Ralph Gräsbeck

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.

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.

Imerslund-Gräsbeck syndrome (selective vitamin B12 malabsorption with proteinuria)

Imerslund-Gräsbeck syndrome (IGS) or selective vitamin B12 (cobalamin) malabsorption with proteinuria is a rare autosomal recessive disorder characterized by vitamin B12 deficiency commonly resulting in megaloblastic anemia, which is responsive to parenteral vitamin B12 therapy and appears in childhood. Other manifestations include failure to thrive and grow, infections and neurological damage. Mild proteinuria (with no signs of kidney disease) is present in about half of the patients. Anatomical anomalies in the urinary tract were observed in some Norwegian patients. Vitamin B12 absorption tests show low absorption, not corrected by administration of intrinsic factor. The symptoms appear from 4 months (not immediately after birth as in transcobalamin deficiency) up to several years after birth. The syndrome was first described in Finland and Norway where the prevalence is about 1:200,000. The cause is a defect in the receptor of the vitamin B12-intrinsic factor complex of the ileal enterocyte. In most cases, the molecular basis of the selective malabsorption and proteinuria involves a mutation in one of two genes, cubilin (CUBN) on chromosome 10 or amnionless (AMN) on chromosome 14. Both proteins are components of the intestinal receptor for the vitamin B12-intrinsic factor complex and the receptor mediating the tubular reabsorption of protein from the primary urine. Management includes life-long vitamin B12 injections, and with this regimen, the patients stay healthy for decades. However, the proteinuria persists. In diagnosing this disease, it is important to be aware that cobalamin deficiency affects enterocyte function; therefore, all tests suggesting general and cobalamin malabsorption should be repeated after abolishment of the deficiency.

Spectral and other studies on the intestinal haem receptor of the pig

We recently demonstrated the presence of a Triton-solubilized high-affinity haem binder on the pig duodenal brush border membrane. The association of haem to the binding factor was determined using radioactive haem and is now studied by a spectrophotometric technique. The binding alters the Soret absorption band of haem from 395 nm to 413 nm. The dissociation constant for the binding of haem to the solubilized binding factor was estimated to be about 10(-9) M by difference spectroscopy. Human serum albumin could not prevent the solubilized binding factor from binding haem. Trypsin digestion destroyed the binder.

The evolution of the reference value concept.

Abstract This manuscript reviews the introduction of the concept of reference values, the corresponding philosophy, and subsequent recommendations reflecting the different subtopics of the field. The generally unrecognised phenomenon that laboratory results of the population tended to be lognormal instead of Gaussian attracted the attention of the author, who became sceptical of the concept of normal values because of its ambiguity. Together with N.-E. Saris a new concept of reference values was launched at a congress in 1969. Briefly, clinical measurements should be interpreted against values from proper control subjects. Subsequently international, regional and national societies established expert panels which produced recommendations covering the general principles and terminology of reference values, the concept of health, standardised specimen collection and preanalytical factors, statistical treatment of collected values, stratification of data, relating observed (patient) values to reference values, etc. The importance of using correct terminology, taking into consideration the aim of ordering the laboratory test and some neglected procedures (e.g., survival values) are emphasised. New developments such as reference changes are mentioned. The field has evolved largely as the result of constructive international teamwork.

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.

Isolation of intrinsic factor and its probable degradation product, as their vitamin B12 complexes, from human gastric juice.

Abstract By a series of chromatographic and gel-filtration steps, the 2 components in human gastric juice carrying intrinsic-factor activity, binders S and I, were prepared as their cyanocobalamin complexes. The former was found to be homogeneous and the latter almos pure. Complex S has a mol. wt. of about 115 000 and contains 2 moles of cobalamin per mole and about 13% carbohydrate. Complex I is somewhat smaller and is apparently a degradation product of S, the original intrinsic factor. Both complexes are probably polymers, most likely dimers.

Proteinuria in cubilin-deficient patients with selective vitamin B12 malabsorption

Abstract.Selective vitamin B12 malabsorption or Gräsbeck-Imerslund disease (megaloblastic anemia 1) is frequently accompanied by proteinuria. The malabsorption-proteinuric syndrome of Finnish patients is caused by a defect in the multiligand receptor cubilin. We studied the urinary proteins of control subjects and 13 adult patients with three defined cubilin mutations (FM1, FM2, FM3), all diagnosed during childhood and subsequently observed. The overall kidney function was unimpaired and did not deteriorate with time. The excretion of total protein and albumin, and to lesser extent of transferrin, immunoglobulin light chains, and α1- and β2-microglobulins, was clearly elevated in 3 patients, mildly elevated in 3, and hardly or not at all increased in the rest. The urinary cobalamin-intrinsic factor receptor was low in 5 patients studied and lowest in the group with clear-cut proteinuria. The proteinuria was not of the classical glomerular or tubular type, but apparently due to the lack of cubilin function needed for tubular reabsorption of some, but not all, proteins of the primary urine.

Vitamin B12-binding Proteins in Normal and Leukemic Human Leukocytes and Sera

The vitamin B12-binding proteins in sera and leukocytes of healthy subjects and patients with chronic myelogenous leukemia (CML) were separated and partially purified by column chromatography as their cyanocobalamin complexes. The molecular weight of transcobalamin I and leukocyte B12-binding protein was 119,000, and that of transcobalamin II was 32,000 as determined by gel filtration. The electrophoretic mobilities were α1 for transcobalamin I, α2 for the leukocyte B12-binding protein, and α2-β for transcobalamin II. After treatment with neuraminidase or chromatographic purification, the mobilities of both transcobalamin I and the leukocyte B12-binding protein became β. Transcobalamin I and the leukocyte B12-binding protein gave a reaction of immunologic identity in immunodiffusion. The fact that leukocytes can produce B12-binding protein in vitro and the similarities between leukocyte B12-binding protein and transcobalamin 1 strongly suggest that CML transcobalamin I is derived from leukocytes.

The theory of reference values: an unfinished symphony

Abstract The history of the theory of reference values can be written as an unfinished symphony. The first movement, allegro con fuoco, played from 1960 to 1980: a mix of themes devoted to the study of biological variability (intra-, inter-individual, short- and long-term), preanalytical conditions, standardization of analytical methods, quality control, statistical tools for deriving reference limits, all of them complex variations developed on a central melody: the new concept of reference values that would replace the notion of normality whose definition was unclear. Additional contributions (multivariate reference values, use of reference limits from broad sets of patient data, drug interferences) conclude the movement on the variability of laboratory tests. The second movement, adagio, from 1980 to 2000, slowly develops and implements initial works. International and national recommendations were published by the IFCC-LM (International Federation of Clinical Chemistry and Laboratory Medicine) and scientific societies [French (SFBC), Spanish (SEQC), Scandinavian societies…]. Reference values are now topics of many textbooks and of several congresses, workshops, and round tables that are organized all over the world. Nowadays, reference values are part of current practice in all clinical laboratories, but not without difficulties, particularly for some laboratories to produce their own reference values and the unsuitability of the concept with respect to new technologies such as HPLC, GCMS, and PCR assays. Clinicians through consensus groups and practice guidelines have introduced their own tools, the decision limits, likelihood ratios and Reference Change Value (RCV), creating confusion among laboratorians and clinicians in substituting reference values and decision limits in laboratory reports. The rapid development of personalized medicine will eventually call for the use of individual reference values. The beginning of the second millennium is played allegro ma non-troppo from 2000 to 2012: the theory of reference values is back into fashion. The need to revise the concept is emerging. The manufacturers make a friendly pressure to facilitate the integration of Reference Intervals (RIs) in their technical documentation. Laboratorians are anxiously awaiting the solutions for what to do. The IFCC-LM creates Reference Intervals and Decision Limits Committee (C-RIDL) in 2005. Simultaneously, a joint working group IFCC-CLSI is created on the same topic. In 2008 the initial recommendations of IFCC-LM are revised and new guidelines are published by the Clinical and Laboratory Standards Institute (CLSI C28-A3). Fundamentals of the theory of reference values are not changed, but new avenues are explored: RIs transference, multicenter reference intervals, and a robust method for deriving RIs from small number of subjects. Concomitantly, other statistical methods are published such as bootstraps calculation and partitioning procedures. An alternative to recruiting healthy subjects proposes the use of biobanks conditional to the availability of controlled preanalytical conditions and of bioclinical data. The scope is also widening to include veterinary biology! During the early 2000s, several groups proposed the concept of ‘Universal RIs’ or ‘Global RIs’. Still controversial, their applications await further investigations. The fourth movement, finale: beyond the methodological issues (statistical and analytical essentially), important questions remain unanswered. Do RIs intervene appropriately in medical decision-making? Are RIs really useful to the clinicians? Are evidence-based decision limits more appropriate? It should be appreciated that many laboratory tests represent a continuum that weakens the relevance of RIs. In addition, the boundaries between healthy and pathological states are shady areas influenced by many biological factors. In such a case the use of a single threshold is questionable. Wherever it will apply, individual reference values and reference change values have their place. A variation on an old theme! It is strange that in the period of personalized medicine (that is more stratified medicine), the concept of reference values which is based on stratification of homogeneous subgroups of healthy people could not be discussed and developed in conjunction with the stratification of sick patients. That is our message for the celebration of the 50th anniversary of Clinical Chemistry and Laboratory Medicine. Prospects are broad, enthusiasm is not lacking: much remains to be done, good luck for the new generations!