Effect of pancreatic extracts on the faecal excretion and on the serum concentration of cobalamin and cobalamin analogues in cystic fibrosis

A malabsorption of crystalline labelled cobalamin is observed in 100% of cystic fibrosis patients. Using radioisotope dilution assays and molecular sieve gel chromatography, we determined the serum concentration and the faecal excretion of cobalamin and cobalamin analogues in nine cystic fibrosis children before and after 4 days' interruption of pancreatic extract treatment. On chromatography, the unsaturated cobalamin binders of the faecal extracts eluted in two positions with molecular masses of 44 300 and 20 300, corresponding mostly to partially degraded R binders. The amounts of the less degraded form of R binder (molecular mass 44 300) increased significantly after interruption of the treatment. The cobalamin concentration in the serum remained normal after interruption of the treatment but the analogue concentrations in the serum decreased and faecal excretion of cobalamin and analogues increased significantly. These results allowed us to suggest that (1) pancreatic insufficiency in cystic fibrosis is responsible for a decrease in the absorption of digestive analogues induced by a defective degradation of R binders, and (2) cobalamin analogues have a short half-life in blood.     

A radioimmunoassay for the R-type binders of cobalamin.

A radioimmunoassay of R-type binders of cobalmin was devised and tested. The assay incorporated an antibody against purified human salivary R binder as the binding reagent. The labeled ligand was cyano[57Co]cobalamin bound to the R binder of pooled human saliva. The standard source of unlabeled ligand was also from human saliva of known R binder content. The assay was responsive to R binders of several sources, to either pure R or that of crude sources and equally to R binder saturated or unsaturated with cobalamin. It was not responsive to transcobalamin II. The assay was reproducible and reliable.     

Activity levels and properties of acid α-glucosidase from liver and neutral α-glucosidase from sera of cystic fibrosis patients and controls

The average activity levels of acid α-glucosidase are comparable in liver supernatant fluids for 15 cystic fibrosis patients and 12 controls (401 ± 131 and 347 ± 109 nmol/h/mg protein, respectively) and no significant differences were found for the cystic fibrosis and control liver acid α-glucosidases in their (a) apparent K_m values for the 4-methylumbelliferyl substrate (1.1 mmol/1), (b) pH optima (4.2) and thermostability curves and (c) isoelectric profiles (one form with an isoelectric point of 4.5 ± 0.2).In contrast, average neutral α-glucosidase activity levels were significantly increased (p < 0.0002) in sera from 21 cystic fibrosis patients compared to 15 controls (10.7 and 2.7 nmol/h/ml). This increased activity is not due to (a) different stability upon storage at ?20°C, (b) the presence of activators in cystic fibrosis sera or inhibitors in normal sera (as determined by mixing studies), (c) altered K_m values or (d) altered pH optima curves. Cystic fibrosis serum neutral α-glucosidase appears to be more thermostable and has a consistently altered isoelectric profile (greater percentage of activity above pI 4.8) when compared to the normal serum enzyme. This altered isoelectric composition may reflect changes in neutral α-glucosidase which contribute to its increased activity in cystic fibrosis sera.     

Cystic fibrosis serum α-l-fucosidase: Confirmation of normal activity levels and normal kinetic and isoelectric focusing properties

Comparable α-l-fucosidase activity was found for normal and cystic fibrosis sera, 7.8 ± 3.3 and 9.8 ± 4.7 nmol/min/ml, respectively. Isoelectric focusing of normal and cystic fibrosis sera α-l-fucosidase revealed similar isoelectric profiles with most activity between isoelectric points of 4.7–5.4. Kinetic analysis of normal and cystic fibrosis sera α-l-fucosidase revealed identical apparent Michaelis constants for the 4-methylumbelliferyl substrate (51 ± 6 μmol/l and 50 ± 8 μmol/1, respectively), similar pH optima curves (both have broad optima between pH 4.8 and 6.5) and identical thermostability curves at three different preincubation temperatures (37°, 45° and 55°C).     

Incorporation of L-leucine and D-glucosamine into skin fibroblasts derived from cystic fibrosis and normal individuals

Cultured skin fibroblasts from patients with diagnosed cystic fibrosis were compared with those from normal individuals in respect to incorporation of l-leucine or d-glucosamine into the subcellular components, to uptake of α-isobutyric acid and calcium, and to (Na⁺-K⁺)ATPase activity, etc. No significant difference which could be attributed to the disease-specific basic metabolic abnormality was demonstrated. Cystic fibrosis skin fibroblasts may not express the primary genetic defect at the level of generalized transport mechanism and overall protein metabolism.     

Excretion of Trypsin-Like Activity,Electrolytes and Protein in Mixed and Parotid Saliva of Patients with Cystic Fibrosis of the Pancreas†

Abstract. The esterolytic activity of mixed and parotid saliva in cystic fibrosis (CF) patients and normal subjects was determined using BAEE (α-Benzoyl-1-arginine-ethylester) as the substrate. Using soybean-trypsin-inhibitor the trypsin-like activity (TLA) was measured and plotted as a function of parotid flow rate. In addition calcium, protein and pH were determined. Trypsin-like activity in mixed and parotid saliva showed large individual variations in CF and normal children. In parotid saliva we could not find any significant difference, whereas a reduction of TLA in mixed saliva of CF patients was observed. The fact that our normal values fell within the range of heterozygotes reported by Rao et al. (19), makes their hypothesis of a close relationship between reduced TLA and the genetic defect very doubtful. Protein, calcium and pH increased with augmented salivation and no difference between CF patients and normal age matched children could be found except for the pH at a flow rate above 0.75 ml/min per m² body surface where significantly lower pH values resulted. The relevance of reduced TLA to the pathogenesis of cystic fibrosis is discussed.     

An enzyme-linked immunosorbent assay to detect IgG antibodies to cell surface components of Pseudomonas cepacia in patients with cystic fibrosis

IgG antibody to the cell surface components of Pseudomonas cepacia was measured in the serum of patients with cystic fibrosis by an enzyme-linked immunosorbent assay with formalinised whole bacteria as antigen. Antibody was detected in the sera of seven patients, known to be colonised with P. cepacia, against antigens from nine different strains of the organism. Absorption studies also showed extensive cross-reactivity. Preliminary studies indicate that the antibody is species specific. Two strains of P. cepacia were selected to provide antigen for screening of sera. Sera from patients with cystic fibrosis, not known to be colonised, showed a rising level of antibody with age. Six out of seven colonised patients had levels clearly raised above the background level for their age-group. These preliminary results suggest that this test may be useful in elucidating the importance of this organism in patients with cystic fibrosis.     

Isoelectric focusing of serum in cystic fibrosis: Failure to distinguish between homozygote and heterozygote sera

Thin-layer isoelectric focusing was performed on samples of sera from patients with Cystic Fibrosis, siblings and obligate heterozygotes (parents), and children without cystic fibrosis (controls). The protein band with an isoelectric point of pH 5.48, previously reported to be absent in homozygote cystic fibrosis sera, was found to have a pI of 5.25. It was present in approximately one-half of the homozygote and heterozygote sera tested, and absent from 18 percent of control sera. The presence of this band is not therefore a reliable marker for the normal gene, and cannot be used to identify the heterozygous carrier for cystic fibrosis.     

Is there a "gold standard" for human serum vitamin B12 assay?

In a study from four laboratories using two commercial vitamin B12 radioassays (impure hog intrinsic factor concentrate containing both intrinsic factor and R binders (IF + R) to measure total corrinoids and the same concentrate presaturated with cobinamide (Cbi) to block B12 binding sites on R binder (IF + R + Cbi) to measure only cobalamins), the rank order of results was generally the same. The concordance between the two tests for classifying sera as normal or deficient was 91% in 311 serum samples. Three percent of sera below the "true B12" (B12 binding to IF + R + Cbi) normal cut-off point were not below the cut-off point for normal "total B12" (B12 binding to IF + R); 6% of sera below the total B12 normal cut-off point were not below true B12 cut-off point. The correlations between Euglena gracilis and the radioassays were 0.80 and 0.83 in the 50 serum samples that also had E. gracilis serum vitamin B12 levels. Lactobacillus leichmannii serum vitamin B12 levels were determined in 49 of the 311 serum samples and results were comparable with results obtained by four radioassay binder systems: IF + R, IF + R + Cbi, highly purified hog IF, and saliva R binder. The closest correlate with L. leichmannii was radioassay using IF + R as binder (r = 0.93), then IF + R + Cbi (r = 0.92), pure IF (r = 0.80), and pure R (r = 0.73). The key to reliable results appears not to reside in a particular assay but rather in determining for each assay its own range of results in participants determined clinically and morphologically normal vs. participants with deficient vitamin B12 (with B12 deficiency defined independently of a serum B12 assay). When laboratory assay results differ from clinical judgment, further evaluation is the appropriate course. There is no "gold standard" for human serum vitamin B12 assay.     

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man. Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with anti-intrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled > 60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels of pancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency. Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation of pancreatic zymogens in an acidic pH of the intestinal juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop imparied cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients.     

Cystic fibrosis-like changes in saliva of healthy persons subjected to anaerobic exercise

The biochemical composition of saliva secreted by healthy persons and by heterozygotes and homozygotes of cystic fibrosis at rest and by healthy persons subjected to aerobic or anaerobic effort were compared. In the saliva from cystic fibrosis homozygotes at rest substantial increases of the activity of ribonuclease (p less than 0.001) and of the concentrations of protein (p less than 0.001), lactate (p less than 0.001), sodium (p less than 0.001), potassium (p less than 0.01) and calcium (p less than 0.05) were found in comparison with saliva from healthy persons at rest. In the saliva from cystic fibrosis heterozygotes at rest similar but less pronounced changes were seen. After anaerobic exercise these biochemical parameters were increased in the saliva of healthy persons and mimicked the values of cystic fibrosis saliva. However, after aerobic effort no changes other than a slightly increased ribonuclease activity were seen in the saliva of healthy persons. This indicates that salivary glands of cystic fibrosis patients, at rest, are in the same state of lactate acidosis and energy depletion as these glands are in healthy persons after anaerobic work.     

Demonstration of serum protein differences in cystic fibrosis by isoelectric focusing in thin-layer polyacrylamide gels

Whole serum samples from cystic fibrosis patients, heterozygotes and normal individuals were analyzed using isoelectric focusing in thin-layer polyacrylamide gels. Using this technique consistent serum protein differences between the three serum types have been demonstrated. A protein with an isoelectric point of 5.48 ± 0.1 was found in 14 out of 17 normal sera and in all heterozygote sera tested but not in 15 out of 16 cystic fibrosis sera tested. A protein with an isoelectric point of 8.41 ± 0.1 was found in 12 out of 13 cystic fibrosis sera and all heterozygote sera but not in 11 out of 12 normal sera tested. The concentration of both of these proteins was demonstrated to be variable among the heterozygotes as shown by electrofocusing of different volumes of serum.Partial characterization of both proteins was achieved using the combined techniques of isoelectric focusing, immunoelectrophoresis, acrylamide to ionagar electrophoresis, ammonium sulfate precipitation and diethylaminoethylcellulose chromatography (pH 8.0). The protein with an isoelectric point at pH 5.48 ± 0.1 was found to be a heat labile beta-globulin and the protein with an isoelectric point of 8.41 ± 0.1 was found to be a heat labile gamma-globulin. The significance of the findings are discussed.     

Oyster ciliary inhibition by cystic fibrosis culture medium

Using the oyster ciliary test, sera and fibroblast culture fluid from certain homozygotes and heterozygotes for cystic fibrosis can be shown to contain an inhibitory factor. The tissue culture fluid derived from the metachromatic fibroblast cultures (cystic fibrosis classes I and II) show ciliary inhibition. The fluid derived from the ametachromatic fibroblast cultures (cystic fibrosis class III), thus far studied, cannot be invariably distinguished from the normal noncarriers. Similarly sera from classes I and II show marked ciliary inhibition, whereas sera from class III individuals do not consistently. These experiments support the notion of genetic heterogeneity in cystic fibrosis. The nature of the inhibitory factor in tissue culture fluid and its relationship to the serum factor remain to be explored.     

Pseudomonas aeruginosa flagellar antibodies in serum,saliva and sputum from patients with cystic fibrosis

Flagellar preparations from Pseudomonas aeruginosa strains were characterized and used in ELISA and immunoblot studies to detect anti-P. aeruginosa flagellar antibodies in sera, saliva and sputum from patients with cystic fibrosis (CF). Serum antiflagellar IgG antibodies were detected, particularly in those CF patients intermittently or chronically colonized by P. aeruginosa. Antibodies to both type-a and -b flagella were detected; however, in some patients a pronounced antibody response to only one of the flagellar types was evident. Raised levels of anti-flagellar antibodies in intermittently and non-P. aeruginosa colonized patients may represent an early antibody response to P. aeruginosa colonization of the CF respiratory tract and implicate instigation of early anti-pseudomonal antibiotic therapy.     

The composition of a mucus glycoprotein from meconium of cystic fibrosis, healthy pre-term and full-term neonates

A high molecular mass mucus glycoprotein fraction (molecular mass greater than 1 million) which is a major component of meconium mucin, has been isolated from individual specimens collected from 20 healthy full-term infants, 19 premature infants and 19 infants with proven cystic fibrosis. The mucus glycoprotein fraction isolated from cystic fibrosis meconium had a significantly lower saccharide content than that isolated from specimens from healthy full-term infants but had a similar composition to that isolated from meconium of premature infants, gestational age 28-32 weeks. The composition of the glycoprotein fraction from the meconium of infants, gestational age 32-36 weeks, lay between that from cystic fibrosis and full-term. There is therefore a change or 'maturation' of epithelial mucin during gestation and it is hypothesised that the cystic fibrosis genetic lesion affects the maturation of epithelial secretion, resulting in the abnormal exocrine secretion associated with the disease.     

Properties of purified salivary ribonuclease,and salivary ribonuclease levels in children with cystic fibrosis and in heterozygous carriers

Alkaline acid- and thermo-stable ribonuclease was isolated from human saliva of healthy subjects, children with cystic fibrosis, and from parents of the latter. The enzyme was purified about 180–200 times, and its ionic requirements, and specificity towards various substrates, characterized and compared with those for human pancreatic ribonuclease.The level of acid- and thermo-stable ribonuclease activity was five times higher in terms of enzyme level per ml saliva, and three times higher in terms of salivary protein, for 62% of children with cystic fibrosis, and for 73% of carrier subjects, relative to control groups. Assay of the level of this ribonuclease activity appears to be a promising diagnostic tool for detection of heterozygote carriers.     

Transcobalamin II in human seminal plasma.

Study of cobalamin-binding proteins revealed seminal plasma to be the most concentrated site of transcobalamin II in man. The next richest normal fluid, blood, has approximately one-tenth its concentration. Normal seminal unsaturated cobalamin-binding capacity averaged 15,030 +/- 7,290 pg/ml, of which 11,550 +/- 6,660 pg/ml was transcobalamin II. Transcobalamin II levels were markedly diminished in subjects lacking seminal vesicles (1520-1660 pg/ml), but not after vasectomy. This suggests that seminal vesicles are the chief source of this protein in semen. R binder concentration was increased in postvasectomy subjects (9,970 +/- 4,900 pg/ml vs. 2,980 +/- 1,370 pg/ml in normals) and varied in other patients. The endogenous cobalamin content of semen was only 88-699 pg/ml, and was carried largely by R binder rather than by transcobalamin II. The function of the unusually large seminal transcobalamin II pool in reproduction is unknown, but seems unlikely to be related solely to cobalamin transport needs, at least within the male reproductive tract itself.     

Sequestration of crystalline and endogenous cobalamin by R binders down to the distal ileum in exocrine pancreatic dysfunction

It has been recently shown that crystalline cyanocobalamin in exocrine pancreatic insufficiency is sequestered by R binders down to the proximal jejunum, and that bile inhibits the binding of cobalamin to intrinsic factor. In freshly collected human bile, we have found a single type of apo R binder, with a relative molecular mass of 128 100, a molecular radius of 4.65 nm, and a mean isoelectric point of 3.72. Salivary and biliary holo R binders were incubated with normal human gastric juice and intestinal juice from healthy subjects and patients having exocrine pancreatic insufficiency. No degradation of these two holo R binders occurs with normal gastric juice and intestinal juice from patients after four hours incubation time at 37 degrees C, but a partial degradation of salivary holo R binders and a complete loss of biliary Cbl binding capacity were observed with normal intestinal juice in the same in vitro conditions. We have confirmed in vivo, using a triple-lumened tube, that a part of the salivary and biliary holo R binders remains undegraded down to the distal ileum in two patients with exocrine pancreatic insufficiency. These findings strongly suggest that the enterohepatic circulation of cobalamin is effective in healthy subjects, whereas it is partially interrupted in the patients. They provide a proof that a part of endogenous and crystalline exogenous cobalamin is sequestered to R binders down to the distal ileum, and confirm that the failure to degrade the digestive R binders is responsible for the malabsorption of crystalline cobalamin in exocrine pancreatic dysfunction.     

Plasma homocysteine in relation to serum cobalamin and blood folate in a psychogeriatric population

Abstract Plasma homocysteine, serum cobalamin and blood folate were analysed in 296 consecutive patients referred to a psychogeriatric department for diagnosis of mental disease. Plasma homocysteine correlated with positive significance with age and serum creatinine, and with negative significance with serum cobalamin and blood folate. Approximately 35–40% of the patients with low serum cobalamin (<150 pmol l^-1) or low blood folates (<150 nmol l^-1) exhibited normal values of plasma homocysteine (<19·9 μmol l^-1). Possibly, these patients do not have a deficiency of the vitamins in the tissue. At least 7·5% of the patients with serum cobalamin levels (>150 pmol l^-1) showed an inexplicably increased level of plasma homocysteine. These patients might have a deficiency of tissue cobalamin despite the normal serum cobalamin levels. The effect of cobalamin supplementation for 7–10 days on plasma homocysteine was tested in 62 patients with different levels of serum cobalamins. We found the most pronounced decrease of plasma homocysteine in the patients with lowest serum cobalamin levels and in the patients with highest plasma homocysteine, indicating that the percentage decrease of the initial concentration of plasma homocysteine could reflect the degree of cobalamin deficiency. Folate supplementation for 7–10 days reduced plasma homocysteine not only in patients with folate deficiency but also in those with a normal folate status, and even in patients with cobalamin deficiency. The latter patients further reduced their plasma homocysteine after additional cobalamin treatment. The findings in this study support the view that, in elderly patients with co-existing organic brain disease, cobalamin substitution should be started when serum cobalamin concentrations are below 200 pmol l^-1 and plasma homocysteine is elevated, to prevent further organic lesions related to cobalamin deficiency.