Bloodlettings in Hemochromatosis Result in Increased Blood Lead (Pb) Concentrations.

Hemochromatosis is a hereditary disorder, most often associated with mutations of the HFE (High FErrum) gene. If left untreated, it can result in severe parenchymal iron accumulation. Bloodletting is the mainstay treatment. We have previously shown that treatment of hemochromatosis by repeated bloodlettings may induce changes in the serum levels of several trace elements. The aim of this work was to evaluate if whole blood concentrations of the environmental pollutants lead (Pb), mercury (Hg), and cadmium (Cd) could be affected by bloodlettings. We recruited 28 patients and 21 healthy individuals (control group). Whole blood and urine levels of Pb, Hg, and Cd were measured before the start and after the completion of treatment using inductively coupled plasma mass spectrometry, together with serum iron and liver function tests. Concentrations of blood Pb, but not Hg or Cd, were significantly increased after treatment. The increase in Pb was higher in C282Y homozygous patients than in the other patients, and it was positively correlated with the serum concentration of alkaline phosphatase. Bloodlettings in hemochromatosis result in an increase in the blood concentration of Pb. Augmented absorption due to iron loss or Pb mobilization from bone may contribute to the higher blood Pb level.

HFE Genotype, Ferritin Levels and Transferrin Saturation in Patients with Suspected Hereditary Hemochromatosis.

HFE hemochromatosis is characterized by increased iron absorption and iron overload due to variants of the iron-regulating HFE gene. Overt disease is mainly associated with homozygosity for the C282Y variant, although the H63D variant in compound heterozygosity with C282Y (C282Y/H63D) contributes to disease manifestation. In this observational study, we describe the association between biochemical findings, age, gender and HFE genotype in patients referred from general practice to a tertiary care referral center for diagnostic workup based on suspected hemochromatosis due to persistent hyperferritinemia and HFE variants. C282Y and H63D homozygosity were, respectively, the most and least prevalent genotypes and we found a considerable variation in transferrin saturation and ferritin levels independent of HFE genotype, which may indeed represent a diagnostic challenge in general practice. While our results confirm C282Y homozygosity as the major cause of iron accumulation, non-C282Y homozygotes also displayed mild to moderate hyperferritinemia with median ferritin levels at 500-700 µg/L, well above the reference cut-off. Such findings have traditionally been ignored in the clinic, and initiation of iron depletion has largely been restricted to C282Y homozygotes. Nevertheless, superfluous iron can aggravate pathogenesis in combination with other diseases and risk factors, such as inflammation, cancer and hepatopathy, and this possibility should not be neglected by clinicians.

Hyperferritinemia-A Clinical Overview.

Ferritin is one of the most frequently requested laboratory tests in primary and secondary care, and levels often deviate from reference ranges. Serving as an indirect marker for total body iron stores, low ferritin is highly specific for iron deficiency. Hyperferritinemia is, however, a non-specific finding, which is frequently overlooked in general practice. In routine medical practice, only 10% of cases are related to an iron overload, whilst the rest is seen as a result of acute phase reactions and reactive increases in ferritin due to underlying conditions. Differentiation of the presence or absence of an associated iron overload upon hyperferritinemia is essential, although often proves to be complex. In this review, we have performed a review of a selection of the literature based on the authors' own experiences and assessments in accordance with international recommendations and guidelines. We address the biology, etiology, and epidemiology of hyperferritinemia. Finally, an algorithm for the diagnostic workup and management of hyperferritinemia is proposed, and general principles regarding the treatment of iron overload are discussed.

Quantitative proteomics suggests decrease in the secretogranin-1 cerebrospinal fluid levels during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing-remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase-3-like protein 1, secretogranin-1 (Sg1), cerebellin-1, neuroserpin, cell surface glycoprotein MUC18, testican-2 and glutamate receptor 4. An independent sample set of 13 early-MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early-MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.

The liver in haemochromatosis.

The review deals with genetic, regulatory and clinical aspects of iron homeostasis and hereditary haemochromatosis. Haemochromatosis was first described in the second half of the 19th century as a clinical entity characterized by excessive iron overload in the liver. Later, increased absorption of iron from the diet was identified as the pathophysiological hallmark. In the 1970s genetic evidence emerged supporting the apparent inheritable feature of the disease. And finally in 1996 a new "haemochromatosis gene" called HFE was described which was mutated in about 85% of the patients. From the year 2000 onward remarkable progress was made in revealing the complex molecular regulation of iron trafficking in the human body and its disturbance in haemochromatosis. The discovery of hepcidin and ferroportin and their interaction in regulating the release of iron from enterocytes and macrophages to plasma were important milestones. The discovery of new, rare variants of non-HFE-haemochromatosis was explained by mutations in the multicomponent signal transduction pathway controlling hepcidin transcription. Inhibited transcription induced by the altered function of mutated gene products, results in low plasma levels of hepcidin which facilitate entry of iron from enterocytes into plasma. In time this leads to progressive accumulation of iron and subsequently development of disease in the liver and other parenchymatous organs. Being the major site of excess iron storage and hepcidin synthesis the liver is a cornerstone in maintaining normal systemic iron homeostasis. Its central pathophysiological role in HFE-haemochromatosis with downgraded hepcidin synthesis, was recently shown by the finding that liver transplantation normalized the hepcidin levels in plasma and there was no sign of iron accumulation in the new liver.

Bloodletting therapy in hemochromatosis: Does it affect trace element homeostasis?

Hemochromatosis is the most common hereditary disorder in the Nordic population, if left untreated it can result in severe parenchymal iron accumulation. Bloodletting is mainstay treatment. Iron and trace elements partially share cellular uptake and transport mechanisms, and the aim of the present study was to see if bloodletting for hemochromatosis affects trace elements homeostasis. We recruited patients referred for diagnosis and treatment of hemochromatosis, four women and 22 men 23-68 years of age. Thirteen were C282Y homozygote, one was C282Y heterozygote, three were H63D homozygote, seven were compound heterozygote and two had none of the mutations above. Iron and liver function tests were performed; serum levels of trace elements were measured using inductively coupled plasma mass spectrometry. Results before the start of treatment and after normalization of iron parameters were compared. On completion of the bloodlettings the following average serum concentrations increased: Co from 5.6 to 11.5 nmol/L, serum Cu 16.2-17.6 µmol/L, Ni increased from 50.0 to 52.6 nmol/L and Sb from 13.2 to 16.3 nmol/L. Average serum Mn concentration declined from 30.2 to 28.3 nmol/L. All changes were statistically significant (by paired t-test). B, Ba, Cs, Mo, Se, Sr and Zn were not significantly changed. We conclude that bloodlettings in hemochromatosis lead to changes in trace element metabolism, including increased absorption of potentially toxic elements.

Iron deficiency is uncommon among lactating women in urban Nepal, despite a high risk of inadequate dietary iron intake.

The main objective of the present study was to examine the association between dietary Fe intake and dietary predictors of Fe status and Hb concentration among lactating women in Bhaktapur, Nepal. We included 500 randomly selected lactating women in a cross-sectional survey. Dietary information was obtained through three interactive 24 h recall interviews including personal recipes. Concentrations of Hb and plasma ferritin and soluble transferrin receptors were measured. The daily median Fe intake from food was 17·5 mg, and 70% of the women were found to be at the risk of inadequate dietary Fe intake. Approximately 90% of the women had taken Fe supplements in pregnancy. The prevalence of anaemia was 20% (Hb levels < 123 g/l) and that of Fe deficiency was 5% (plasma ferritin levels < 15 µg/l). In multiple regression analyses, there was a weak positive association between dietary Fe intake and body Fe (ß 0·03, 95% CI 0·014, 0·045). Among the women with children aged < 6 months, but not those with older infants, intake of Fe supplements in pregnancy for at least 6 months was positively associated with body Fe (P for interaction < 0·01). Due to a relatively high dietary intake of non-haem Fe combined with low bioavailability, a high proportion of the women in the present study were at the risk of inadequate intake of Fe. The low prevalence of anaemia and Fe deficiency may be explained by the majority of the women consuming Fe supplements in pregnancy.

Erythrocytapheresis compared with whole blood phlebotomy for the treatment of hereditary haemochromatosis.

BACKGROUND: Hereditary haemochromatosis may result in severe organ damage which can be prevented by therapy. We studied the possible advantages and disadvantages of erythrocytapheresis as compared with phlebotomy in patients with hereditary haemochromatosis. MATERIALS AND METHODS: In a prospective, randomised, open-label study, patients with hereditary haemochromatosis were randomised to bi-weekly apheresis or weekly whole blood phlebotomy. Primary end-points were decrease in ferritin levels and transferrin saturation. Secondary endpoints were decrease in haemoglobin levels, discomfort during the therapeutic procedure, costs and technicians' working time. RESULTS: Sixty-two patients were included. Thirty patients were randomised to apheresis and 32 to whole blood phlebotomy. Initially, ferritin levels declined more rapidly in the apheresis group, and the difference became statistically highly significant at 11 weeks; however, time to normalisation of ferritin level was equal in the two groups. We observed no significant differences in decline of transferrin saturation, haemoglobin levels or discomfort. The mean cumulative technician time consumption until the ferritin level reached 50 µg/L was longer in the apheresis group, but the difference was not statistically significant. The cumulative costs for materials until achievement of the desired ferritin levels were three-fold higher in the apheresis group. CONCLUSION: Treatment of hereditary haemochromatosis with erythrocytapheresis instead of whole blood phlebotomy results in a more rapid initial decline in ferritin levels and a reduced number of procedures per patient, but not in earlier achievement of target ferritin level. The frequency of discomfort was equally low with the two methods. The costs and, probably, technician time consumption were higher in the apheresis group.

Quantitative proteomics comparison of arachnoid cyst fluid and cerebrospinal fluid collected perioperatively from arachnoid cyst patients.

BACKGROUND: There is little knowledge concerning the content and the mechanisms of filling of arachnoid cysts. The aim of this study was to compare the protein content of arachnoid cysts and cerebrospinal fluid by quantitative proteomics to increase the understanding of arachnoid cysts. METHODS: Arachnoid cyst fluid and cerebrospinal fluid from five patients were analyzed by quantitative proteomics in two separate experiments.In a label-free experiment arachnoid cyst fluid and cerebrospinal fluid samples from individual patients were trypsin digested and analyzed by Orbitrap mass spectrometry in a label-free manner followed by data analysis using the Progenesis software.In the second proteomics experiment, a patient sample pooling strategy was followed by MARS-14 immunodepletion of high abundant proteins, trypsin digestion, iTRAQ labelling, and peptide separation by mix-phase chromatography followed by Orbitrap mass spectrometry analysis. The results from these analyzes were compared to previously published mRNA microarray data obtained from arachnoid membranes. RESULTS: We quantified 348 proteins by the label-free individual patient approach and 1425 proteins in the iTRAQ experiment using a pool from five patients of arachnoid cyst fluid and cerebrospinal fluid. This is by far the largest number of arachnoid cyst fluid proteins ever identified, and the first large-scale quantitative comparison between the protein content of arachnoid cyst fluid and cerebrospinal fluid from the same patients at the same time. Consistently in both experiment, we found 22 proteins with significantly increased abundance in arachnoid cysts compared to cerebrospinal fluid and 24 proteins with significantly decreased abundance. We did not observe any molecular weight gradient over the arachnoid cyst membrane. Of the 46 proteins we identified as differentially abundant in our study, 45 were also detected from the mRNA expression level study. None of them were previously reported as differentially expressed. We did not quantify any of the proteins corresponding to gene products from the ten genes previously reported as differentially abundant between arachnoid cysts and control arachnoid membranes. CONCLUSIONS: From our experiments, the protein content of arachnoid cyst fluid and cerebrospinal fluid appears to be similar. There were, however, proteins that were significantly differentially abundant between arachnoid cyst fluid and cerebrospinal fluid. This could reflect the possibility that these proteins are affected by the filling mechanism of arachnoid cysts or are shed from the membranes into arachnoid cyst fluid. Our results do not support the proposed filling mechanisms of oncotic pressure or valves.

[Dietary supplement of iron for iron deficiency]. / Kosttilskudd med jern ved jernmangel.

BACKGROUND: A low supply of iron in the diet may result in iron deficiency and mild iron-deficiency anaemia in healthy individuals. Women are more susceptible than men because of menstrual iron loss. We compared the effect of a low dose of iron, administered as a dietary supplement, with a high pharmacological dose of iron to otherwise healthy individuals with iron deficiency and mild iron deficiency anaemia. MATERIAL AND METHOD: In a randomised, double-blind trial conducted in 2000-2001, 73 women and three men with iron deficiency received either 27.6 mg of iron consisting of ferrous fumarate enriched with 13% haem iron, or 100 mg ferrosulphate daily for 12 weeks. Blood samples were analysed four times in the course of the treatment. RESULTS: The median ferritin value rose by 13 and 7 µg/l in the high-dose and low-dose group, respectively. The increase in ferritin was significantly higher in the high-dose than in the low dose group ( < 0.001). There was no statistically significant difference between the groups in the change in Hb, serum-iron or serum-iron binding capacity. The median haemoglobin value increased by 0.4 g/100 ml in both groups. Gastrointestinal side effects were experienced by 58% in the high-dose group and 35% in the low-dose group. Four subjects in the high-dose group and one in the low-dose group broke off the treatment because of side effects. INTERPRETATION: A supplement of low-dose iron is enough to increase iron stores in cases of nutritional iron deficiency in healthy individuals and to optimise haemoglobin. High-dose iron caused the largest increase in iron stores. Low-dose iron resulted in the least side effects.

Discovery and initial verification of differentially abundant proteins between multiple sclerosis patients and controls using iTRAQ and SID-SRM.

In the present study, we aimed to discover cerebrospinal fluid (CSF) proteins with significant abundance difference between early multiple sclerosis patients and controls, and do an initial verification of these proteins using selected reaction monitoring (SRM). iTRAQ and Orbitrap MS were used to compare the CSF proteome of patients with clinically isolated syndrome (CIS) (n=5), patients with relapsing-remitting multiple sclerosis that had CIS at the time of lumbar puncture (n=5), and controls with other inflammatory neurological disease (n=5). Of more than 1200 identified proteins, five proteins were identified with significant abundance difference between the patients and controls. In the initial verification using SRM we analyzed a larger patient and control cohort (n=132) and also included proteins reported as differentially abundant in multiple sclerosis in the literature. We found significant abundance difference for 11 proteins after verification, of which the five proteins alpha-1-antichymotrypsin, contactin-1, apolipoprotein D, clusterin, and kallikrein-6 were significantly differentially abundant in several of the group comparisons. This initial study form the basis for further biomarker verification studies in even larger sample cohorts, to determine if these proteins have relevance as diagnostic or prognostic biomarkers for multiple sclerosis.

Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid.

BACKGROUND: The mechanisms behind formation and filling of intracranial arachnoid cysts (AC) are poorly understood. The aim of this study was to evaluate AC fluid by proteomics to gain further knowledge about ACs. Two goals were set: 1) Comparison of AC fluid from individual patients to determine whether or not temporal AC is a homogenous condition; and 2) Evaluate the protein content of a pool of AC fluid from several patients and qualitatively compare this with published protein lists of cerebrospinal fluid (CSF) and plasma. METHODS: AC fluid from 15 patients with temporal AC was included in this study. In the AC protein comparison experiment, AC fluid from 14 patients was digested, analyzed by LC-MS/MS using a semi-quantitative label-free approach and the data were compared by principal component analysis (PCA) to gain knowledge of protein homogeneity of AC. In the AC proteome evaluation experiment, AC fluid from 11 patients was pooled, digested, and fractionated by SCX chromatography prior to analysis by LC-MS/MS. Proteins identified were compared to published databases of proteins identified from CSF and plasma. AC fluid proteins not found in these two databases were experimentally searched for in lumbar CSF taken from neurologically-normal patients, by a targeted protein identification approach called MIDAS (Multiple Reaction Monitoring (MRM) initiated detection and sequence analysis). RESULTS: We did not identify systematic trends or grouping of data in the AC protein comparison experiment, implying low variability between individual proteomic profiles of AC.In the AC proteome evaluation experiment, we identified 199 proteins. When compared to previously published lists of proteins identified from CSF and plasma, 15 of the AC proteins had not been reported in either of these datasets. By a targeted protein identification approach, we identified 11 of these 15 proteins in pooled CSF from neurologically-normal patients, demonstrating that the majority of abundant proteins in AC fluid also can be found in CSF. Compared to plasma, as many as 104 proteins in AC were not found in the list of 3017 plasma proteins. CONCLUSIONS: Based on the protein content of AC fluid, our data indicate that temporal AC is a homogenous condition, pointing towards a similar AC filling mechanism for the 14 patients examined. Most of the proteins identified in AC fluid have been identified in CSF, indicating high similarity in the qualitative protein content of AC to CSF, whereas this was not the case between AC and plasma. This indicates that AC is filled with a liquid similar to CSF. As far as we know, this is the first proteomics study that explores the AC fluid proteome.

Anti-inflammatory and hypolipidemic effects of the modified fatty acid tetradecylthioacetic acid in psoriasis--a pilot study.

Tetradecylthioacetic acid (TTA) is a bioactive 3-thia fatty acid, giving hypolipidemic response, inhibiting the proliferation and increasing the differentiation of normal adult epidermal keratinocytes and showing anti-oxidant and anti-inflammatory effects. Psoriasis is an inflammatory disease associated with abnormalities in lipid profile, lipid peroxidation, antioxidant capacity, eicosanoid metabolism and increased frequency of cardiovascular events. On this background we have conducted a pilot study to explore the hypothesis that this modified fatty acid could improve dyslipidemia and reduce inflammation in psoriatic patients. In this double-blinded, placebo-controlled study, we assessed the metabolic effects of systemic TTA in a limited number of patients with mild to moderate psoriasis, 1000 mg TTA daily for 28 days. The most important findings were: (i) TTA reduced plasma total cholesterol, non HDL-cholesterol, LDL/HDL cholesterol ratio, triglycerides and total fatty acids; (ii) TTA decreased plasma TNF-α, IL-8 and VCAM-1; and (iii) plasma fatty acid composition changed with an increased level of monounsaturated fatty acids and decreased n-3 polyunsaturated fatty acids. In conclusion TTA exerts both hypolipidemic and anti-inflammatory effects in psoriasis patients. The results further indicate that TTA can be of therapeutic benefit for a subgroup of psoriatic patients.

Arachnoid cysts do not contain cerebrospinal fluid: A comparative chemical analysis of arachnoid cyst fluid and cerebrospinal fluid in adults.

BACKGROUND: Arachnoid cyst (AC) fluid has not previously been compared with cerebrospinal fluid (CSF) from the same patient. ACs are commonly referred to as containing "CSF-like fluid". The objective of this study was to characterize AC fluid by clinical chemistry and to compare AC fluid to CSF drawn from the same patient. Such comparative analysis can shed further light on the mechanisms for filling and sustaining of ACs. METHODS: Cyst fluid from 15 adult patients with unilateral temporal AC (9 female, 6 male, age 22-77y) was compared with CSF from the same patients by clinical chemical analysis. RESULTS: AC fluid and CSF had the same osmolarity. There were no significant differences in the concentrations of sodium, potassium, chloride, calcium, magnesium or glucose. We found significant elevated concentration of phosphate in AC fluid (0.39 versus 0.35 mmol/L in CSF; p = 0.02), and significantly reduced concentrations of total protein (0.30 versus 0.41 g/L; p = 0.004), of ferritin (7.8 versus 25.5 ug/L; p = 0.001) and of lactate dehydrogenase (17.9 versus 35.6 U/L; p = 0.002) in AC fluid relative to CSF. CONCLUSIONS: AC fluid is not identical to CSF. The differential composition of AC fluid relative to CSF supports secretion or active transport as the mechanism underlying cyst filling. Oncotic pressure gradients or slit-valves as mechanisms for generating fluid in temporal ACs are not supported by these results.

Natural killer cells as biomarkers of hyperbaric stress during a dry heliox saturation dive.

INTRODUCTION: Diving, hyperbaric oxygen, and decompression have been described as inducers of alterations in various components of the human immune system, such as the distribution of circulating lymphocytes. Hypothetically, the monitoring of specific lymphocyte subsets during hyperbaric exposure, including T- and NK-cell subsets, can serve as biomarkers of hyperbaric stress. METHODS: Eight experienced saturation divers and eight reference subjects, naive to deep saturation diving, were examined. Peripheral blood mononuclear cells were isolated before and at different points during a 19.3-d dry heliox saturation dive to 2.64 MPa (254 msw). The NK cell cytotoxicity was estimated in a 4-h 51Cr-release assay using the NK cell sensitive tumor cell-line K562 as target cells. The major lymphocyte subpopulations, with special emphasis on the NK cell subsets, were phenotypically delineated by the use of 4-color flow cytometry. RESULTS: Although NK cell cytotoxicity increased significantly in the divers during the compression phase and the reference subjects who remained in normoxic conditions outside the chamber, the NK cell cytotoxicity was significantly higher in the divers. DISCUSSION: This finding, together with augmentation in the absolute number of circulating NK cells in the divers due to a possible activation of specific parts of the innate cellular immune system during hyperbaric exposure, suggests the monitoring of specific immune functions can be useful as biomarkers of hyperbaric-induced inflammatory stress.