Intramuscular autotransplantation of pancreatic islets in a 7-year-old child: a 2-year follow-up.

A 7-year-old girl with severe hereditary pancreatitis underwent total pancreatectomy. A total of 160,000 islet equivalents (6400 islet/kg) were transplanted to the brachioradialis muscle of the right forearm. Her plasma C-peptide level was undetectable after pancreatectomy but increased to 1.37 ng/mL after 17 days; at this time point, her insulin requirement was 0.75 units of insulin/kg/day. At 5- and 27-months, her hemoglobin A1c (HbA1c) and insulin requirements were 4.5 and 5.3% and 0.3 and 0.18 units/kg/day, respectively. Basal and stimulated C-peptide levels were 0.67 +/- 0.07 and 3.36 +/- 1.37 ng/mL, respectively. Stimulated insulin levels were 30% higher in the islet-bearing arm compared to the contralateral arm after glucagon stimulation. After surgery and islet transplantation, the quality of life improved dramatically and she gained 8 kg of weight. In summary, a normal HbA1c, a low insulin requirement and the absence of recurrent hypoglycemia and the gradient of insulin between the arms indicate that the intramuscularly transplanted islets contribute to a long-term clinically significant metabolic control.

Hydrolysis of milk fat globules by pancreatic lipase. Role of colipase, phospholipase A2, and bile salts.

Human milk fat globules require colipase to be hydrolyzed by pancreatic lipase in the presence of bile salts. This is contrary to a recent report in this Journal (J. Clin. Invest. 67: 1748-1752.) according to which inhibition of lipase by bile salt could be overcome by the addition of colipase or phospholipase A2. This latter finding is shown to be due to contamination of commercially available pancreatic phospholipase A2 by colipase.

Mode of action of tetrahydrolipstatin: a derivative of the naturally occurring lipase inhibitor lipstatin.

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme.substrate.inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the beta-lactone reacting with the active site of the enzyme.

The temperature-dependent interfacial inactivation of porcine pancreatic lipase. Effect of colipase and bile salts.

This paper confirms and extends the previous observation that colipase and bile salts stabilize pancreatic lipase against inactivation at its water/substrate interface. It is shown that colipase and bile salts above their critical micellar concentration offer better protection than either of them alone. Colipase has no effect on the catalytic efficiency of lipase against an emulsified substrate in the absence or presence of bile salts. Its reported activation of pancreatic lipolysis at high temperatures in the absence of bile salts is, most likely, fully explained by its protective effect on lipase inactivation. Colipase at high concentrations relative to lipase inhibits the enzyme activity in a competitive fashion. The temperature-dependent surface inactivation of lipase has certain consequences for the methodology of lipase activity determination.

Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat.

Tetrahydrolipstatin (THL) derived by hydrogenation from lipstatin, a lipase inhibitor produced by Streptomyces toxytricini, has been shown to inhibit in vitro the activity of all three lipases secreted to the gastro-intestinal tract; gastric lipase, pancreatic lipase and carboxylester lipase (cholesterol ester hydrolase). The effects of THL on intestinal absorption of fat (transport to the thoracic duct chyle) has now been investigated after intraduodenal infusion in a rat model. Absorption of label from oleic acid when administered with monoolein in micellar bile salt solution was not affected by THL in concentrations up to 10(-4) M calculated on the volume of the aqueous phase. Absorption of free cholesterol in micellar bile salt solution of the lipolytic products of triolein; oleic acid and monoolein, is not significantly affected at a concentration of THL of 10(-4) M. Absorption of cholesterol from cholesteryl oleate under the same conditions is almost completely inhibited. The results indicate that absorption of free cholesterol is not dependent on the activity of pancreatic cholesterol ester hydrolase. The absorption of emulsified triolein was not significantly affected by 10(-5) M THL but decreased to around 30% of the controls by a concentration 10-times higher. There was no significant decrease of cholesterol absorption when administered in emulsified triolein while absorption of cholesteryl oleate was reduced at both concentrations of THL and almost completely at 10(-4) M. Radioactivity from [2-14C]THL when administered emulsified in triolein was recovered in urine, bile and thoracic duct lymph to 10-14, 8-13 and 1-3%, respectively, largely independent on dose administered. Label from [1"-14C] THL was recovered in the same amounts in lymph but much less in bile and urine indicating that the amino acid moiety has been split off early in the absorption process.

One-step purification of procolipase from human pancreatic juice by immobilized antibodies against human colipase86.

Purified antibodies to human colipase86 were coupled to CNBr-activated Sepharose 4B. The immunoadsorption column thus obtained was used to purify procolipase from human pancreatic juice in one step by immunoaffinity chromatography. A single form of procolipase was obtained, having similar biological properties as previously characterized procolipases from horse and pig. The sequence of the N-terminal propeptide was determined to be Ala-Pro-Gly-Pro-Arg. In bovine, equine and porcine procolipases the corresponding N-terminal sequence is Val-Pro-Asp-Pro-Arg.

On the binding of bile salt to pancreatic lipase.

The binding of taurodeoxycholate to pancreatic lipase and a few other proteins has been studied with equilibrium dialysis and in gel filtration experiments. A three compartment dialysis cell has been used; with this cell, complete equilibration is not necessary for calculation of the binding even at bile salt concentrations above the critical micellar concentration. The results indicate that taurodeoxycholate does not bind to lipase below the critical micellar concentration, that the binding starts in the critical micellar concentration range of the bile salt and reaches around 12 mol taurodeoxycholate per mol of lipase at taurodeoxycholate concentrations well above the critical micellar concentration. Previous results indicating a binding of maximally 1-2 mol taurodeoxycholate/mol lipase were too low, depending on the experimental conditions in which complete equilibration was not obtained. The binding isotherm for taurodeoxycholate to lipase is similar to that for co-lipase; colipase and lipase in mixture bind as much taurodeoxycholate as the sum for the single proteins. Taurodeoxycholate binds to ribonuclease and chymotrypsinogen to a similar extent as to lipase.

Purification and characterization of human pancreatic colipase.

Two colipases, named colipase I and colipase II, have been isolated from extracts of human pancreatic gland. The two proteins can be separated by ion-exchange chromatography, isoelectric focusing and slab technique gel electrophoresis. The result of this study indicates that the two colipases, both of which are glycoproteins, have identical amino acid compositions. The pI values were found to be 6.1 for colipase I and 5.8 for colipase II. The different colipases have also been found in human pancreatic juice. The N-terminal amino acid was glycine for both colipase I (gland) and colipase II (juice). Only minor differences were found between the colipases isolated from gland and juice, and colipase I from gland alone was examined in detail.

Evolutionary studies on pancreatic colipase.

In this evolutionary study the following criteria have been used to prove the existence of colipase: 1. It restores the activity of human and porcine pancreatic lipase inhibited by bile salt. 2. It cross-reacts with antisera to human and porcine colipases. 3. Its restoration of lipase activity, inhibited by bile salt, in the tributyrin assay system, is prevented by antiserum to colipase. 4. It is a heat-stable, low-molecular-weight protein (molecular weight about 10 000 by gel-filtration). The occurrence of colipase has been verified in the exocrine pancreatic cells from hagfish (Myxine glutinosa), ratfish (Chimaera monstrosa), rayfish (Raja radiata). Greenland shark (Somnius microcephalus) and dogfish (Squalus acanthius). No colipase activity could be found in the gastric juice of crayfish (Pacifastacus leniusculus). These results indicate that colipase envolved in the vertebrates before the organized exocrine pancreatic gland and occurred simultaneously with the bile salts/bile alcohols.

Is there a specific lysophospholipase in human pancreatic juice?

The existence of a specific lysophospholipase in human pancreatic juice was evaluated. The proteins were separated by a series of chromatographic steps including Sephacryl S-200, cholate-Sepharose 4B, Sephadex G-100 and CM-Sephadex G-50. The enzyme activities against 1-palmitoyl lysolecithin (LL) as well as tributyrin (TB) and p-nitrophenyl butyrate (PNPB) were determined in all the fractions of these purification procedures. Enzyme activity against LL was always eluted in parallel with activities against TB and PNPB, and no unique activity against LL could be found. The specific activity against LL was 40-times lower than that against PNPB and 200-times lower than that against TB. It is concluded that there is no unique lysophospholipase in human pancreatic juice and that the hydrolysis of lysolecithin is most likely performed by carboxyl ester lipase.

Concerted action of human carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro: importance of the physicochemical state of the substrate.

The pancreatic enzyme carboxyl ester lipase (CEL) has been shown to hydrolyse a large number of different esters, including triacylglycerols, cholesteryl esters and retinyl esters with an absolute requirement for bile salts. Some of the lipids that are substrates for CEL can also be hydrolysed by pancreatic lipase. In order to investigate the relative roles of human CEL and pancreatic lipase, the two enzymes were incubated on a pH-stat with isotope-labelled lipid substrate mixtures in physicochemical forms resembling the state of the dietary lipids in human intestinal contents. In the first set of experiments, cholesteryl oleate (CO) and retinyl palmitate (RP) were solubilised in an emulsion of triolein (TO) stabilised by egg phosphatidylcholine and bile salts. Lipase (always added together with its cofactor, colipase) hydrolysed TO, with monoolein and oleic acid as end-products, whereas CEL alone could not hydrolyse TO in the presence of phosphatidylcholine (PC). Lipase alone did not hydrolyse CO or RP, but CEL did hydrolyse these esters if lipase was present. Release of [3H]glycerol from labelled TO increased only slightly if CEL was added compared to lipase alone, suggesting that monoolein hydrolysis was slow under these conditions. In the second set of experiments, CO and RP were dissolved in bile salt/monoolein/oleic acid dispersions with varying bile salt concentrations. CEL hydrolysed CO and RP more rapidly in a system with a high bile salt concentration containing mixed micelles than in a system with a low bile salt concentration, where the lipids were dispersed in the form of mixed micellar and non-micellar aggregates; both types of aggregate have been reported to exist in human intestinal contents. In conclusion, these data suggest that the main function of CEL under physiological conditions is to hydrolyse cholesteryl and retinyl esters, provided that the triacylglycerol oil phase is hydrolysed by pancreatic lipase, which probably causes a transfer of the substrate lipids of CEL from the oil emulsion phase to an aqueous bile salt/lipolytic product phase. Depending on the bile salt/lipolytic product ratio, the substrate will reside in either micellar or non-micellar lipid aggregates, of which the micellar state is preferred by CEL.

Lipase-colipase interactions during gel filtration. High and low affinity binding situations.

The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.

Factors regulating the formation of chylomicrons and very-low-density lipoproteins by the rat small intestine.

The aim of this study was to investigate how the relationship between chylomicron and very-low-density lipoprotein (VLDL) transport of fatty acid into lymph was affected by the total amount of lipid transported via the intestinal lymphatics in the rat. Two different experimental conditions were employed. First, intestinal lymph fistula rats were infused with four different levels of [3H]oleic acid (15, 30, 60 and 120 mumol per h) at a constant rate for 8 h. Lymphatic transport of [3H]oleic acid via chylomicrons and VLDLs was measured in lymph collected during the seventh h. Within the dose range studied chylomicron increased exponentially, while the output in VLDL reached a plateau at a total lymph [3H]oleic acid output of approx. 60 mumol/h. A linear regression analysis of the ln(chylomicron/VLDL) versus the total output in lymph yielded a coefficient of correlation of 0.95. Second, we utilized the fact that intraduodenal infusion of the nonionic detergent Pluronic L-81 (L-81) inhibits chylomicron transport and that this inhibition is reversed by the cessation of L-81 infusion (unblocking). A linear regression analysis of the ln(chylomicron/VLDL) versus total lymph [3H]oleic acid output during the first 4 h of unblocking gave a coefficient of correlation of 0.79. Statistical analysis of the regression equations from the two experiments showed that for the same lymphatic [3H]oleic acid output, the chylomicron/VLDL ratio was significantly lower in the L-81 experiment, indicating that the relative rates of formation of chylomicron to VLDL were different under these two experimental conditions. However, the principal pattern was the same, i.e., chylomicron production increased, while VLDL production became saturated when the amount of oleic acid transported to the lymph was increased.

Effect of pancreatic phospholipase A2 and gastric lipase on the action of pancreatic carboxyl ester lipase against lipid substrates in vitro.

Preincubation of a triolein/phospholipid/cholesteryl oleate-emulsion in vitro with either pancreatic phospholipase A2 (PLA2) or gastric lipase (GL) resulted in hydrolysis (measured by pH-stat-titration) of cholesteryl [3H]oleate only after human pancreatic carboxyl ester lipase (CEL) was added to the system. No appreciable hydrolysis was observed when CEL was added alone. Consequently, a concerted action either of PLA2 and CEL or of GL and CEL made the substrate cholesteryl oleate available for hydrolysis by CEL. This was the case when cholesteryl oleate was solubilised in a phospholipid-stabilised triglyceride emulsion, which is the physico-chemical form in which the major part of dietary cholesteryl esters are presented to the gastro-intestinal tract of man.

Lipid binding and activating properties of porcine pancreatic colipase split at the Ile79-Thr80 bond.

Porcine colipase, the protein cofactor of pancreatic lipase, was isolated from pancreas freshly collected on animals and from a side fraction from the production of insulin (Novo Nordisk A/S). Samples of purified colipase were analyzed for homogeneity by polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography (RPLC), quantitative N-terminal sequence determination and mass spectrometry. The activating properties of colipase preparations were assayed against tributyrin, triolein or the commercial Intralipid emulsion, in presence of bile salt. Two fractions of colipase with the same specific activity were purified from fresh pancreas. The major fraction (85%) contained one single protein corresponding to fragment 1-93 of the 95-residue form of colipase (procolipase) previously characterized in porcine pancreatic juice. The other fraction (15%) corresponded to fragment 1-91 of procolipase. Also, two fractions of colipase were purified from the side fraction supplied by Novo. These fractions consisted of the 95-residue proform of colipase and of fragment 1-93, respectively, both specifically cleaved at the Ile79-Thr80 peptide bond with partial removal of isoleucine at position 79 and serine at position 78. Procolipase split at the 79-80 bond retained full activity on tributyrin and triolein and on the Intralipid emulsion but the kinetics of hydrolysis of triacylglycerol substrates showed much longer lag periods than those observed with native procolipase. Also, all forms of procolipase split at the 79-80 bond showed one peak in RPLC but their retention time was markedly decreased as compared to that of native procolipase which indicated a weaker hydrophobic binding capacity. The value of the retention time was of the same order of magnitude as that of inactive reduced procolipase. Treatment of native procolipase by pancreatic endopeptidases showed that elastase is likely responsible for specific cleavage at the 79-80 bond of procolipase purified from the Novo extract. Limited proteolysis by trypsin of the proforms of colipase split at the 79-80 bond reduced the lag period. Results presented in this communication provide the first direct evidence showing that the finger-shaped peptide segment between half-cystine residues at positions 69 and 87 is involved in colipase-lipid interaction as previously hypothesized from the three-dimensional structure of the protein.

The primary sequence of human pancreatic colipase.

The amino acid sequence of an activated colipase purified from human pancreas was determined. The protein consists of a single polypeptide chain of 86 amino acids (human colipase86) and has a molecular weight of 9289. The sequence was determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of two CNBr peptides. Sequence determination of porcine procolipase II was also performed, which showed that in the original sequence determination apparently two residues were missed. These residues were determined to be a leucine at position 37 and a serine in position 50. For comparison with porcine and equine procolipases, the residues composing human colipase are numbered from 6 to 91. No human procolipase has been isolated so far. The colipases from man, pig, horse and chicken show a high degree of homology: human colipase differs from the other proteins by substitutions of 19 (porcine), 24 (equine A) and 21 (equine B) residues, respectively.

Adverse health events and late mortality after pediatric allogeneic hematopoietic SCT-two decades of longitudinal follow-up.

Treatment-related late toxicities after pediatric allogeneic hematopoietic SCT (allo-HSCT) are increasingly important as long-term survival has become an expected outcome for many transplanted children and adolescents. In a retrospective cohort study, we assessed long-term health outcomes in 204 allo-HSCT survivors transplanted in childhood or adolescence (<20 years) between 1978 through 2000 after a median follow-up time of 12 (range 4-28) years. Data on conditioning regimen, adverse health events (AE) and growth and hormonal substitutions (hormone replacement therapies (HRTs)) were obtained from medical records. AEs were graded retrospectively according to Common Terminology Criteria for Adverse Events v3.0. Late deaths (⩾48 months after allo-HSCT) were evaluated separately. Multivariate analysis demonstrated that chronic GVHD (P<0.000) and longer follow-up time (P<0.05) correlated with AEs, whereas CY-based conditioning was inversely correlated (P<0.002). TBI and longer follow-up duration predicted more severe AEs (P<0.001 and P<0.001, respectively). HRTs were more frequent after TBI. Diabetes type II, dyslipidemia and hypertension were detected in 9, 7 and 7% of the survivors, respectively. Late deaths (n=22) were most frequently due to pulmonary failure (n=7), followed by secondary malignancy (n=5). The occurrence of AEs after pediatric allo-HSCT is high and likely to increase during extended follow-up, particularly in patients who have received TBI.

Mutations in pea seedborne mosaic virus genome-linked protein VPg after pathotype-specific virulence in Pisum sativum.

Pisum sativum plant introduction (PI) line 269818 is resistant to potyvirus pea seedborne mosaic virus (PSbMV) isolates, categorized as pathotype P1, and is susceptible to pathotype P4 isolates. This difference in infectivity is determined by the viral genome-linked protein (VPg) cistron. Mutational analysis of VPg of PSbMV isolates DPD1 and NY representing pathotypes P1 and P4 revealed that codon changes affecting amino acids 105 to 117 in the central region of VPg influenced virulence on PI 269818. In contrast, infectivity on pea cultivar Dark Skinned Perfection, which is susceptible to both pathotypes, was not affected by the mutations. Mutants overcoming resistance in PI 269818 were analyzed for changes in the VPg coding region upon passage through PI 269818 and Dark Skinned Perfection. Adaptive changes were observed only upon passage through PI 269818 and only at codons from amino acid 105 to 117. Expression of DPD1 VPg in PI 269818 did not affect infection by NY, which suggests that VPg from DPD1 is not an elicitor of a general resistance response. The results are compatible with the hypothesis that viral amplification depends upon the interaction between VPg and a host factor.

Thyroid function in children after allogeneic bone marrow transplantation.

Thyroid function was investigated in 35 children after allogeneic BMT. The study was longitudinal and all patients were followed for at least 5 years. Once a year TSH, T4, T3 and the TRH test were performed. Patients with severe aplastic anemia (n = 6) were transplanted without total body irradiation (TBI) and they had no detectable alterations in thyroid function. Patients with leukemia (n = 27) were conditioned with 10 Gy TBI in one fraction. The accumulated frequencies of thyroid dysfunction were 3 of 27 (11%) with high TSH and low T3 or T4 levels, and 10 of 27 (37%) with high basal TSH and normal T3 and T4 levels. An additional 11 of 27 (41%) had an exaggerated TSH response in the TRH test and normal basal TSH and T3/T4 levels. Only 3 of 27 (11%) continued to have normal values. Treatment with levo-thyroxine (L-T4) was given to the patients with a high basal TSH level. As 24 of 27 (89%) children had signs of disturbance in the thyroid axis, prophylactic L-T4 treatment for a few years after BMT with TBI may be of value. The main cause of a change in thyroid function after BMT seems to be conditioning with TBI.

Phosphatidylcholine as substrate for human pancreatic phospholipase A2. Importance of the physical state of the substrate.

The long-chain phosphatidylcholine/sodium cholate aqueous system as substrate for human pancreatic phospholipase A2 (PLA2) was investigated. At a constant phosphatidylcholine (PC) concentration of 8 mM, the enzyme activity increased with a decrease in cholate (C) concentration up to a PC/C ratio of approximately 0.8 and then rather abruptly decreased to lower values at a ratio above 1.5. At ratios between 0.8 and 1.5, an increasing lag phase in the PLA2 activity was seen, indicating a progressive decrease in substrate availability to the enzyme. Reaction mixtures with a PC/C ratio of up to 0.67 were optically clear solutions composed of mixed bile salt/PC micelles of increasing mixed micellar aggregate size. Ratios between 0.67 and 1.5 were characterized by an increase in turbidity (at 330 and 450 nm) due to increasing formation of vesicles or liposomes. Above a PC/C ratio of 1.5, a sharp increase in turbidity was seen due to increasing formation of bilayer structures other than vesicles. Pure vesicles obtained by dialysis of mixed micellar solutions were not hydrolyzed by the enzyme. Addition of bile salts reversed the inhibition which was accompanied by a decrease in turbidity. Phosphatidylcholine was preferred as substrate for human PLA2 when present in large mixed disc-like bile salt micelles. Vesicular or other types of lamellar liquid-crystalline phases of long-chain phosphatidylcholine did not serve as substrate for PLA2.