Influence of ß-casein genotype on physicochemical properties and functionality of bovine milk.

Several studies have been focused on the effect of milk protein genetic variants on milk physicochemical properties and functionality in recent years. ß-casein, an important protein related to milk processibility, has been reported to have 2 main genetic variants A1 and A2, for which cows may be homozygous or heterozygous. In this study, several physicochemical properties of milk with ß-casein variants A1A1, A1A2, and A2A2 from 3 collection occasions were analyzed. Higher manganese content and lower pH were found to be associated with the A1A1 variant compared with the other 2 genotypes. Better rennet and acid coagulation were found in A1A1 milk compared with A1A2 and A2A2 milk (although P > 0.05), whereas A2A2 milk was more stable to creaming compared with the other 2 genotypes, which may be linked to its smaller fat globule size. Thus, milk from cows with A1A1 genotype could be preferable for cheese making, while that with A2A2 variant can be used in formulations requiring good stability against creaming, and for example, yogurt making, where the softer yogurt texture may be easier to digest.

Application of hot melt extrusion for improving bioavailability of artemisinin a thermolabile drug.

Hot melt extrusion has been used to produce a solid dispersion of the thermolabile drug artemisinin. Formulation and process conditions were optimized prior to evaluation of dissolution and biopharmaceutical performance. Soluplus®, a low Tg amphiphilic polymer especially designed for solid dispersions enabled melt extrusion at 110 °C although some drug-polymer incompatibility was observed. Addition of 5% citric acid as a pH modifier was found to suppress the degradation. The area under plasma concentration time curve (AUC0-24h) and peak plasma concentration (Cmax) were four times higher for the modified solid dispersion compared to that of pure artemisinin.

In-process rheometry as a PAT tool for hot melt extrusion.

Real time measurement of melt rheology has been investigated as a Process Analytical Technology (PAT) to monitor hot melt extrusion of an Active Pharmaceutical Ingredient (API) in a polymer matrix. A developmental API was melt mixed with a commercial copolymer using a heated twin screw extruder at different API loadings and set temperatures. The extruder was equipped with an instrumented rheological slit die which incorporated three pressure transducers flush mounted to the die surface. Pressure drop measurements within the die at a range of extrusion throughputs were used to calculate rheological parameters, such as shear viscosity and exit pressure, related to shear and elastic melt flow properties, respectively. Results showed that the melt exhibited shear thinning behavior whereby viscosity decreased with increasing flow rate. Increase in drug loading and set extrusion temperature resulted in a reduction in melt viscosity. Shear viscosity and exit pressure measurements were found to be sensitive to API loading. These findings suggest that this technique could be used as a simple tool to measure material attributes in-line, to build better overall process understanding for hot melt extrusion.

The effect of storage conditions on the composition and functional properties of blended bulk tank milk.

The objective of this study was to investigate the effects of storage temperature and duration on the composition and functional properties of bulk tank milk when fresh milk was added to the bulk tank twice daily. The bulk tank milk temperature was set at each of 3 temperatures (2, 4, and 6°C) in each of 3 tanks on 2 occasions during two 6-wk periods. Period 1 was undertaken in August and September when all cows were in mid lactation, and period 2 was undertaken in October and November when all cows were in late lactation. Bulk tank milk stored at the 3 temperatures was sampled at 24-h intervals during storage periods of 0 to 96 h. Compositional parameters were measured for all bulk tank milk samples, including gross composition and quantification of nitrogen compounds, casein fractions, free amino acids, and Ca and P contents. The somatic cell count, heat stability, titratable acidity, and rennetability of bulk tank milk samples were also assessed. Almost all parameters differed between mid and late lactation; however, the interaction between lactation, storage temperature, and storage duration was significant for only 3 parameters: protein content and concentrations of free cysteic acid and free glutamic acid. The interaction between storage temperature and storage time was not significant for any parameter measured, and temperature had no effect on any parameter except lysine: lysine content was higher at 6°C than at 2°C. During 96 h of storage, the concentrations of some free amino acids (glutamic acid, lysine, and arginine) increased, which may indicate proteolytic activity during storage. Between 0 and 96 h, minimal deterioration was observed in functional properties (rennet coagulation time, curd firmness, and heat stability), which was most likely due to the dissociation of ß-casein from the casein micelle, which can be reversed upon pasteurization. Thus, this study suggests that blended milk can be stored for up to 96 h at temperatures between 2°C and 6°C with little effect on its composition or functional properties.

Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; <700,000 cells/mL), medium (M-SCC; from 701,000 to 1,500,000 cells/mL), and high (H-SCC; >1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and ß-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein based on proteolysis patterns of sodium caseinate incubated with isolated and lysed leukocyte types.

A descriptive study of the multidisciplinary healthcare experiences of inpatient resuscitation events.

Background: In-hospital resuscitation events have complex and enduring effects on clinicians, with implications for job satisfaction, performance, and burnout. Ethically ambiguous cases are associated with increased moral distress. We aim to quantitatively describe the multidisciplinary resuscitation experience. Methods: Multidisciplinary in-hospital healthcare professionals at an adult academic health center in the Midwestern United States completed surveys one and six weeks after a resuscitation event. Surveys included demographic data, task load (NASA-TLX), overall and moral distress, anxiety, depression, and spiritual peace. Spearman's rank correlation was computed to assess task load and distress. Results: During the 5-month study period, the study included 12 resuscitation events across six inpatient units. Of 82 in-hospital healthcare professionals eligible for recruitment, 44 (53.7%) completed the one-week post-resuscitation event survey. Of those, 37 (84.1%) completed the six-week survey. Highest median task load burden at one week was seen for temporal demand, effort, and mental demand. Median moral distress scores were low, while "at peace" median scores tended to be high. There were no significant non-zero changes in task load or distress scores from weeks 1-6. Mental demand (r = 0.545, p < 0.001), physical demand (r = 0.464, p = 0.005), performance (r = -0.539, p < 0.001), and frustration (r = 0.545, p < 0.001) significantly correlated with overall distress. Performance (r = -0.371, p = 0.028) and frustration (r = 0.480, p = 0.004) also significantly correlated with moral distress. Conclusions: In-hospital healthcare professionals' experiences of resuscitation events are varied and complex. Aspects of task load burden including mental and physical demand, performance, and frustration contribute to overall and moral distress, deserving greater attention in clinical contexts.

Proteolytic and proteomic changes in milk at quarter level following infusion with Escherichia coli lipopolysaccharide.

Mastitis is a major disease in dairy cattle, which causes significant economic losses due to decreased milk production, veterinary costs, and discarded milk. Escherichia coli is one of the most prevalent species of gram-negative bacteria that induce clinical mastitis. The objective of the present study was to characterize the proteolytic and proteomic changes in milk in response to infusion with lipopolysaccharide (LPS) at quarter level in a model mastitis system. One quarter of each of 2 cows was infused with 0.1 or 5 µg of LPS. The somatic cell count of the infused quarters reached a peak 6 h after infusion to a greater extent in the cow infused with 5 µg of LPS and changes in plasmin activity in milk differed between the 2 animals. Urea-polyacrylamide gel electrophoretograms of milk samples of the cow infused with 5 µg of LPS obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of ß- and α(s1)-casein during incubation of milk samples due to indigenous proteolytic activity. Two-dimensional gel electrophoretograms of milk at 0, 6, or 12 h after infusion with LPS showed hydrolysis of α(s)-casein and ß-casein as well as the appearance of lower molecular weight products. Eleven fragments from proteolysis of the caseins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and, in addition, proteolysis patterns of casein by the indigenous bovine milk proteases plasmin and cathepsin D were studied in model studies using 2-dimensional gel electrophoretograms. Twelve hours after infusion, lower abundance markers of inflammation were identified, including serotransferrin, fibrinogen ß chain, protein S100 A12, and the antimicrobial polypeptide cathelicidin.

Effect of fat content and homogenization under conventional or ultra-high-pressure conditions on interactions between proteins in rennet curds.

The objective of this study was to investigate the influence of conventional and ultra-high-pressure homogenization on interactions between proteins within drained rennet curds. The effect of fat content of milk (0.0, 1.8, or 3.6%) and homogenization treatment on dissociation of proteins by different chemical agents was thus studied. Increasing the fat content of raw milk increased levels of unbound whey proteins and calcium-bonded caseins in curds; in contrast, hydrophobic interactions and hydrogen bonds were inhibited. Both homogenization treatments triggered the incorporation of unbound whey proteins in the curd, and of caseins through ionic bonds involving calcium salts. Conventional homogenization-pasteurization enhanced interactions between caseins through hydrogen bonds and hydrophobic interactions. In contrast, ultra-high-pressure homogenization impaired hydrogen bonding, led to the incorporation of both whey proteins and caseins through hydrophobic interactions and increased the amount of unbound caseins. Thus, both homogenization treatments provoked changes in the protein interactions within rennet curds; however, the nature of the changes depended on the homogenization conditions.

Novel listerial genetic loci conferring enhanced barotolerance in Escherichia coli.

AIMS: To identify Listeria monocytogenes genes with a role in high-pressure (HP) resistance. METHODS AND RESULTS: A L. monocytogenes genomic library constructed in an Escherichia coli background was screened for loci conferring increased HP resistance. Pressure treatments at 400 megapascals for 5 min in Luria-Bertani (LB) agar were used to screen for increased resistance to pressure. Colonies arising on the treated agar plates were isolated, the plasmid extracted and the inserts sequenced to identify the genetic loci conferring HP resistance. Seven different genetic regions were identified, which encoded proteins similar to an inorganic polyphosphate/ATP-NAD kinase, the septation ring formation regulator EzrA, flagellar motor proteins MotA and MotB, proteins similar to the quorum sensing Agr system from Staphylococcus (AgrA, AgrC and AgrD), proteins similar to a transcription regulator (RpiR family) and a fructose phosphotransferase system, proteins of unknown function, and a Fur regulator. Of the seven loci confirmed, three (EzrA, MotA/B and the Agr system) maintained significantly reproducible HP tolerance when expressed in a different E. coli background. CONCLUSIONS: Novel genetic loci from the L. monocytogenes genome confer increased HP resistance when heterologously expressed in an E. coli background. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular and functional approaches to the screening of genetic elements linked to HP resistance provide greater insights into microbial inactivation and/or survival mechanisms when using HP as a means of controlling/eliminating bacterial growth. This information will ultimately have significant implications for the use of HP processing in the food industry, in terms of both food quality and safety.

A role for proline synthesis and transport in Listeria monocytogenes barotolerance.

AIMS: To assess the contribution of proline biosynthesis to listerial barotolerance. METHODS AND RESULTS: Using a Listeria monocytogenes proBA deletion mutant, incapable of synthesizing proline, together with a proline-overproducing strain, the contribution of proline synthesis to listerial barotolerance was determined. The ΔproBA strain does not survive as well as the wild type when subjected to treatment of 500 MPa in rich media and 400 MPa in minimal media (c. 1 log lower survival in both conditions). Betaine and carnitine decrease the ability of the wild type to survive at low pressures (300 MPa), but confer normal or slightly increased levels of protection at higher pressures (350 and 400 MPa). CONCLUSIONS: A functional proline synthesis system is required for optimal survival of Listeria following treatment at high-pressure (HP) levels (500 MPa in brain heart infusion and 400 MPa in defined medium), particularly where other compatible solutes are absent or limiting. SIGNIFICANCE AND IMPACT OF THE STUDY: Given the potential of HP processing as an effective food processing/safety strategy, understanding how pathogens such as Listeria have evolved to cope with such stresses is an important food safety consideration. In this context, the work presented here may help to develop safer and more effective processing regimens.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus.

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of ß- and α(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of ß-casein being faster than that of α(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of ß- and α(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α(s1)- and ß-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Haemochromatosis: an inherited metal and toxicity syndrome.

A newly-identified major histocompatibility Class I-like gene, HFE (originally HLA-H) located approximately 3.5 Mb telomeric to the Class I cluster on chromosome 6p 21.3 harbours mutations in haemochromatosis. Two of these, Cys282Tyr (C282Y) and His63Asp (H63D, a minor determinant) have diagnostic utility as approximately 90% of adults are homozygous or compound heterozygotes for these alleles. The pathophysiological role of HFE is unclear: it is expressed as a surface molecule on many cells and the C282Y mutation disrupts interactions with beta 2-microglobulin, thus preventing surface expression. Lately, there has been experimental evidence that HFE protein interacts with the transferrin-receptor, affecting receptor turnover or its affinity for ligand.

The combined effects of high pressure and nisin on germination and inactivation of Bacillus spores in milk.

AIMS: The aim of this work was to investigate the germination and inactivation of spores of Bacillus species in buffer and milk subjected to high pressure (HP) and nisin. METHODS AND RESULTS: Spores of Bacillus subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or 500 MPa at 40 degrees C with or without 500 IU ml(-1) of nisin. Treatment at 500 MPa resulted in high levels of germination (4 log units) of B. subtilis spores in both milk and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability of B. subtilis spores in milk and buffer was reduced by 2.5 logs by cycled HP, while the addition of nisin (500 IU ml(-1)) prior to HP treatment resulted in log reductions of 5.7 and 5.9 in phosphate buffered saline and milk, respectively. Physical damage of spores of B. subtilis following HP was apparent using scanning electron microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40 degrees C in the presence of 500 IU ml(-1) nisin proved less effective at inactivating the spores of these isolates compared with B. subtilis and some strain-to-strain variability was observed. CONCLUSIONS: Although high levels of germination of Bacillus spores could be achieved by combining HP and nisin, complete inactivation was not achieved using the aforementioned treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of HP treatment and nisin may be an appealing alternative to heat pasteurization of milk.

Stability of casein micelles cross-linked by transglutaminase.

In this study, caseins micelles were internally cross-linked using the enzyme transglutaminase (TGase). The integrity of the micelles was examined on solubilization of micellar calcium phosphate (MCP) or on disruption of hydrophobic interactions and breakage of hydrogen bonds. The level of monomeric caseins, determined electrophoretically, decreased with increasing time of incubation with TGase at 30 degrees C; after incubation for 24 h, no monomeric beta- or kappa-caseins were detected, whereas only a small level of monomeric alphaS1-casein remained, suggesting near complete intramicellar cross-linking. The ability of casein micelles to maintain structural integrity on disruption of hydrophobic interactions (using urea, sodium dodecyl sulfate, or heating in the presence of ethanol), solubilization of MCP (using the calcium-chelating agent trisodium citrate) or high-pressure treatment was estimated by measurement of the L*-value of milk; i.e., the amount of back-scattered light. The amount of light scattered by casein micelles in noncross-linked milk was reduced by >95% on complete disruption of hydrophobic interactions or complete solubilization of MCP; treatment of milk with TGase increased the stability of casein micelles against disruption by all methods studied and stability increased progressively with incubation time. After 24 h of cross-linking, reductions in the extent of light scattering were still apparent in the presence of high levels of dissociating agents, possibly through citrate-induced removal of MCP nanoclusters from the micelles, or urea- or sodium dodecyl sulfate-induced increases in solvent refractive index, which reduce the extent of light-scattering.

Classification and genetic features of neonatal haemochromatosis: a study of 27 affected pedigrees and molecular analysis of genes implicated in iron metabolism.

Neonatal haemochromatosis (NH) is a severe and newly recognised syndrome of uncertain aetiology, characterised by congenital cirrhosis or fulminant hepatitis and widespread tissue iron deposition. NH occurs in the context of maternal disease including viral infection, as a complication of metabolic disease in the fetus, and sporadically or recurrently, without overt cause, in sibs. Although an underlying genetic basis for NH has been suspected, no test is available for predictive analysis in at risk pregnancies. As a first step towards an understanding of the putative genetic basis for neonatal haemochromatosis, we have conducted a systematic study of the mode of transmission of this disorder in a total of 40 infants born to 27 families. We have moreover carried out a molecular analysis of candidate genes (beta(2)-microglobulin, HFE, and haem oxygenases 1 and 2) implicated in iron metabolism. No pathogenic mutations in these genes were identified that segregate consistently with the disease phenotype in multiplex pedigrees. However, excluding four pedigrees with clear evidence of maternal infection associated with NH, a pedigree showing transmission of maternal antinuclear factor and ribonucleoprotein antibodies to the affected infants, and two families with possible matrilineal inheritance of disease in maternal half sibs, a large subgroup of the affected pedigrees point to the inheritance of an autosomal recessive trait. This included 14 pedigrees with affected and unaffected infants and a single pedigree where all four affected infants were the sole offspring of consanguineous but otherwise healthy parents. We thus report three distinct patterns of disease transmission in neonatal haemochromatosis. In the differentiation of a large subgroup showing transmission of disease in a manner suggesting autosomal recessive inheritance, we also provide the basis for further genome wide studies to define chromosomal determinants of iron storage disease in the newborn.

Influence of starter culture on flavor and headspace volatile profiles of fermented whey and whey produced from fermented milk.

Rennet whey and skim milk were compared as media for fermentation by commercial cheese, yogurt, and probiotic starter cultures. Effect of culture, medium, and their interaction on flavor was assessed and compared by sensory descriptive analysis and headspace volatile analysis by proton transfer reaction-mass spectrometry. In general, the aroma of fermented whey was similar to that of whey separated from fermented milk, indicating a favorable possibility of substituting milk with whey in the manufacture of fermented milk-like beverages. Starter culture significantly affected most sensory characteristics of the products. Key volatile compounds for the characteristic flavor of yogurt, such as acetaldehyde and diacetyl, were not significantly affected by medium when fermented with the yogurt culture, and reached similar levels in both systems. Volatile analysis results were consistent with the results of the sensory evaluation, indicating the high reliability of proton transfer reaction-mass spectrometry in detecting important volatile compounds for aroma. Integration of this sensory and chemical information allows a better understanding of how flavor and related compounds are affected by ingredients or processing, which may be useful for the development of value-added whey products.

Sensory characteristics and related volatile flavor compound profiles of different types of whey.

To characterize the flavor of liquid whey, 11 samples of whey representing a wide range of types were sourced from cheese and casein-making procedures, either industrial or from pilot-plant facilities. Whey samples were assessed for flavor by descriptive sensory evaluation and analyzed for headspace volatile composition by proton transfer reaction-mass spectrometry (PTR-MS). The sensory data clearly distinguished between the samples in relation to the processes of manufacture; that is, significant differences were apparent between cheese, rennet, and acid wheys. For Mozzarella and Quarg wheys, in which fermentation progressed to low pH values, the starter cultures used for cheese making had a significant influence on flavor. In comparison, Cheddar and Gouda wheys were described by milk-like flavors, and rennet casein wheys were described by "sweet" (oat-like and "sweet") and thermally induced flavors. The volatile compound data obtained by PTR-MS differentiated the samples as distinctive and reproducible "chemical fingerprints". On applying partial least squares regression to determine relationships between sensory and volatile composition data, sensory characteristics such as "rancid" and cheese-like odors and "caramelized milk," yogurt-like, "sweet," and oat-like flavors were found to be related to the presence and absence of specific volatile compounds.

A novel transflectance near infrared spectroscopy technique for monitoring hot melt extrusion.

A transflectance near infra red (NIR) spectroscopy approach has been used to simultaneously measure drug and plasticiser content of polymer melts with varying opacity during hot melt extrusion. A high temperature reflectance NIR probe was mounted in the extruder die directly opposed to a highly reflective surface. Carbamazepine (CBZ) was used as a model drug, with polyvinyl pyrollidone-vinyl acetate co-polymer (PVP-VA) as a matrix and polyethylene glycol (PEG) as a plasticiser. The opacity of the molten extrudate varied from transparent at low CBZ loading to opaque at high CBZ loading. Particulate amorphous API and voids formed around these particles were found to cause the opacity. The extrusion process was monitored in real time using transflectance NIR; calibration and validation runs were performed using a wide range of drug and plasticiser loadings. Once calibrated, the technique was used to simultaneously track drug and plasticiser content during applied step changes in feedstock material. Rheological and thermal characterisations were used to help understand the morphology of extruded material. The study has shown that it is possible to use a single NIR spectroscopy technique to monitor opaque and transparent melts during HME, and to simultaneously monitor two distinct components within a formulation.

Hereditary juvenile haemochromatosis: a genetically heterogeneous life-threatening iron-storage disease.

Juvenile haemochromatosis is a rare inborn error of iron metabolism with clinical manifestations before 30 years of age. Unlike adult haemochromatosis which principally affects men, juvenile haemochromatosis affects the sexes equally; it causes early endocrine failure, dilated cardiomyopathy and joint disease. We report four patients (two of each sex) from three pedigrees affected by juvenile haemochromatosis with a mean onset at 22 years (range 14-30). All had endocrine deficiency with postpubertal gonadal failure secondary to pituitary disease; two suffered near-fatal cardiomyopathy with heart failure. Mean time to diagnosis from the first clinical signs of disease was 9.8 years (range 0.5-20) but general health and parameters of iron storage responded favourably to iron-depletion therapy. A 24-year-old man listed for heart transplantation because of cardiomyopathy [left ventricular (LV) ejection fraction 16%] responded to intravenous iron chelation with desferrioxamine combined with phlebotomy (ejection fraction 31%). A 27-year-old woman with subacute biventricular heart failure refractory to medication required orthotopic cardiac transplantation before the diagnosis was established (LV ejection fraction 25%). Genetic studies showed that these two patients with cardiomyopathy from unrelated families were heterozygous for the HFE 845G-->A (C282Y) mutation and wild-type at the H63D locus: complete sequencing of the intron-exon boundaries and entire coding sequence of the HFE gene failed to identify additional lesions. Two siblings in a pedigree without cardiomyopathy were wild-type at the HFE C282Y locus; although the brother harboured a single copy of the 187C-->G (H63D) allele, segregation analysis showed that in neither sibling was the iron-storage disease linked to MHC Class I markers on chromosome 6p. Juvenile haemochromatosis is thus a genetically heterogenous disorder distinct from the common adult variant.

Localization of iron transport and regulatory proteins in human cells.

The recent discovery of HFE, the MHC-Class-I-like gene mutated in up to 90% of patients with hereditary haemochromatosis, and the gene encoding the Nramp2/divalent metal transporter-1 (DMT-1) implicated in ferrous iron transport holds promise for a greater understanding of human iron metabolism. Since the HFE protein can be crystallized as a ternary complex with the transferrin receptor and iron-saturated transferrin, and DMT-1 expression is up-regulated in hereditary haemochromatosis, these proteins are likely to interact in a common pathway for human iron homeostasis. To investigate the cellular interactions between the cognate proteins encoded by these genes, we generated a panel of rabbit and avian antisera from human HFE and DMT-1 derived peptides. The antibodies were characterized by ELISA reactions and Western immunoblotting. Immunohistochemical staining showed that DMT-1 protein localized to the brush border of human duodenum where it is predicted to serve as the principal transporter of ferrous iron from the intestinal lumen. In the human cell lines, Caco-2 (small intestinal phenotype upon differentiation) and K562 (erythroleukaemic) HFE, in the presence of iron-saturated transferrin, co-localized with transferrin receptors in an early endosome compartment using confocal immunofluorescence microscopy. This interaction may be critical in small-intestinal crypt cells which express HFE, where it may function to modulate their intrinsic iron status thereby programming iron absorption by DMT-1 in the mature enterocyte. In undifferentiated Caco-2 cells, DMT-1 localized to a discrete late endosome compartment distinct from that occupied by HFE where, in addition to brush-border iron uptake, it may function to regulate the availability of iron delivery to intracellular iron pools. Disruption of the HFE gene as a result of mutations associated with hereditary haemochromatosis may thus impair homeostatic mechanisms controlling iron absorption within the small-intestine epithelium by a direct interaction with transferrin receptors and by subsequent alteration of DMT-1 expression. Identification of the molecular interactions of HFE with DMT-1 and other key components of the iron transport pathway has implications for a mechanistic understanding of the pathophysiology of human iron storage diseases as well as the regulation of normal iron balance.