RS are known to lower the pH in the colon which leads to less production , and/or accumulation of potentially harmful by-products of protein fermentation , for example , ammonia or phenols which may promote tumorigenesis -LSB- 6 -RSB- . To estimate potential health benefits of RS it is important to be able to predict its behavior in the human gastrointestinal tract , in particular the amount and the structure of starch reaching the large intestine . The digestion of food and absorption of nutrients are spatiotemporal and dynamic processes involving complex enzymatic systems and transport reactions . Thus , the simulation of all these biochemical and physiological events in a single model is illusive . Nevertheless , several in vitro digestion methods , from basic batch systems to sophisticated dynamic models have been developed in order to quantify digestibility of food -LSB- 7 -- 9 -RSB- . A realistic approach implies a well-defined system that takes into account the specific contributions of oral , gastric and intestinal digestion . Advantages of using in vitro instead of in vivo models are low costs , relatively easy performance , no limitations by ethical constraints , and the possibility to compare different substrates when applying standardized conditions . Therefore , the aim of this study was to compare a rather simple and a more sophisticated in vitro model as well as the Megazyme RS method to determine the indigestible fractions of two RS preparations containing RS2 and RS3 . The digestion residues obtained by the two models were compared and characterized . Furthermore , the RS3 fractions were compared with ileostomy effluents obtained in an in vivo study -LSB- 10 -RSB- . Materials and methods Substrates The RS3 containing carbohydrate source , C ⋆ Actistar ® , is a retrograded long chain maltodextrin product obtained after partial enzymatic hydrolysis , and subsequent retrogradation of tapioca starch -LRB- RTmd -RRB- , produced according to United States Patent 6 043 229 -LSB- 11 -RSB- and obtained from Cerestar-Cargill -LRB- Vilvoorde , Belgium -RRB- . Physico-chemical characteristics of RTmd were described recently by Pohu -LSB- 12 -RSB- . The RS2 containing carbohydrate source is native high amylose maize -LRB- HAM -RRB- starch and was obtained from Cerestar International -LRB- Neuilly-sur-Seine , France -RRB- . Batch in vitro digestion model The batch model used is a three step incubation at 37 °C simulating the digestion in mouth , stomach and small intestine . The method was carried out as described by Lebet et al. -LSB- 7 -RSB- and modified by Jörger -LSB- 13 -RSB- . A sample of 30 g substrate was suspended in 500 ml phosphate buffer -LRB- 20 mM , pH 6.9 , Na2HPO4 1.42 g/l , KH2PO4 1.36 g/l , NaCl 0.58 g/l -RRB- , and incubated stepwise with -LRB- a -RRB- 0.5 ml human salivary α-amylase solution -LRB- Sigma A1031 , Buchs , CH , 10 mg/ml in CaCl2 1 mM -RRB- at pH 6.9 for 15 min , -LRB- b -RRB- 1.25 ml porcine pepsin suspension -LRB- Sigma P7012 , 1 mg/ml in NaCl 9 g/l -RRB- at pH 2.0 for 30 min and -LRB- c -RRB- 10 ml porcine pancreatin -LRB- Sigma P7545 , 0.5 mg/ml in CaCl2 25 mM -RRB- at pH 6.9 for 3 h in the presence of 12 g bovine bile -LRB- Sigma B8381 -RRB- . Degradation products were removed by dialysis -LRB- Servapor 44146 , Serva Feinbiochemica GmbH & Co. , Heidelberg , Germany ; cut-off 12 -- 14 kDa -RRB- overnight under continuous movements against running deionized water < 20 °C . The retentate was freeze-dried . Based on total starch -LRB- TS -RRB- determinations in the starting materials and digestion residues , the amount of indigestible starch was calculated . Dynamic in vitro digestion model -LRB- TIM-1 -RRB- The dynamic model used in this study has been described by Minekus et al. -LSB- 8 -RSB- . The model comprises four serial compartments simulating stomach , duodenum , jejunum and ileum . The in vitro digestion was performed for 6 h at 37 °C . A mixture of 60 g substrate , 180 g electrolyte solution -LRB- NaCl 5 g/l , KCl 0.6 g/l , CaCl2 · 2H2O 0.3 g/l , NaHCO3 0.6 g/l -RRB- , 60 g water , 5 g pepsin solution -LRB- Sigma P7012 , 0.28 g/l in solution A : NaCl 3.1 g/l , KCl 1.1 g/l , CaCl2 · 2H2O 0.15 g/l , NaHCO3 7.1 g/l -RRB- and 5 g lipase solution -LRB- Rhizopus lipase , Amano Pharmaceutical Co. . F-AP 15 , Ltd. . Japan , 0.25 g/l in solution A -RRB- were introduced into the gastric compartment . Computer controlled peristaltic valve pumps controlled meal transit through the individual compartments . The pH value was computer monitored by adding HCl -LRB- 1 M -RRB- or NaHCO3 -LRB- 1 M -RRB- , respectively . In the stomach the values were preset to pH 4.5 , 2.8 , 1.8 , 1.7 and 1.5 at 0 , 20 , 40 , 60 and 90 min , respectively . In the small intestine , the pH was maintained at 6.5 , 6.8 and 7.2 in the duodenum , jejunum and ileum , respectively . About 1 ml trypsin solution -LRB- Sigma T4665 , 2 g/l in solution A -RRB- was added to the duodenum at the beginning of the experiment . Secretions of porcine bile 4 g/100 g in water -LRB- Sigma B8631 -RRB- and pancreatic solution 7 g/100 g in water -LRB- Pancrex-V powder , Paines & Byrne , Greenford , UK -RRB- entered the duodenal compartment at 0.5 and 0.25 ml/min , respectively . The absorption of water and digestive products from the jejunal and ileal compartments was simulated using hollow-fiber devices -LRB- cut-off 5 -- 10 kDa -RRB- . Ileal effluents -LRB- indigestible fraction -RRB- were collected after 2 , 4 and 6 h , pooled and freeze-dried . Substrate remaining in the jejunum and ileum at the end of the experiment was considered to be indigestible as well and was therefore mixed with the ileal effluent pool . TS determinations of the starting materials , the digestion residues and the remaining substrate in the model after the experiment were carried out . Starch degradation products -LRB- up to DP 7 -RRB- were quantified in dialysates by high-performance anion-exchange chromatography with pulsed amperometric detection -LRB- HPAEC-PAD -RRB- in a BioLC System -LRB- Dionex Corp. , Sunnyvale CA , USA -RRB- using a Carbo-Pac PA1 column and applying a sodium acetate gradient -LRB- unpublished , internal method -RRB- . TS and oligosaccharide quantifications were recalculated as glucose content . The recovered glucose after the experiment -LRB- in the digestible and indigestible residues -RRB- was assumed as 100 % , of which the indigestible fraction is expressed as a fraction . Characterization Both RS preparations were characterized before and after digestion . Additionally , freeze dried RTmd fractions from an ileostomy study feeding RTmd were pooled -LRB- 7 subjects -RRB- and used for characterization analyses -LSB- 10 -RSB- . TS was determined using the Megazyme TS assay kit -LRB- Megazyme , Co. . Wicklow , Ireland -RRB- -LSB- 14 -RSB- . In brief , starch was hydrolyzed in two phases . In phase 1 , starch was pre-treated with dimethyl-sulfoxide -LRB- DMSO -RRB- , totally solubilized and partially hydrolyzed with α-amylase . In phase 2 , dextrins were quantitatively hydrolyzed to glucose with amyloglucosidase -LRB- AMG -RRB- . Glucose was subsequently determined enzymatically using the Hexokinase/Glucose -6-phosphate-dehydrogenase assay according to Boehringer -LSB- 15 -RSB- . The RS fraction was determined using the Megazyme RS assay kit -LRB- Megazyme , Co. . Wicklow , Ireland -RRB- -LSB- 16 -RSB- . The samples were incubated in a shaking water bath with pancreatic α-amylase and AMG at 37 °C for 16 h , during which time non-resistant starch was solubilized and hydrolyzed to glucose . RS was recovered as a pellet after centrifugation , which was dissolved by stirring in KOH 2 M and quantitatively hydrolyzed to glucose with AMG -LSB- 16 -RSB- . Glucose was determined as described above . Protein was determined as sum of amino acids after acid hydrolysis and ion-exchange chromatography with post-column ninhydrin detection -LSB- 17 -RSB- . Wide-angle X-Ray powder diffraction -LRB- X-Ray -RRB- measurements were performed using a Scintag PADX Diffractometer -LRB- PANAlytical , Almelo , NL -RRB- operating at 45 mA and 40 kV at an angular range of 2θ from 1 to 40 ° with a step size of 0.03 ° . Counting time was 5 s on each step . Differential scanning calorimetry -LRB- DSC -RRB- measurements were performed using the Thermal Analyst system 2000 -LRB- DSC 2910 , TA Instument Ltd , Newcastle , UK -RRB- . Samples at a starch : water ratio of 1:4 were prepared and heated from 4 °C to 200 °C at 10 °C / min , thereafter the samples were cooled at 20 °C / min to 4 °C and heated again at the same heating rate . All the DSC results were evaluated from the mean of three separate determinations for each sample , unless stated otherwise . Statistical analysis Statistical analyses were carried out using SPSS , version 12.0.1 for Windows -LRB- one-way ANOVA and Post-Hoc Tukey -RRB- . Differences were considered to be significant at P < 0.05 . Results Composition and digestibility of RS containing preparations In Table 1 the average TS contents and indigestible starch fractions of RTmd and HAM are shown . All data are mean values of at least five repetitions . Data resulting from the dynamic and the batch model are based on the TS content before and after digestion . Both preparations contain identical amounts of TS . The two in vitro digestion models as well as the Megazyme RS method which is accepted as the official AOAC method for RS determination , led to similar amounts of indigestible RS3 fractions . Digestibility of RS2 was similar using the dynamic model , and the Megazyme RS method , whereas the batch model led to a lower digestibility . Expressing the RS results obtained by the Megayzme RS method based on substrate fresh weight -LRB- instead of g/100 g TS as shown in Table 1 -RRB- yielded to 50.2 ± 2.6 , and 45.3 ± 4.1 g/100 g for RTmd and HAM , respectively . This is very well in accordance with respectively , 48.3 ± 2.8 and 46.3 ± 3.9 g/100 g found in a collaborative study published earlier by McCleary et al. -LSB- 16 -RSB- . Standard deviations after HAM digestion were found to be higher compared to RTmd digestion . The batch system showed a better reproducibility than the dynamic model and Megazyme RS method for both substrates . Table 1Average total starch -LRB- TS -RRB- content of retrograded tapioca maltodextrins -LRB- RTmd -RRB- and high amylose maize starch -LRB- HAM -RRB- and their indigestible starch fractions obtained by dynamic digestion , batch digestion and the Megazyme resistant starch -LRB- RS -RRB- method -LRB- mean values ± SD -RRB- SampleTS -LRB- g/100 g dm -RRB- Digestion methodIndigestible starch fraction -LRB- g/100 g TS dm -RRB- RTmd94 .3 ± 1.8 -LRB- n = 17 -RRB- Dynamic59 .6 ± 2.1 -LRB- n = 6 -RRB- a , bBatch60 .6 ± 0.9 -LRB- n = 5 -RRB- aMegazyme57 .7 ± 2.9 -LRB- n = 12 -RRB- bHAM94 .3 ± 1.1 -LRB- n = 7 -RRB- Dynamic57 .5 ± 4.7 -LRB- n = 5 -RRB- dBatch65 .8 ± 3.5 -LRB- n = 10 -RRB- cMegazyme54 .8 ± 5.0 -LRB- n = 17 -RRB- dMeans with the same letters are not significantly different -LRB- Tukey P < 0.05 -RRB- Composition of digestion residues In Table 2 the average TS and RS contents of RTmd and HAM digestion residues are shown . TS quantifications of in vitro digestion residues were carried out after each single experiment in duplicate . In contrast , RS contents as well as TS in ileostomy effluents were measured in the pooled fractions only . TS contents in digestion residues turned out to be lower than in corresponding starting materials -LRB- Table 1 -RRB- , which are due to additional enzymes and bile added during digestion . Moreover , residues obtained from the batch digestion contained more starch than those from the dynamic model . The digestion residues of RTmd and HAM obtained from the dynamic model contained 1.5 g protein/100 g dm and 1.8 g protein/100 g dm , respectively , whereas in the batch digestion residues only 0.6 g protein/100 g dm and 1.2 g protein/100 g dm were determined . Bile contains large amounts of glycine that was correspondingly found in higher amounts in the dynamically digested residues -LRB- 0.2 and 0.3 g/100 g dm for RTmd and HAM , respectively -RRB- compared to the batch digested residues -LRB- 0.3 and 0.4 g/100 g dm for RTmd and HAM , respectively -RRB- . Also other amino acids like asparagine , glutamine , alanine , proline , and leucine were found in higher amounts in dynamically digested preparations . The lower amount of TS found in the ileal effluent pool compared to the in vitro digested samples is due to the dilution by additional endogenous and exogenous material such as diet derived protein , dietary fiber and fat . Table 2Total starch -LRB- TS -RRB- and resistant starch -LRB- RS -RRB- contents of in vitro and in vivo digestion residues of retrograded tapioca maltodextrins -LRB- RTmd -RRB- and high amylose maize starch -LRB- HAM -RRB- -LRB- mean values ± SD -RRB- SampleTS -LRB- g/100 g dm -RRB- RS -LRB- g/100 g dm -RRB- RTmd dynamic72 .4 ± 3.7 -LRB- n = 12 -RRB- 36.4 ± 1.0 -LRB- n = 2 -RRB- RTmd batch88 .6 ± 0.6 -LRB- n = 10 -RRB- 61.4 ± 1.2 -LRB- n = 3 -RRB- RTmd in vivo48 .4 ± 0.0 -LRB- n = 2 -RRB- 31.3 ± 1.1 -LRB- n = 2 -RRB- HAM dynamic74 .9 ± 1.7 -LRB- n = 10 -RRB- 27.6 ± 2.0 -LRB- n = 2 -RRB- HAM batch80 .9 ± 2.6 -LRB- n = 12 -RRB- 35.7 ± 0.7 -LRB- n = 3 -RRB- It is expected that RS values close to 100 % are found in the in vivo as well as the in vitro digestion residues . As shown in Table 2 lower amounts of RS -LRB- 28 -- 62 % -RRB- were found in the digestion residues . This can be explained by the fact that the digestion method carried out prior to the analytical RS determination has an influence on the starch structures leading to less resistant starch . Therefore , analysing RS in digestion residues may be interpreted as characterization method , indicating the highly resistant fraction . Different amounts of RS were found because of additional non-starch material present in the samples as described above . Calculating RS recoveries based on TS led to 50 g/100 g TS for the dynamically digested RTmd fractions , whereas after batch and in vivo digestion similar amounts -LRB- 69 and 65 g/100 g TS , respectively -RRB- were found . In contrast , clearly lower RS amounts were found in both HAM digestion residues -LRB- 37 and 44 g/100 g -RRB- after dynamic and batch digestion , respectively . In Fig. 1 the absorption of digestion products from the jejunum and ileum compartments during the dynamic experiment can be followed . The main digestion products found were maltose and maltotriose despite the fact that larger molecules are able to pass the hollow fiber devices as well . Starch digestion products up to DP3 were found when RTmd was being digested , whereas after HAM digestion minor amounts of maltotetraose -LRB- less than 0.1 % of total digestible glucose -RRB- were detected as well . The kinetics of RTmd and HAM digestion differed considerably . The digestible fraction of RTmd was degraded completely after 4 h , whereas degradation of HAM still continued after 4 h . The lower amounts of degradation products collected during the first 4 h from HAM digestion , and the higher total amounts separated from the ileum compartment show that HAM under these experimental conditions is being digested more slowly compared to RTmd . The enzyme used for starch digestion in the dynamic model is a α-amylase , therefore maltose is theoretically expected to be the smallest degradation product . As can be seen in Fig. 1 , glucose was found as well . Glucose could originate from added compounds during the dynamic digestion experiment , such as bile and enzymes . Moreover , a small contamination of α-amylase by AMG or α-amylase containing small amounts of side-activity can not be excluded . The total amount of starch degradation products absorbed during the dynamic in vitro digestion experiment is slightly higher for HAM compared to RTmd . This is in accordance with a somewhat lower non-significant indigestible residue of HAM compared to RTmd as shown in Table 1 . Fig. 1Digestible starch degradation products -LRB- up to DP3 -RRB- of retrograded tapioca maltodextrins -LRB- RTmd -RRB- , and high amylose maize starch -LRB- HAM -RRB- recovered from the jejunum -LRB- a -RRB- and the ileum -LRB- b -RRB- compartments Characterization of digestion residues X-ray diffraction X-ray diffraction patterns of RTmd and its dynamic , batch and in vivo digestion residues are presented in Fig. 2 . RTmd and indigestible RTmd fractions all led to a typical A-type pattern . However , the diffraction patterns , with exception of RTmd dynamically digested , exhibited an additional peak at θ2 = 20 ° which can be interpreted as V-form due to the presence of amylose -- lipid complexes . RTmd and RTmd batch digested residues were very similar concerning their crystallinity . The only differences were the peaks at 2θ = 20 ° , and 2θ = 23 ° , which were slightly lower and higher , respectively in the batch digested sample . In contrast to RTmd and its batch digested fraction , dynamically digested RTmd showed no peak at 2θ = 27 °C and no pronounced double peak at 2θ = 17 ° and 2θ = 18 ° -LRB- both typical for A-type structure -RRB- , which indicate a loss in crystallinity due to digestion . The in vivo digestion residue showed an additional peak at 2θ = 29 ° ; this peak could be due to additional structures of proteins or lipids present in this sample . Fig. 2X-ray diffraction patterns of retrograded tapioca maltodextrins -LRB- RTmd -RRB- and their digestion residues HAM containing samples led to lower crystallinity compared to RTmd -LRB- Fig. 3 -RRB- , which correspond to Gerard et al. -LSB- 18 -RSB- , showing that high amylose maize starches have low crystallinity . HAM and its digestion residues led to B-type pattern , although the peak at 2θ = 20 ° observed in HAM and batch digested HAM indicated a mixture between B - and V-type which was described by Shi et al. -LSB- 19 -RSB- as well . Due to in vitro digestion of HAM and dynamically digestion of RTmd the peak θ2 = 26 ° disappeared which points to a loss in crystallinity . Similar as for RTmd -LRB- Fig. 2 -RRB- the dynamic digestion led to higher changes in crystallinity compared to batch digestion . Fig. 3X-ray diffraction patterns of high amylose maize starch -LRB- HAM -RRB- and their digestion residues DSC The thermal behavior of the substrate melting was studied using DSC measurements . The results from the first and second heating are shown in Table 3 . During the first heating , endothermic peak temperatures -LRB- Tp -RRB- turned out to be slightly lower for RS2 compared to RS3 . The peak ranges within the RTmd and HAM containing preparations were found to be similar with exception of a slightly narrower peak of in vivo digested RTmd . Enthalpies were found to be lower for HAM containing preparations compared to RTmd containing preparations . The melting transition of RTmd was separated by an exothermic effect -LRB- not shown -RRB- . Such a melting characteristic can be interpreted as partial melting -LRB- endothermic -RRB- , followed by recrystallization -LRB- exothermic -RRB- and final melting -LRB- endothermic -RRB- -LSB- 20 , 21 -RSB- . This exothermic peak was not visible in the digestion residues , which indicates that due to digestion the fractions responsible for these rearrangements were removed . Within the RS3 fractions , in vivo digestion led to significantly lower enthalpies . Dynamic digestion of both preparations led to an exothermic peak when the sample was reheated -LRB- Table 3 -RRB- . This indicates that the reorganization of the crystalline structures takes place during the second heating . RTmd and its batch and in vivo digested fractions did not show any heat flow during the second heating -LRB- therefore not shown in Table 3 -RRB- , it can be interpreted that recrystallization was completed during cooling in these samples already . HAM containing samples , excluding the dynamically digested fraction , all led to an endothermic peak during reheating which indicates a reversible reconstruction of the amylose -- lipid complexes -LSB- 20 -RSB- . Table 3Average results -LRB- n = 3 -RRB- from differential scanning calorimetry -LRB- DSC -RRB- for retrograded tapioca maltodextrins -LRB- RTmd -RRB- and high amylose maize starch -LRB- HAM -RRB- and their digestion residues . T0 : onset temperatures -LRB- °C -RRB- , Tp : peak temperatures -LRB- °C -RRB- , Tc : completion temperatures -LRB- °C -RRB- , ΔH : transition enthalpy ± SD -LRB- J/g -RRB- -LRB- endothermic if nothing else stated -RRB- SampleT0 -LRB- °C -RRB- Tp -LRB- °C -RRB- Tc -LRB- °C -RRB- ΔH -LRB- J/g -RRB- First heatingRTmd -LRB- n = 2 -RRB- 60.7112.8140.49.0 ± 1.1 aRTmd dynamic58 .4108.6132.49.1 ± 0.2 aRTmd batch61 .8109.1136.410.8 ± 2.0 aRTmd in vivo58 .4105.0123.55.2 ± 1.1 bHAM61 .896.9110.02.6 ± 0.3 cHAM dynamic55 .598.7127.04.2 ± 2.2 cHAM batch -LRB- n = 2 -RRB- 59.991.5110.53.6 ± 0.7 cSecond heatingRTmd dynamic79 .787.9144.0 * 2.4 ± 0.4 HAM60 .088.2109.62.7 ± 0.9 dHAM dynamic84 .994.2146.1 * 2.8 ± 0.2 dHAM batch59 .883.2101.90.9 ± 0.0 e * Exothermic , means with the same letters are not significantly different -LRB- Tukey P < 0.05 -RRB- Discussion Digestibility of RS preparations In vitro digestion models use a strictly standardized procedure based on an average human digestibility . Therefore standard deviations of in vitro experiments are expected to be low . In vitro obtained HAM fractions showed higher standard deviations compared to in vitro digested RTmd fractions . Possibly the molecular structures of HAM have been more sensitive to small differences in experimental conditions such as mechanical stress , temperature or pH between experiments . RTmd in vitro digestion residues result in quantitatively similar RS fractions -LRB- Table 1 -RRB- which are very well in accordance with 59 g/100 g found in vivo after feeding RTmd to ileostomy patients -LSB- 10 -RSB- . In contrast , in vitro digestion using HAM as a substrate led to different amounts of RS . These differences may be explained by methodological variations in the in vitro models used , suggesting that HAM was more affected by the experimental conditions . During the dynamic digestion experiment the substrate is exposed to the movements of the flexible walls -LSB- 8 -RSB- , in the batch model the slurry is stirred -LSB- 7 -RSB- , and in the Megazyme RS method the sample is shaken -LSB- 16 -RSB- . An effect of mechanical treatment on starch degradation of HAM but not of RTmd was shown by McCleary and Monaghan -LSB- 22 -RSB- before . They performed the Megazyme RS method comparing shaking and stirring . Stirring led to a higher digestibility of HAM , whereas RTmd did not show any differences . In the present study however , stirring applied in the batch model led to a lower digestibility compared to shaking as used in the Megazyme RS method . This shows that apart from mechanical forces , also other factors may have a significant impact on starch degradation . For example , different amounts and types of enzymes , their accessibilities to the substrate as well as the formation of the enzyme -- substrate complexes could have a larger influence on starch degradation of HAM compared to RTmd using in vitro digestion models . Furthermore , the incubation time during the dynamic digestion is nearly twice as long compared to the batch digestion and over four times as long in the Megazyme RS method . As pointed out in Fig. 1 , 99 % of in vitro digestible RTmd fractions were degraded after 4 h , whereas only 86 % of those of HAM were digested at this stage . If the dynamic digestion experiment would have been stopped after 4 h -LRB- the duration of the batch digestion experiment is 3.75 h -RRB- , the remaining indigestible fraction of HAM would be identical to that obtained by the batch in vitro digestion . Whether a prolongation of the batch incubation would lead to HAM fractions similar to those obtained with the dynamic model , remains to be elucidated . The analytical RS contents found in the dynamically digested residues was lower in both preparations when compared to the batch digested fractions -LRB- Table 2 -RRB- . This leads to the assumption that in the dynamic approach starch structures are attacked to a higher extent compared to the batch model . Nevertheless , the amount of TS recovered after digestion of RTmd was not affected , whereas the TS recovery of HAM possibly was influenced by the different amount of analytical RS present in the sample ; the results strongly indicate that structural features play an important role in digestibility . Structural aspects of the RS preparations X-Ray diffraction A mixture between A - and V-type structures for RTmd , and its in vivo digestion residues -LRB- Fig. 2 -RRB- was found by Pohu -LSB- 12 -RSB- as well . The tightly packed A-type structure is known to be very heat stable . Shamai et al. -LSB- 23 -RSB- investigated RS3 fractions from high amylose maize , maize flour and wheat starch , respectively . They found that RS3 produced at low retrogradation temperatures of 40 °C lead to B-type patterns , whereas incubation at 95 °C produced a mixture of A - and V-type polymorphs . This is in contrast to the present study , where RS3 produced from long chain tapioca maltodextrins was found to consist of a mixture between A - and V-type crystal structures , even though rather low retrogradation temperatures of 52 -- 54 °C were applied -LSB- 11 -RSB- . This shows that the structural behavior of RS3 can not be predicted without knowing the exact process parameters and these parameters are rarely disclosed , particularly in patents . HAM led to a mixture of B - and V-type polymorphs , which is characteristic for native high amylose maize starch , as shown by several research groups -LSB- 23 -- 25 -RSB- . The dynamically digested fraction did not show the V-type characteristic peak at 2θ = 20 ° . This peak is known to represent amylose -- lipid complexes . It is possible that the extra-addition of lipase in the dynamic digestion model was able to degrade lipids present in HAM and RTmd to an extent where no complexation was possible anymore . During batch digestion no additional lipase was used since this enzyme is present in the pancreatin preparation . In earlier experiments it has been shown that the pH-optimum of the pancreatic lipase is approximatively 9 -LSB- 26 -RSB- . Therefore , the incubation at pH 6.9 in the batch model is expected to lead to a small lipid degradation only . This could explain that the peak at 2θ = 20 ° was only reduced during batch procedure , but disappeared using the dynamic digestion model . In vivo digested RTmd showed amylose -- lipid complexes -LRB- Fig. 2 -RRB- as well . However , it can not be stated whether the lipid degradation in the dynamic model was more efficient compared to in vivo digestion because diet-derived lipids led to a much higher lipid content before in vivo digestion -LRB- results not shown -RRB- . The X-ray patterns of RTmd and HAM samples indicate that the molecules within the crystals are not packed in the same way which leads to different functional properties of HAM , and RTmd . Batch digestion of both preparations did not lead to a distinct change in crystallinity . However , in vivo digestion caused slightly broader , and dynamic digestion distinctly broader diffraction peaks , which indicate the presence of either imperfect or relatively small crystallites -LSB- 27 -RSB- . The latter could have occurred due to the mechanical stress which underlines that the dynamic model attacks the starch structures more strongly . DSC Endothermic transitions of the investigated samples took place at temperatures which are known to destroy the amylose -- lipid complexes . At lower temperatures -LRB- 45 -- 60 °C -RRB- no peak was detected indicating that amylopectin is neither present in RTmd nor in HAM -LSB- 20 -RSB- . RTmd in vivo digested showed the lowest peak maximum and enthalpy of all the digestion residues . This may be due to the higher amount of non-starch material in the sample -LRB- Table 1 -RRB- which may compete for water binding and thus reduce the starch swelling . Dynamically digested HAM showed a slightly broader endothermic peak compared to the other samples which indicates a broader polydispersity -LSB- 28 -RSB- . This observation coincides well with the statement that starch is degraded differently , and probably more strongly in the dynamic model . In vitro digestion of HAM led to slightly but not significantly higher enthalpies , especially when dynamically digested . Cooke et al. -LSB- 27 -RSB- suggested that the enthalpy of gelatinization primarily reflects the loss of the double helical order which could occur to a higher extent during dynamic digestion . Conclusions The results of this study show that both , a simple and a more sophisticated in vitro digestion model and the ileostomy study led to similar amounts of starch escaping digestion when using a RS3 containing carbohydrate source as substrate . In contrast , when the two in vitro digestion models were compared with a RS2 containing preparation , different amounts of indigestible starch were found . It is not possible to predict which model simulated the in vivo RS2 fraction more precisely . The more sophisticated in vitro digestion model attacked starch probably more strongly compared to the batch in vitro digestion model using an RS2 and an RS3 containing substrate . This was confirmed by differences in crystalline fractions and structural rearrangements as measured by X-ray and DSC measurements . These structural differences , caused by different experimental parameters influenced starch digestibility of HAM but not of RTmd . The behavior of other RS preparations in the two in vitro models described here can not be deduced from the obtained results and has to be examined case by case .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

resistant starch type 2
resistant starch type 3
x-ray


Incidence of late vitamin K deficiency bleeding in newborns in the Netherlands in 2005 : evaluation of the current guideline Vitamin K prophylaxis is recommended to prevent the hazard of haemorrhage caused by vitamin K deficiency in newborns . The present Dutch guideline recommends 1 mg of vitamin K1 orally at birth , followed by a daily dose of 25 μg of vitamin K1 from 1 to 13 weeks of age for breastfed infants . Since the introduction of this prophylaxis , the incidence of vitamin K deficiency bleeding -LRB- VKDB -RRB- has decreased ; however , late VKDB is still reported . From 1 January to 31 December 2005 , a nationwide active surveillance was performed by the Netherlands Paediatric Surveillance Unit -LRB- NSCK -RRB- to study the current incidence and aetiology of late VKDB in infants . Six cases could be validated as late VKDB : all were breastfed , one fatal idiopathic intracranial haemorrhage at the age of 5 weeks and five bleedings secondary to an underlying cholestatic liver disease between the age of 3 and 7 weeks . The total incidence of late VKDB and idiopathic late VKDB was calculated to be 3.2 -LRB- 95 % CI : 1.2 -- 6.9 -RRB- and 0.5 -LRB- 95 % CI : 0 -- 2.9 -RRB- per 100,000 live births , respectively . With the current Dutch guideline , idiopathic late VKDB is rare but late VKDB secondary to cholestasis still occurs in breastfed infants . Doubling the daily dose of vitamin K1 to 50 μg , as is comparable to formula-feeding , may possibly prevent VKDB in this group . Further research , however , is needed to prove this hypothesis . Introduction Vitamin K deficiency can cause severe haemorrhage in the newborn . The term `` haemorrhagic disease of the newborn '' -LRB- HDN -RRB- was coined by Charles Townsend in 1894 to describe an acquired bleeding disorder in the newborn due to vitamin K deficiency -LSB- 26 -RSB- . Nowadays , the term HDN is replaced by vitamin K deficiency bleeding -LRB- VKDB -RRB- , as neonatal bleeding is often not due to vitamin K deficiency and VKDB often occurs after the 4-week neonatal period -LSB- 23 -RSB- . VKDB can be classified according to the time of presentation after birth into early -LRB- 0 -- 24 h -RRB- , classical -LRB- 1 -- 7 days -RRB- and late -LRB- 2 -- 12 weeks -RRB- VKDB . Late VKDB occurs primarily in exclusively breastfed infants who have received no or inadequate vitamin K prophylaxis . In addition , intestinal malabsorption defects -LRB- cholestatic jaundice , cystic fibrosis , α-1-antitrypsin deficiency etc. -RRB- are a well known frequent cause of late VKDB -LSB- 13 -RSB- . In some children , `` warning bleeds '' such as mild bruises , nose bleeds or umbilical oozing are the first manifestation of VKDB . In about 50 % of the cases in late VKDB , intracranial haemorrhage occurs , accounting for death and serious neurological and mental handicaps -LSB- 22 -RSB- . The efficacy of neonatal vitamin K prophylaxis -LRB- oral or parenteral -RRB- in the prevention of classic VKDB is firmly established -LSB- 16 -RSB- . It has been the standard of care since the Committee on Nutrition of the American Academy of Paediatrics recommended to administer vitamin K prophylaxis to all newborn infants in 1961 -LSB- 1 -RSB- . Since then , many countries adopted this recommendation , although controversies concerning the best dose , route and frequency of administration still exist . In 1990 , the Dutch Paediatric Association recommended the administration of a single oral dose of 1 mg of vitamin K1 at birth to all healthy infants . Parenteral administration of vitamin K1 at birth was recommended for newborns especially at risk -LRB- preterm babies , babies born with instrumental help or caesarean section , asphyxia or if maternal medication was interfering with coagulation -RRB- . For infants who are largely or fully breastfed , a daily dose of 25 μg of vitamin K1 orally was recommended from 1 to 13 weeks of age . In case of additional risk factors like a known malabsorption , this dose should be increased to 50 μg per day -LSB- 29 -RSB- . After the introduction of this guideline , the incidence of late VKDB , which was studied by a nationwide active surveillance performed by the Netherlands Paediatric Surveillance Unit -LRB- NSCK -RRB- in 1992 to 1994 , decreased from about 7.0 to 1.1 / 100,000 live births -LSB- 2 -RSB- . However , still , some cases of late VKDB have been reported in the Netherlands , merely due to an underlying cholestatic liver disease -LSB- 7 , 10 , 11 -RSB- . Therefore , a one-year surveillance was started again to study the current incidence and the aetiology of late VKDB in the Netherlands . Discussion will rise about the efficacy of the current guideline in order to completely prevent late VKDB . Methods From 1 January to 31 December 2005 , an active surveillance programme for VKDB was in operation in the Netherlands , using methods developed by the British Paediatric Association Surveillance Unit -LSB- 8 -RSB- . Surveillance was based on monthly report cards sent to paediatricians responsible for in - and outpatient care in all hospitals by the NSCK . A `` nothing to report '' option was included . The return rate of these report cards was 94.7 % in 2004 -LSB- 17 -RSB- . The paediatricians were asked to report all infants in whom bleeding may have resulted from VKDB . The reported cases were validated with a questionnaire asking for information about the infant , type of feeding , clinical presentation , dose of vitamin K prophylaxis , route of vitamin K prophylaxis , associated diseases and laboratory data . For all reported cases , we asked for an anonymous hospital discharge letter to verify the diagnosis of late-onset VKDB . The case definition of late-onset VKDB was as internationally defined : any infant between 8 days to 26 weeks of age , with spontaneous bruising , bleeding or intracranial haemorrhage associated with a grossly prolonged prothrombin assay , not due to an inherited coagulopathy or disseminated intravascular coagulation -LSB- 23 -RSB- . Confirmed VKDB was diagnosed when the prothrombin assay results were grossly abnormal compared with the standards for age : international normalized ratio -LRB- INR -RRB- ≥ 4 control value , prothrombin time -LRB- PT -RRB- ≥ 4 control value and at least one of the following was present : Platelet count normal or raised and normal fibrinogenProthrombin assay returned to normal after vitamin K administrationConcentration of proteins induced by vitamin K absence -LRB- PIVKA -RRB- exceeded normal controls PIVKA is a sensitive indicator of vitamin K status , allowing the detection of subclinical deficiency states which would not be detected by conventional coagulation assays -LSB- 20 -RSB- . Idiopathic cases were defined as cases in whom no factor predisposing to vitamin K deficiency was identified . Secondary cases were those in whom an underlying condition such as cholestasis or other causes of malabsorption of vitamin K could be identified . Vitamin K prophylaxis was considered to be `` as recommended '' if the Dutch guideline was followed at the time of bleeding : 1 mg of vitamin K1 -LRB- oral , intravenous , intramuscular -RRB- directly after birth , followed by a daily dose of 25 μg of vitamin K1 from 1 to 13 weeks of age for all breastfed babies . The Konakion mixed micellar preparate -LRB- Phytomenadion 10 mg/ml , Hoffmann-La Roche , Basel , Switzerland -RRB- , which can be used for both oral , intramuscular and intravenous administration , an oral preparate called Davitamon K -LRB- marketed by Chefaro , Rotterdam , the Netherlands , 25 μg/5gtt -RRB- and an oral preparate , Phytomenadion , which is made by the Dutch pharmacist Mixtura -LRB- Phytomenadion 10 mg/ml FNA -RRB- were the only vitamin K preparations licensed in the Netherlands . The 95 % confidence intervals -LRB- CI -RRB- were calculated according to a binomial distribution and the chi-squared test was used for the comparison of incidences between different periods , with a value of P < 0.05 being considered as significant . Results The response rate to the surveillance amounted to 93.4 % and the response to our questionnaires was 100 % . In total , seven cases of late VKDB were reported : four reports from academic paediatric centres and three from general district hospitals . A hospital discharge letter was obtained in six cases . In all cases , the diagnosis could be verified by the completed questionnaire or the additional discharge letter . One case did not fulfil the criteria of the case definition because there were no clinical signs of bleeding , only biochemical clotting disorders due to a vitamin K deficiency . Six cases fulfilled the criteria of the case definition and could be validated as late VKDB . The surveillance data are shown in Table 1 . The mean age of presentation was 5 weeks and 2 days , and all of the cases were exclusively breastfed . In all cases , the PT time or INR was at least four times greater than normal and vitamin K deficiency was confirmed by a documented PT or INR normalisation after the administration of vitamin K. Five of the cases also received fresh frozen plasma . Table 1Confirmed cases of late vitamin K deficiency bleeding -LRB- VKDB -RRB- in the Netherlands from 1 January to 31 December 2005Patient ; sex ; age -LRB- weeks -RRB- PresentationFeedingVitamin K prophylaxisProthrombin assay -LRB- after vitamin K administration -RRB- Additional morbidityOutcome of bleedingA ; female ; 5 + 1/7Intracranial bleedingBreastfeedingBirth : 1 mg p.o. → 25 μg/dayPT > 120 s -LRB- 13.3 -RRB- ; APTT > 120 s -LRB- 37 -RRB- NoDiedB ; female ; 6 + 6/7Intracranial bleedingBreastfeedingBirth : 1 mg p.o. → 25 μg/dayINR > 8 -LRB- INR < 2 -RRB- ; APTT > 240 s -LRB- 49 -RRB- Biliary atresiaHemipareses rightC ; male ; 4 + 4/7Intracranial bleedingBreastfeedingBirth : 1 mg p.o. → 25 μg/dayPT > 90 s -LRB- 30 -RRB- ; APTT > 120 s -LRB- 38 -RRB- ; Vitamin K : 0.1 nmol/lBiliary atresiaHemipareses rightD ; male ; 5 + 2/7Nasal bleedingBreastfeedingBirth : 1 mg p.o. → 25 μg/dayPT > 75 s -LRB- normal -RRB- ; APTT : 118 s -LRB- normal -RRB- Biliary atresiaNo sequelaeE ; male ; 6 + 2/7Nasal bleedingBreastfeedingBirth : 1 mg p.o. → 25 μg/dayPT > 165 s -LRB- normal -RRB- ; APTT > 240 s -LRB- normal -RRB- Biliary atresiaNo sequelaeF ; male ; 3 + 6/7Nasal bleedingBreastfeedingBirth : 1 mg p.o. → 25 μg/dayPT > 71 s -LRB- 12.7 -RRB- ; APTT : 128 s -LRB- 39.1 -RRB- CholestasisNo sequelae Concerning the cause of VKDB , there was one case of idiopathic VKDB , who presented with signs of bleeding at the age of 5 weeks . She was breastfed and died due to the consequence of an intracranial bleeding . She received exactly the recommended prophylaxis . Unfortunately , no post-mortem obduction was performed but , biochemically , there were no signs of cholestasis . Five out of six cases could be validated as secondary late VKDB . The age of presentation in this group was between 3 and 7 weeks of age . All of these bleedings were due to an underlying cholestasis which was diagnosed after the first presentation of bleeding . In two cases , an intracranial bleeding was the first presenting sign . Four cases were diagnosed as having a bile duct atresia . All infants had been exclusively breastfed and all had received exactly the recommended prophylaxis . In the study period , the number of live births amounted to 187,910 -LSB- 21 -RSB- . Therefore , the total incidence of late VKDB and idiopathic late VKDB was calculated to be 3.2 -LRB- 95 % CI : 1.2 -- 6.9 -RRB- and 0.5 -LRB- 95 % CI : 0 -- 2.9 -RRB- per 100,000 live births , respectively . Discussion Data from the United Kingdom , Germany and Switzerland prove that intramuscular vitamin K1 prophylaxis -LRB- 1 mg -RRB- is highly effective in the prevention of late VKDB . This is supported by data from Australia , which demonstrate a higher incidence of late VKDB in babies who received oral vitamin K compared to those with parenteral vitamin K at birth . However , most of these failures of oral vitamin K were in cases reported with an underlying cholestatic liver disease -LSB- 3 , 12 , 14 , 25 , 32 , 33 -RSB- . In 1992 , Golding et al. -LSB- 5 -RSB- reported an increased risk of developing childhood cancer after parenteral vitamin K1 prophylaxis . Recent studies , however , show that solid tumours are no commoner in children who had parenteral vitamin K. Although no convincing evidence between parenteral vitamin K and childhood leukaemia was found in these studies , it can not be fully excluded that there is no risk -LSB- 4 , 18 -RSB- . Since the report of Golding et al. , there has been an increasing trend towards oral vitamin K administration and many oral prophylaxis regimes have been developed . In 1997 , we summarised the results of surveillance studies of over 2 million infants in four different countries using different multiple oral dose schedules and parenteral administration of vitamin K . A daily low oral dose of 25 μg of vitamin K1 following an initial oral dose of 1 mg vitamin K1 directly after birth for exclusively breastfed infants , as was used in the Netherlands since 1990 , seemed to be as effective as parenteral vitamin K1 prophylaxis -LSB- 3 -RSB- . The present surveillance study shows that the current Dutch guideline still seems quite effective in preventing idiopathic late VKDB , but it is insufficient against secondary late VKDB . In our surveillance , the incidence of all late VKDB and idiopathic late VKDB was 3.2 -LRB- 95 % CI : 1.2 -- 6.9 -RRB- and 0.5 -LRB- 95 % CI : 0 -- 2.9 -RRB- per 100,000 live births , respectively . The incidence of the latter is comparable to 0.5 -LRB- 95 % CI : 0.1 -- 1.6 -RRB- per 100,000 live births reported in our surveillance study performed by the same Netherlands Paediatric Surveillance Unit -LRB- NSCK -RRB- in 1992 -- 1994 -LSB- 2 -RSB- . The incidence of late VKDB secondary to an underlying cholestatic liver disease is , however , significantly higher -LRB- P < 0.05 -RRB- ; 2.7 -LRB- 95 % CI : 0.9 -- 6.2 -RRB- compared to 0.7 -LRB- 95 % CI : 0.1 -- 2.0 -RRB- in 1992 -- 1994 . A higher incidence of cholestatic liver diseases in general or a delayed recognition of symptoms can be explanations for this difference . Unfortunately , data about the incidence or recognition of cholestatic liver diseases in the Netherlands are not available . In this surveillance , there were no reports of late VKDB after parenteral administration of vitamin K1 . This can be explained by the assumption of the higher efficacy of parenteral vitamin K1 or the low use of intramuscular and intravenous vitamin K1 these days . Since 1994 , a new preparate , Konakion mixed micellar , has been available . This mixed micellar formulation is prepared with the phospholipids lecithin and the bile salt glycocholic acid , and it was presumed that this preparate would reduce the incidence of late VKDB due to a better absorption of oral vitamin K. However , a recent randomised controlled trial shows that the mixed micellar vitamin K gives unreliable and still severely impaired intestinal absorption in infants with cholestasis -LSB- 15 -RSB- . Furthermore , von Kries et al. -LSB- 31 -RSB- reported no significant reduction in the incidence of late VKDB in healthy infants and infants with an underlying cholestatic liver disease with this preparate compared to the use of the original Konakion -LRB- Cremophor EL -RRB- and Kanavit -LRB- Polysorbat 80 -RRB- . In view of the fact that vitamin K has limited tissue reserves and is rapidly catabolised , with 60 -- 70 % of a single dose being excreted via the urine and bile in about 3 days , it seems reasonable that repeated doses rather than a higher single dose is more effective in preventing late VKDB -LSB- 15 -RSB- . An important study from Denmark has established that 1 mg of oral vitamin K1 given weekly during the first 3 months of life , following an initial oral dose of 2 mg of vitamin K1 directly after birth , is as effective as 1 mg of vitamin K1 given intramuscularly at birth -LSB- 9 -RSB- . Therefore , we hypothesise that the dosage of 25 μg of vitamin K1 daily is not enough to prevent the secondary cases of late VKDB , as we encountered in this study . In most co-operative studies , the upper age limit for inclusion was set arbitrarily at the end of 12 weeks of age -LSB- 32 -RSB- . In our study , we included infants presenting with VKDB up to the age of 26 weeks of age , as suggested by Tripp et al. -LSB- 27 -RSB- . However , no infant between 12 and 26 weeks of age was reported . The mean age of all infants with late VKDB was 5 weeks , which is comparable to other reports -LSB- 22 -RSB- . In our study , the boy : girl ratio was 2.5:1 . Why late VKDB occurs more in boys than girls is unknown , but this is consistent with other European reports -LSB- 22 , 24 -RSB- . Unfortunately , this study has some limitations . First , this surveillance was based on one data source only : the Netherlands Paediatric Surveillance Unit . In spite of the high return rate of the report cards -LRB- 93.4 % -RRB- , it is plausible that dramatic conditions , such as intracranial bleedings , are more likely to be reported , as also noted by von Kries et al. -LSB- 31 -RSB- . For this reason , our data could give an under-reporting of the true incidence of late VKDB in the Netherlands . However , the fact that 50 % of late VKDB cases were intracranial bleedings is comparable to other , larger reports -LSB- 3 , 13 , 14 , 19 , 22 , 30 , 31 , 33 -RSB- . Secondly , the surveillance lasted only 1 year . In all probability , the confidence interval would be smaller when the length of the surveillance was prolonged . Third , the proportion of infants that is breastfed is unknown , and , additionally , we lack data about compliance to the prophylaxis . It is likely , however , that less than 100 % of the eligible cases received the recommended prophylaxis . Prophylaxis failure , therefore , is likely to be underestimated due to a greater denominator than the true numbers exposed . We would suggest to double the daily dose of vitamin K1 to 50 μg for all breastfed babies from 1 to 13 weeks of age to prevent bleedings in infants with an underlying cholestatic liver disease as well . This amount is comparable to the ingested dose of vitamin K1 by formula-feeding -LSB- 6 -RSB- . Formula is supplemented with vitamin K1 to a minimal concentration of 50 μg/l . VKDB are not reported in formula-fed babies and there is no evidence that they are at an increased risk of childhood cancer -LSB- 28 -RSB- . Studies with such a concentration of vitamin K1 have never been performed and further research is necessary to study the efficacy of this prophylaxis in infants with underlying cholestatic liver disease . Due to the low incidence of late VKDB , it would be difficult to perform a randomised trial of 25 μg versus 50 μg of vitamin K1 daily . Alternatively , a new surveillance for 1 or 2 years after implementing 50 μg of vitamin K1 as the prophylaxis could give an indication of the efficacy of this dose . Furthermore , an earlier recognition of symptoms associated with cholestatic liver disease and immediate investigation and treatment of `` warning bleeds '' helps to prevent the severe consequences of secondary late VKDB . In conclusion , with the current guideline of vitamin K prophylaxis in the Netherlands , idiopathic late VKDB is rare , but late VKDB secondary to an underlying cholestatic liver disease still occurs . We suggest to double the daily maintenance dose of vitamin K1 to 50 μg per day for all breastfed infants from 1 to 13 weeks of age in order to prevent bleedings in children with an underlying cholestatic liver disease as well . Further research , however , is recommended .

O O O B I O O O O O O O O O O O O O O O B I B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I B O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


What

is

new

in

iron

overload

Children

with

severe

chronic

hemolytic

anemia

or

congenital

erythroblastopenia

are

transfusion

dependent

Long-term

transfusion

therapy

prolongs

life

but

results

in

a

toxic

accumulation

of

iron

in

the

organs

The

human

body

can

not

actively

eliminate

excess

iron

Therefore

the

use

of

a

chelating

agent

is

required

to

promote

excretion

of

iron

So

far

iron

chelation

has

been

done

by

subcutaneous

infusion

of

deferoxamine

given

over

h

days

per

week

Compliance

is

poor

and

chelation

often

insufficient

Ferritin

measurements

and

sometimes

liver

biopsies

are

used

to

evaluate

the

iron

burden

in

the

body

At

the

present

time

new

iron

chelators

that

can

be

given

orally

are

available

Furthermore

magnetic

resonance

imaging

-LRB-

MRI

-RRB-

assessment

of

tissue

iron

is

a

noninvasive

and

highly

reproducible

method

which

is

able

to

quantitate

organ

iron

burden

In

conclusion

iron

overload

can

be

measured

more

accurately

with

noninvasive

methods

such

as

MRI

Deferasirox

is

a

once-daily

oral

therapy

for

treating

transfusional

iron

overload

which

improves

patient

compliance

and

quality

of

life

Introduction

Children

suffering

from

severe

chronic

hemolytic

anemia

such

as

β-thalassemia

or

congenital

erythroblastopenia

are

at

risk

of

developing

severe

iron

overload

Regular

blood

transfusions

are

potentially

life

saving

However

every

single

transfusion

of

one

unit

of

blood

brings

mg

of

iron

into

the

body

of

the

recipient

Patients

receiving

U

every

weeks

-LRB-

U

per

year

-RRB-

will

accumulate

g

of

iron

by

the

end

of

the

year

in

addition

to

the

amount

absorbed

by

the

gut

The

human

body

has

no

mechanism

for

active

excretion

of

excess

iron

Repeated

transfusions

lead

to

elevated

iron

levels

that

result

in

major

organ

damage

Excess

iron

is

deposited

largely

in

the

liver

spleen

myocardium

and

several

endocrine

organs

In

young

patients

growth

and

maturation

is

slowed

and

in

all

age

groups

liver

disease

diabetes

mellitus

and

other

complications

develop

Once

the

body

's

storage

capacity

is

exceeded

free

iron

catalyses

the

formation

of

highly

reactive

hydroxyl

radicals

which

cause

membrane

damage

and

protein

denaturation

This

process

leads

to

tissue

damage

and

ultimately

to

significant

morbidity

and

mortality

-LSB-

-RSB-

Measuring

iron

levels

-LRB-

Table

-RRB-

Table

of

methods

for

evaluating

iron

overload

InvasiveCostLimitationValidationFerritinNoLowInfection/inflammationYesLICYesMedianFibrosis/cirrhosisYesSQUIDNoHighNot

easily

availableYesMRINoHighDedicated

personYesLIC

liver

iron

content

SQUID

superconducting

quantum

interference

device

MRI

magnetic

resonance

imaging

There

are

several

methods

of

assessing

body

iron

load

As

of

excess

iron

is

deposited

in

the

liver

most

techniques

focus

on

measuring

liver

iron

levels

and

it

is

widely

accepted

that

liver

iron

content

-LRB-

LIC

-RRB-

provides

an

accurate

measure

of

whole-body

iron

concentration

The

invasive

nature

of

the

liver

biopsy

means

that

other

markers

such

as

serum

ferritin

levels

are

frequently

employed

Other

approaches

using

biomagnetic

susceptometry

and

magnetic

resonance

imaging

-LRB-

MRI

-RRB-

are

also

being

assessed

in

order

to

identify

an

accurate

low-risk

and

convenient

approach

to

assessing

patient

iron

status

Ferritin

Serum

ferritin

is

easily

measured

using

a

commercially

available

kit

A

ferritin

constantly

below

mg/l

has

been

shown

to

reduce

the

risk

of

cardiac

complications

but

a

target

value

of

mg/l

is

recommended

Factors

such

as

inflammation

ascorbate

status

and

hepatitis

can

affect

serum

ferritin

levels

Therefore

results

should

be

interpreted

with

caution

and

a

trend

in

the

evolution

of

serial

measurements

is

a

better

index

than

day-to-day

variation

In

patients

with

transfusion-dependent

diseases

chelation

should

be

initiated

after

blood

transfusions

or

when

ferritin

level

rises

above

mg/l

Liver

biopsy

LIC

measurement

is

the

most

accurate

method

for

assessing

body

iron

-LRB-

Fig.

-RRB-

It

also

provides

information

about

the

severity

of

the

liver

disease

-LSB-

-RSB-

Liver

samples

can

be

obtained

by

percutaneous

or

transjugular

access

LIC

measurement

from

a

needle

biopsy

has

been

associated

with

a

coefficient

of

variation

of

less

than

in

livers

with

no

advanced

disease

However

it

has

been

shown

that

variability

can

be

much

higher

in

fibrotic

and

cirrhotic

livers

Values

of

LIC

are

expressed

as

milligrams

of

iron

per

gram

of

dry

liver

weight

Fig.

biopsy

-LRB-

Perls

stain

-RRB-

iron

deposits

are

in

blue

Courtesy

of

B.

Turlin

Pathology

Department

University

Hospital

of

Rennes

Liver

biopsy

is

an

invasive

technique

that

is

associated

with

some

pain

and

at

risk

of

hemorrhage

and

infection

It

is

not

indicated

for

routine

assessment

Superconducting

quantum

interference

device

-LRB-

SQUID

-RRB-

SQUID

is

capable

of

measuring

very

small

changes

in

magnetic

flux

Iron

stored

as

ferritin

and

hemosiderin

is

the

only

relevant

paramagnetic

material

in

the

human

body

The

magnitude

of

paramagnetic

response

is

directly

related

to

the

amount

of

iron

in

a

certain

volume

of

tissue

This

is

a

noninvasive

method

with

a

linear

correlation

with

LIC

assessed

by

biopsy

However

equipment

availability

is

extremely

limited

-LSB-

-RSB-

Magnetic

resonance

imaging

-LRB-

MRI

-RRB-

MRI

measures

tissue

iron

concentration

indirectly

by

detecting

the

paramagnetic

influences

of

storage

iron

-LRB-

ferritin

and

hemosiderin

-RRB-

on

proton

resonance

behavior

With

MRI

the

transmitted

signal

is

a

microwave

which

excites

water

protons

in

the

body

to

higher

magnetic

energy

states

As

these

water

protons

relax

back

to

the

unexcited

state

they

emit

microwaves

that

are

received

and

interpreted

by

the

scanner

Iron

deposits

act

like

little

magnets

when

placed

in

a

strong

magnetic

field

disrupting

coherence

among

the

protons

and

darkening

the

image

more

quickly

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

LIC

determined

using

MRI

shows

excellent

correlation

with

that

obtained

from

liver

biopsy

Furthermore

MRI

has

the

ability

to

evaluate

the

entire

organ

and

gives

more

accurate

measurement

of

LIC

particularly

in

patients

with

heterogeneous

iron

content

-LRB-

Fig.

-RRB-

It

requires

a

dedicated

imaging

method

and

equipment

operator

-LSB-

-RSB-

Fig.

echo

images

of

liver

collected

by

J.

Wood

-LSB-

-RSB-

at

four

different

echo

times

The

top

four

images

were

collected

from

a

patient

having

a

liver

iron

of

mg/g

The

bottom

four

images

were

collected

from

a

normal

volunteer

All

images

darken

as

the

echo

time

-LRB-

TE

-RRB-

lengthens

but

the

iron-heavy

tissue

darkens

faster

The

half-life

of

this

process

is

called

and

the

rate

is

called

-LRB-

-RRB-

Fig.

echo

imaging

illustrating

discordant

iron

loading

of

the

liver

and

heart

a

Heavy

liver

iron

loading

-LRB-

dark

tissue

-RRB-

with

heart

sparing

image

b

Heavy

cardiac

iron

loading

with

no

liver

deposition

From

-LSB-

-RSB-

Iron

chelation

therapy

Within

years

of

initiation

of

regular

blood

transfusions

iron

starts

to

accumulate

in

the

body

The

use

of

a

chelating

agent

then

becomes

mandatory

-LSB-

-RSB-

At

the

present

time

three

different

therapies

are

available

-LRB-

Table

-RRB-

Table

between

iron

chelators

-LRB-

adapted

from

-LSB-

-RSB-

-RRB-

CharacteristicsDeferoxamine

-LRB-

Desferal

-RRB-

Deferiprone

-LRB-

Ferriprox

-RRB-

Deferasirox

-LRB-

Exjade

-RRB-

Route

of

administrationSubcutaneous

or

hRoutes

of

iron

excretionUrine/stoolUrinestoolDose

mg/kg

per

mg/kg

per

mg/kg/dayGuidelines

for

monitoring

therapyAudiometry

and

eye

exams

annuallyComplete

blood

count

weekly

ALT

level

monthly

for

first

months

then

every

monthsSerum

creatinine

level

monthly

ALT

level

monthlySerum

ferritinSerum

ferritinSerum

ferritinAssessment

of

liver

iron

annuallyAssessment

of

liver

iron

annuallyAssessment

of

liver

iron

annuallyAssessment

of

cardiac

iron

annually

after

years

of

ageAssessment

of

cardiac

iron

annually

after

years

of

ageAssessment

of

cardiac

iron

annually

after

years

of

ageAdvantagesLong-term

experienceOrally

activeOrally

activeEffective

in

maintaining

normal

or

near-normal

iron

storesSafety

profile

well

establishedOnce-daily

administrationReversal

of

cardiac

disease

with

intensive

therapyEnhanced

removal

of

cardiac

ironDemonstrated

equivalency

to

deferoxamine

at

higher

dosesMay

be

combined

with

deferiproneMay

be

combined

with

deferoxamineTrials

in

several

hematologic

disordersDisadvantagesRequires

parenteral

infusionMay

not

achieve

negative

iron

balance

in

all

patients

at

mg/kg

per

dayLimited

long-term

dataEar

eye

bone

toxicityRisk

of

agranulocytosisNeed

to

monitor

renal

functionPoor

complianceMay

not

achieve

negative

iron

balance

in

all

patients

at

highest

dose

Deferoxamine

mesylate

-LRB-

Desferal

-RRB-

is

the

current

standard

for

iron

chelation

therapy

The

long-term

efficacy

of

this

approach

has

been

demonstrated

limiting

organ

damage

and

preventing

premature

death

Deferoxamine

has

a

very

short

half-life

in

plasma

-LRB-

min

-RRB-

Therefore

deferoxamine

is

administered

parenterally

either

subcutaneously

h

a

day

at

least

days

a

week

or

intravenously

through

a

Port-a-Cath

for

continuous

infusion

Low

compliance

with

the

prolonged

subcutaneous

administration

is

the

main

reason

of

ineffective

treatment

Toxic

side

effects

such

as

auditory

ocular

and

neurotoxic

abnormalities

are

also

drawbacks

in

the

therapy

Deferiprone

-LRB-

Ferriprox

-RRB-

is

one

of

the

hundreds

of

oral

chelators

that

have

been

tested

It

was

shown

to

have

comparable

efficacy

to

subcutaneous

deferoxamine

It

has

been

used

as

a

monotherapy

or

in

combination

with

deferoxamine

-LSB-

-RSB-

It

increases

urinary

excretion

of

iron

and

is

more

effective

than

deferoxamine

in

the

removal

of

excess

iron

from

the

heart

as

shown

from

MRI

studies

-LSB-

-RSB-

Deferiprone

and

its

iron

complex

are

cleared

from

the

plasma

with

a

half-life

of

min

Therefore

it

should

be

taken

three

times

a

day

The

major

toxic

side

effects

are

agranulocytosis

musculoskeletal

and

joint

pains

gastric

intolerance

and

zinc

deficiency

Those

side

effects

are

considered

to

be

reversible

The

combination

of

deferoxamine

and

deferiprone

is

most

effective

when

used

in

the

following

way

deferiprone

-LRB-

mg/kg

per

day

-RRB-

during

the

day

and

deferoxamine

-LRB-

mg/kg

per

day

-RRB-

at

least

nights

a

week

No

new

toxic

effects

have

been

reported

for

the

combination

Deferasirox

-LRB-

Exjade

-RRB-

has

been

tested

recently

in

clinical

trials

-LSB-

-RSB-

Its

long

half-life

makes

it

suitable

for

once-daily

oral

administration

Efficacy

at

a

dose

of

mg/kg

per

day

is

at

least

equivalent

to

that

of

deferoxamine

-LRB-

mg/kg

per

day

days

weekly

-RRB-

in

a

subgroup

of

patients

with

higher

hepatic

iron

burdens

The

most

common

toxic

side

effects

are

skin

rash

increase

in

transaminases

and

creatinine

and

gastrointestinal

symptoms

such

as

nausea

vomiting

diarrhea

and

abdominal

pain

The

skin

rash

and

gastrointestinal

symptoms

tend

to

decrease

spontaneously

with

time

and

an

increase

in

creatinine

may

require

a

dose

adjustment

The

increase

in

transaminases

is

usually

linked

to

an

insufficient

dose

of

deferasirox

and

an

increase

in

the

hepatic

iron

overload

A

higher

dose

of

deferasirox

will

result

in

an

improvement

in

transaminases

Discussion

Chronic

iron

overload

due

to

blood

transfusions

leads

to

significant

morbidity

and

early

mortality

unless

adequate

chelation

therapy

is

administered

Deferoxamine

is

the

reference

chelation

therapy

but

compliance

to

the

treatment

is

often

poor

because

it

must

be

administered

by

prolonged

subcutaneous

or

intravenous

infusion

Oral

chelators

are

now

available

Deferasirox

has

the

advantage

of

being

administered

once

daily

with

a

low

toxicity

profile

Furthermore

its

cost

effectiveness

has

also

been

demonstrated

-LSB-

-RSB-

MRI

has

emerged

as

the

dominant

technique

by

which

to

evaluate

iron

overload

because

of

its

sensitivity

reproducibility

and

ability

to

image

multiple

organs

in

the

body

during

a

single

imaging

session

O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O