Biosynthesis of 32P-labelled hydroxocobalamin and a study of its behaviour in rats.

Using Propionibacterium freudenreichii and 32P-ATP, batches of 32P-labelled cobalamin (Cbl) were biosynthesized with a maximum specific activity of 61 microCi/mg, i.e. about 100 times higher than previously reported. Pharmacological doses mixed with 57Co-Cbl were injected subcutaneously in the form of hydroxo-Cbl into rats subsequently killed 5-20 days later. The two labelled Cbls were distributed in approximately the same way, the highest concentration being found in kidney (typical for rats) and about one-fifth of that in liver. These findings tallied with previous observations with radioactive cyano-Cbl and microbiological assay. In all injected rats, the 57Co/32P ratio was lower in liver than in kidney. Drugs eradicating the intestinal flora had no influence. In rats receiving the vitamin orally, the ratio was higher in liver than in kidney. All of our findings could be due to formation of a cobinamide-like compound lacking phosphorus. It is concluded that we have produced radiophosphorus-labelled Cbl that enables studies in vivo.

The driving license examination as a stress model: effects on blood picture, serum cortisol and the production of interleukins in man.

We have studied the following stress model: the tension caused by sitting for the theoretical part of the driving license examination. Volunteers were investigated twice, after their driving license examination and after a (stress-free) control session. The effects of the stress were investigated by studying the blood picture (differential counts), serum concentration of cortisol, and cytokine production in stimulated blood cells. Relationships between the subjective perception of stress and the physiological reaction were also investigated. This stress induced significant increase in the concentrations of cortisol and hemoglobin, and in the values of hematocrit and MCV, and in the lipopolysaccharide-induced release of IL-1beta and -6. The subjective feelings of irritability and wakefulness were also significantly higher after the exam. A significant relationship was found between the changes in the stimulated production of IL-1beta and irritability. The responsiveness to psychological stress might be influenced by the temporary mood of the subjects.

Preanalytical factors (biological variation) and the measurement of serum soluble intercellular adhesion molecule-1 in humans: influence of the time of day, food intake, and physical and psychological stress.

BACKGROUND: Adhesion proteins such as soluble intercellular adhesion molecule-1 (sICAM-1) may be important markers for early atherosclerosis and some other diseases. To devise optimum specimen collection procedures, we investigated the effects on serum sICAM-1 of several preanalytical factors (factors that occur before a specimen is analyzed) such as the time of the day, food ingestion, and physical and psychological stress. METHODS: Three sets of experiments were conducted: (a) 30 subjects were investigated during the morning, after an overnight fast, and then after the usual breakfast and at 1200; (b) 20 subjects were studied before and after exposure to thermal stress (sauna + swimming in ice-cold water); and (c) 15 volunteers were investigated after their driving license examination and during a (stress-free) control session. Conventional methods and kits were used to determine the blood picture and serum sICAM-1. RESULTS: All of these preanalytical factors induced a significant increase ( approximately 10%) in the concentration of sICAM-1. CONCLUSION: It is advisable to consider timing, food intake, and stress when collecting specimens and analyzing data on the concentration of sICAM-1 in serum.

Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1.

Megaloblastic anaemia 1 (MGA1, OMIM 261100) is a rare, autosomal recessive disorder characterized by juvenile megaloblastic anaemia, as well as neurological symptoms that may be the only manifestations. At the cellular level, MGA1 is characterized by selective intestinal vitamin B12 (B12, cobalamin) malabsorption. MGA1 occurs worldwide, but its prevalence is higher in several Middle Eastern countries and Norway, and highest in Finland (0.8/100,000). We previously mapped the MGA1 locus by linkage analysis in Finnish and Norwegian families to a 6-cM region on chromosome 10p12.1 (ref. 8). A functional candidate gene encoding the intrinsic factor (IF)-B12 receptor, cubilin, was recently cloned; the human homologue, CUBN, was mapped to the same region. We have now refined the MGA1 region by linkage disequilibrium (LD) mapping, fine-mapped CUBN and identified two independent disease-specific CUBN mutations in 17 Finnish MGA1 families. Our genetic and molecular data indicate that mutations in CUBN cause MGA1.

A urinary radioisotope-binding assay to diagnose Gräsbeck-Imerslund disease.

BACKGROUND: Gräsbeck-Imerslund disease (congenital familial selective vitamin B12-malabsorption with proteinuria, MGA1, MIM No. 261100) is a rare disorder displaying autosomal recessive inheritance. This study was designed to investigate the usefulness of measuring the activity of the urinary receptor for the intrinsic factor-cobalamin complex as a tool to diagnose this disease. METHODS: The receptor activity was measured by a radioisotope-binding assay, using phenyl-Sepharose gel as the adsorbant solid phase of the receptor. RESULTS: In 10 Finnish patients, urinary receptor activity was on the average 640 times (15-1400 times) lower than that in 13 healthy control subjects: mean values of 0.1 nmol/mol (range, 0.01-0.32 nmol/mol) and 6.4 nmol/mol (range, 3.8-12.4 nmol/mol) creatinine, respectively. The mean value of urinary receptor activity in 11 first-degree, healthy relatives of the patients was 4.6 nmol/mol (range, 1.1-10.4 nmol/mol) creatinine, a difference from levels in control subjects that is not statistically significant. When the first-degree relatives were divided into heterozygotes (parents and siblings heterozygous for the haplotype of genetic markers associated with the disease gene) and wild-type homozygotes (siblings not displaying the disease haplotype), no difference was seen. CONCLUSION: Determination of receptor activity in the urine is a highly accurate method for diagnosis of Gräsbeck-Imerslund disease at an early stage, but it does not detect carriers of the disorder.

Selective cobalamin malabsorption and the cobalamin-intrinsic factor receptor.

The disease is characterised by cobalamin (Cbl) deficiency in children 0-5 years old, causing failure to thrive, infections, megaloblastic anaemia, neuropathy, and mild general malabsorption; slight proteinuria is common. Cbl injections produce remission, but Cbl malabsorption and proteinuria persist. About 250 cases have been reported. Dogs also have it. The heredity is autosomal and recessive. The physiological and pathological absorption mechanisms are described: Cbl liberated from food by digestion is first bound to haptocorrin, but in the intestine it is transferred to intrinsic factor. In the ileum the complex attaches to a receptor on the enterocytes; this requires neutral pH and Ca2+. The receptor is a membrane-bound glycoprotein consisting of multiple subunits. The receptor-ligand complex is endocytosed and degraded in lysosomes, and the vitamin is transferred to transcobalamin which carries it to tissues. The same receptor is strongly expressed in the kidneys, but urine also contains its activity which can be assayed for diagnosis. The basic lesion is an error in the ileal receptor. In the affected dogs the synthesised receptor is retained intracellularly. Urine and ileal biopsies from human cases contained little receptor but it had conserved affinity for the ligand. Recently examined Arab patients did not excrete reduced amounts of the receptor. Apparently, the disease has subsets, such as different structural errors in the receptor and possibly faulty transport inside the enterocyte. The cause of the proteinuria is unknown but kidney damage due to severe Cbl deficiency and an error in a multiligand renal receptor are among the possibilities.

Preanalytical factors and the measurement of cytokines in human subjects.

Cytokines are widely measured in research. However, cytokine analyses are influenced by a myriad of factors. For instance, a delay in the separation of plasma from cells may lead to a 50% decrease in the concentration of tumor necrosis factor in plasma. Another example is the secretion of interleukin-1 beta in women which can be twice as high during the follicular phase as in the luteal phase. The factors influencing the outcome of these tests can be divided into in vivo preanalytical factors (e.g., aging, chronobiological rhythms, diet, etc), in vitro preanalytical factors (e.g., specimen collection, equipment, transport, storage, etc), and analytical factors. To improve the value of the cytokine tests, factors strongly influencing the results have to be controlled. This can be done by using standardized assays and specimen collection procedures. In general, sufficient attention is not given to the preanalytical factors, especially in the measurement of cytokines. This article reviews the preanalytical factors which may influence the outcome of these tests in human subjects.

Selective intestinal malabsorption of vitamin B12 displays recessive mendelian inheritance: assignment of a locus to chromosome 10 by linkage.

Juvenile megaloblastic anemia caused by selective intestinal malabsorption of vitamin B12 has been considered a distinct condition displaying autosomal recessive inheritance. It appears to have a worldwide distribution, and comparatively high incidences were reported 30 years ago in Finland and Norway. More recently, the Mendelian inheritance of the condition has been questioned because almost no new cases have occurred in these populations. Here we report linkage studies assigning a recessive-gene locus for the disease to chromosome 10 in previously diagnosed multiplex families from Finland and Norway, proving the Mendelian mode of inheritance. The locus is tentatively assigned to the 6-cM interval between markers D10S548 and D10S466, with a multipoint maximum lod score (Zmax) of 5.36 near marker D10S1477. By haplotype analysis, the healthy sibs in these families did not appear to constitute any examples of nonpenetrance. We hypothesize that the paucity of new cases in these populations is due either to a dietary effect on the gene penetrance that has changed with time, or to a drop in the birth rate in subpopulations showing enrichment of the mutation, or to both of these causes.

Decreased activity of intestinal and urinary intrinsic factor receptor in Gräsbeck-Imerslund disease [corrected].

BACKGROUND/AIMS: The pathogenesis of inherited intestinal cobalamin malabsorption (Gräsbeck-Imerslund disease) remains unknown. The authors studied whether the disease corresponds to a defective expression and/or function of the intrinsic factor-cobalamin receptor in the ileum. METHODS: Intrinsic factor-cobalamin receptor activity was measured using radioisotope assay and gel-filtration exclusion chromatography in ileal biopsy specimens and urine concentrates from 4 patients with Gräsbeck-Imerslund disease and 5 controls. RESULTS: Receptor activity was 164 +/- 13 fmol/mg of protein in control biopsy specimens and < 2.6 fmol/mg protein in specimens from patients. The association constant was estimated to be 3.8 +/- 0.4 (nmol/L)-1 in controls. A dramatic decrease in receptor activity was also observed in urine concentrate from patients with an association constant of 1.9 and 3.3 (nmol/L)-1. Isoelectrofocusing of the cross-linked intrinsic factor-cobalamin receptor complex showed an isoelectric point at 4.8 in a patient as well as in control samples. CONCLUSIONS: It is concluded that Gräsbeck-Imerslund disease is related to decreased intrinsic factor-receptor activity in intestinal mucosa; the receptor assay in urine can be helpful for diagnosis.

How to diagnose cobalamin deficiency.

Cobalamin deficiency must be suspected in all patients with unexplained neuropsychiatric symptoms or unexplained anemia. Special attention should be paid to patients at risk of developing cobalamin deficiency such as elderly people, vegetarians, HIV-infected patients, patients with gastrointestinal diseases and patients with autoimmunity or a family history of pernicious anemia. The assays aimed to answer the question: does this patient suffer from cobalamin deficiency, include analysis of P--cobalamins and analyses of the metabolites that accumulate upon cellular cobalamin deficiency, P--methylmalonate and P--homocysteine. P--cobalamins or especially a fraction of P--cobalamins, P--TC cobalamins are markers for latent cobalamin deficiency. An increased concentration of P--methylmalonate that decreases upon injection of cobalamin indicates overt metabolic cobalamin deficiency. The same holds for P--homocysteine but this analysis is less specific than P--methylmalonate. We suggest that either assay of P--cobalamins or P--methylmalonate is employed as screening test for cobalamin deficiency, and that further tests are performed only if the initial test in combination with the clinical picture gives an unclear answer. Once cobalamin deficiency has been diagnosed, the cause for the deficiency should be sought and the patient should be treated for life. Cobalamin absorption tests such as the Schilling test are considered of limited use. Gastric atrophy is likely to be present in patients with increased P--gastrin or decreased P--pepsinogen A. However, this condition can be diagnosed also by upper gastrointestinal endoscopy.

Effects of psychological stress on plasma interleukins-1 beta and 6, C-reactive protein, tumour necrosis factor alpha, anti-diuretic hormone and serum cortisol.

The study was undertaken to determine whether psychological stress influences immunobiological functions and is an important preanalytical factor to be considered in connection with blood specimen collection. Two kinds of stress were applied, the Stroop colour conflict test and the thrill of a novice about to make the first jump with a parachute. In both test situations, the level of the stress indicators cortisol or anti-diuretic hormone rose significantly. The concentrations of the cytokines studied did not change significantly. However, in the parachute test significant positive correlations were found, e.g. between the changes of cortisol and C-reactive protein and between anti-diuretic hormone and interleukin-1 beta. This suggests that there is an interaction between the endocrine and the immune systems in the response to a psychological stress.

Establishing reference values from adults: recommendation on procedures for the preparation of individuals, collection of blood, and handling and storage of specimens. Committee on Reference Values of the Scandinavian Society for Clinical Chemistry.

The concentrations of blood components are influenced by a number of preanalytical factors, the importance of which varies [1]. Therefore it is necessary to standardize the specimen collection, as was done by the Committee on Reference Values of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, which in 1975 published its recommendation on the production of reference values in clinical chemistry [2]. It was the very first recommendation of its kind. Since then, new information has accumulated, and it has become necessary to revise the part concerning the preparation of subjects for blood collection, the collection procedure itself, and the subsequent handling of the specimen. Also, The International Federation of Clinical Chemistry (Expert Panel on Theory of Reference Values) has produced its own recommendation [3]. The recommendation described below replaces pp. 39-44 of the former Scandinavian recommendation [2]. The procedure described below is designed for the collection of specimens for the measurement of the majority of components in blood and specimen collection from the cubital vein is described. It can be easily adapted for the collection of blood from other vessels [4]. Certain laboratory investigations require a special protocol. In that case, the preparation of the subject prior to and during specimen collection as well as the procedure itself should be described in sufficient detail to permit reproduction by adequately trained personnel. In any project designed to produce reference values the same protocol for the preparation of individuals and specimen collection should be used throughout the project. The protocol should be described in detail. Thus, it is not satisfactory to state only that the present recommendation was followed. Each section of the recommendation is followed by a check list for points which should be taken into account.

Preanalytical factors and standardized specimen collection: influence of psychological stress.

In order to devise and evaluate standardized specimen collection procedures, we studied the influence of psychological stress on the results of commonly analysed blood components: creatine kinase, lactate dehydrogenase, total protein and albumin in serum and blood picture. In addition, serum cortisol was assayed. Two kinds of stress were used: the Stroop test, a colour conflict task, and the thrill caused by the first jump of new parachutists. More changes were observed after the parachutist test than after the Stroop test. There was a difference in the responses of males and females. Females were more sensitive, especially to the parachutist test. Most of the changes observed were interpreted as being caused by haemoconcentration, possibly related to muscular tension. Cortisol, commonly used to indicate the level of stress, did not react much and is therefore not a good index of psychological stress.

Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.

A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.