PET imaging of patients with non-small cell lung cancer employing an EGF receptor targeting drug as tracer.

BACKGROUND: We have previously developed (11)C-erlotinib as a new positron emission tomography (PET) tracer and shown that it accumulates in epidermal growth factor receptor (EGFR)-positive lung cancer xenografts in mice. Here, we present a study in patients with non-small cell lung cancer (NSCLC) investigating the feasibility of (11)C-erlotinib PET as a potential method for the identification of lung tumours accumulating erlotinib. METHODS: Thirteen patients with NSCLC destined for erlotinib treatment were examined by contrast-enhanced computed tomography (CT), (11)C-erlotinib PET/low-dose CT and (18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) PET/low-dose CT before start of the erlotinib treatment. After 12 weeks treatment, they were examined by (18)F-FDG PET/contrast-enhanced CT for the assessment of clinical response. RESULTS: Of the 13 patients included, 4 accumulated (11)C-erlotinib in one or more of their lung tumours or lymph-node metastases. Moreover, (11)C-erlotinib PET/CT identified lesions that were not visible on (18)F-FDG PET/CT. Of the four patients with accumulation of (11)C-erlotinib, one died before follow-up, whereas the other three showed a positive response to erlotinib treatment. Three of the nine patients with no accumulation died before follow-up, four showed progressive disease while two had stable disease after 12 weeks of treatment. CONCLUSION: Our data show a potential for (11)C-erlotinib PET/CT for visualizing NSCLC lung tumours, including lymph nodes not identified by (18)F-FDG PET/CT. Large clinical studies are now needed to explore to which extent pre-treatment (11)C-erlotinib PET/CT can predict erlotinib treatment response.

Increased prevalence of autoimmunity in Turner syndrome--influence of age.

Individuals with Turner syndrome (TS) are prone to develop autoimmune conditions such as coeliac disease (CD), thyroiditis and type 1 diabetes (T1DM). The objective of the present study was to examine TS of various karyotypes for autoantibodies and corresponding diseases. This was investigated in a prospective cross-sectional study of Danish TS patients (n = 107, median age 36.7 years, range: 6-60 years). A medical history was recorded and a blood sample was analysed for autoantibodies against gliadin, transglutaminase, adrenal cortex, intrinsic factor, anti-thyroid peroxidase (anti-TPO) and glutamic-acid-decarboxylase 65 (GAD-65). Autoantibodies were present in 58% (n = 61) of all patients, whereof 18% (11) had autoantibodies targeting more than one organ. Patients with autoantibodies were significantly older than those without (P = 0.001). Anti-TPO was present in 45% (48) of patients, of whom 33% (16) were hypothyroid. Overall, 18% (19) presented with CD autoantibodies, of whom 26% (five) had CD. Anti-TPO and CD autoantibodies co-existed in 9% (10). Immunoglobulin A deficiency was found in 3% (three) of patients, who all had CD autoantibodies without disease. Among four patients with anti-GAD-65 none had T1DM, but two were classified as having T2DM. One patient had adrenocortical autoantibodies but not adrenal failure. Autoantibodies against intrinsic factor were absent. Anti-GAD-65 was increased in isochromosomal karyotypes (3/23 versus 1/84, P = 0.008) with no other association found between autoantibodies and karyotype. In conclusion, TS girls and women face a high prevalence of autoimmunity and associated disease with a preponderance towards hypothyroidism and CD. Thus, health care providers dealing with this patient group should be observant and test liberally for these conditions even before clinical symptoms emerge.

Early changes in vitamin B12 uptake and biomarker status following Roux-en-Y gastric bypass and sleeve gastrectomy.

BACKGROUND & AIMS: Bariatric surgery increases the risk of micronutrient deficiencies, including vitamin B12 (B12) deficiency. We analysed early changes in biomarkers of B12 status following bariatric surgery. METHODS: We prospectively included adult patients (n = 27) referred for either Roux-en-Y Gastric Bypass (RYGB) (n = 19) or Sleeve Gastrectomy (SG) (n = 8). Blood samples were drawn before surgery and 2 and 6 months following surgery for measurement of B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA). The B12 absorption capacity was estimated from the increase in plasma holoTC two days after a standardised oral B12 challenge. RESULTS: B12 status decreased following both RYBG and SG. While a decrease in plasma B12 was not evident until 6 months postoperatively, we observed a statistically significant decrease in plasma holoTC and increase in MMA already 2 months postoperatively. These changes were more pronounced at 6 months post surgery. Correspondingly, the B12 absorption capacity was decreased following surgery. CONCLUSIONS: HoloTC and MMA were superior to B12 to detect early changes in B12 status following bariatric surgery. Our data challenge the current concept that liver B12 stores secure long-term maintenance of B12 status. They indicate that B12 treatment in pharmacological doses may be warranted immediately after surgery.

A longitudinal study of serum cobalamins and its binding proteins in lactating women.

OBJECTIVE: To examine longitudinal changes in serum cobalamins, transcobalamin (TC) and haptocorrin (HC) during lactation and to investigate the influence of vitamin B12 supplementation on these parameters. DESIGN: A 9-month follow-up study. SUBJECTS AND METHODS: Lactating mothers (N=89) including 23 supplemented with vitamin B12 (1-18 microg/daily), 41 partly supplemented and 25 not supplemented. Blood samples collected 3 weeks (baseline) and 4 and 9 months post-partum were analysed for cobalamins, TC and HC. Both the total concentration and the cobalamin-saturated form (holo) of TC and HC were analysed. RESULTS: No significant differences were observed in serum cobalamins or its binding proteins related to supplementation with vitamin B12 or the duration of lactation. Serum cobalamins remained unchanged from 3 weeks to 9 months post-partum. Total TC (holoTC) (median+/-s.e. pmol/l) decreased between 3 weeks (710+/-23 (85+/-12)) and 9 months (602+/-21 (76+/-11)) (P<0.0001 (P=0.0002)), whereas total HC (holoHC) increased from (422+/-11 (300+/-9)) at 4 months to (455+/-13 (317+/-10)) to 9 months post-partum (P<0.0001 (P<0.0001)). CONCLUSION: We report a decrease in TC and an increase in HC during a 9-month period post-partum. No differences were observed between the vitamin B12-supplemented and the unsupplemented groups. Thus, supplementation with vitamin B12 has no impact on the circulating level of serum cobalamins or its binding proteins in a Danish population of lactating mothers.

Systemically administered trefoil factors are secreted into the gastric lumen and increase the viscosity of gastric contents.

BACKGROUND AND PURPOSE: Trefoil factors (TFFs) secreted by mucus-producing cells are essential for the defence of the gastrointestinal mucosa. TFFs probably influence the viscoelastic properties of mucus, but this has not been demonstrated in vivo. We therefore studied the gastric secretion of systemically administered TFF2 and TFF3, and their influence on the viscosity of the secretions. EXPERIMENTAL APPROACH: Mice and rats under general anaesthesia were injected intravenously with human (h) TFF2, hTFF3 (5 mg kg(-1) to mice and 25 mg kg(-1) to rats), murine (m) (125)I-TFF3, or (125)I-hTFF3 (300,000 cpm, mice only). The appearance of TFFs in the gastric mucosa and luminal secretions was analysed by autoradiography, gamma-counting, and ELISA, and the viscosity by rheometry. KEY RESULTS: (125)I-mTFF3 and (125)I-hTFF3 were taken up by secretory cells of the gastrointestinal tract and detected at the gastric mucosal surface 15 min after injection. Stressing the stomach by carbachol (3.5 microg kg(-1)) and pyloric ligation significantly increased the uptake. Injected hTFF2, hTFF3, and mTFF3 were retrieved from the gastric contents after 4 h. In rats, an approximately seven-fold increase in the viscosity was detected after injection of TFF2 compared to the controls, whereas TFF3 did not increase the viscosity. In mice, TFF2 increased the viscosity approximately 4-fold. CONCLUSIONS: These data indicate that systemically administered TFFs are transferred to the gastric lumen in a biologically active form.

Folate and vitamin B12 in relation to lactation: a 9-month postpartum follow-up study.

OBJECTIVE: To investigate the relation between lactation and markers of folate and vitamin B12 (B12) deficiency in women with and without vitamin supplementation. DESIGN: A 9-month follow-up study. SUBJECTS AND METHODS: Blood samples from 91 women, who gave birth to a single healthy child, were collected 3 weeks, 4 and 9 months postpartum and analysed for circulating level of homocysteine (tHcy), methylmalonic acid (MMA), folate and B12. The participants were categorized as exclusively, partly or not breast-feeding dependent on the degree of lactation 4 months postpartum. During follow-up, lifestyle factors were recorded by structured interviews. RESULTS: Among 72 exclusively breast-feeding women, the median (10-90% percentile) tHcy was 5.8 (3.1-8.3) micromol/l 3 weeks postpartum, 6.1 (4.1-10.3) micromol/l 4 months postpartum and 5.3 (3.6-8.7) micromol/I 9 months postpartum. At 9 months postpartum, none of the women breast-fed exclusively. No significant change occurred in the concentration of B12 and folate. Exclusively breast-feeding women without vitamin supplementation had higher median tHcy than supplemented exclusively breast-feeding women 4 and 9 months postpartum (7.0 vs 5.4 micromol/l (P < 0.001) and 5.8 vs 4.5 micromol/l (P = 0.003), respectively). Six women had increased (>15 micromol/l) tHcy; four of these were unsupplemented and exclusively breast-feeding. CONCLUSION: We found no overall indication of depletion of the folate and B12 stores during the lactation period in this population. However, folate-supplemented women had lower tHcy and higher folate levels, suggesting a beneficial effect of supplementation with folate throughout lactation.

A Mr 43,000 epidermal growth factor-related protein purified from the urine of breast cancer patients.

A tumor-associated epidermal growth factor (EGF)-like activity was detected in the urine of breast cancer patients by means of an EGF radioreceptor assay and an anchorage-independent growth assay. The clonogenic growth factor activity of pooled void volume eluate fractions from a Bio-Gel P-30 column was completely neutralized by an anti-human epidermal growth factor antiserum but not by an anti-transforming growth factor alpha antiserum. This activity was determined in the urine of 71 breast cancer patients. A statistically significant correlation was found between EGF-like clonogenic activity and axillary lymph node status, tumor size, stage of disease, and grade of differentiation of the primary tumor. The Bio-Gel P-30 void volume fraction was used to purify the EGF-related polypeptide to apparent homogeneity by subsequent binding to and elution from A431 cells followed by isoelectric focusing. A polypeptide of a pI of approximately 3.4 was identified to be related to EGF by neutralization and immunoprecipitation experiments with anti-human epidermal growth factor antisera. This polypeptide migrated as a single band of Mr 43,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Partial purification of transforming growth factors from human milk.

Crude, delipidated milk and the acid:ethanol extracts of primary human breast tumors contain several activities that biologically resemble transforming growth factors (TGFs) in that they promote the anchorage-independent growth of normal rat kidney and Mm5mt/c1 mouse mammary tumor cells in soft agar. Three major TGF species with isoelectric points (pl) of about 4.0, 6.0-6.5, and 7.0 have been detected in both tumors and milk. The pl 4.0 species from milk has been purified about 10,000-fold by isoelectric focusing and high-performance liquid chromatography. This species, designated milk-derived growth factor II (MDGFII), coelutes from gel filtration columns with an authentic human epidermal growth factor standard when using a low ionic strength eluting buffer. However, on the same column, MDGFII is completely resolved from human epidermal growth factor with high ionic strength eluting buffers. Nevertheless, MDGFII purified by the latter technique still competes with 125I-epidermal growth factor for receptor binding to A431 cell membranes. Additionally the TGF activity of MDGFII present in the pl 4.0 fraction of milk is markedly inhibited by anti-epidermal growth factor receptor antibody preparations. Consequently MDGFII appears to be an alpha-TGF. MDGFII is a pepsin-sensitive, disulfide reducing agent-sensitive, heat-stable protein that may be physiologically important for the mammary gland or the neonate.

A subclass of HER1 ligands are prognostic markers for survival in bladder cancer patients.

Members of the epidermal growth factor (EGF) family have been suggested as prognostic markers in patients with bladder cancer. Thus far, there has been no consensus on their usefulness. We report an analysis of six ligands and two receptors of which a subset correlate to tumor stage and survival. Biopsies from bladder cancer tumors were obtained from 73 patients followed for a median of 28 months. The mRNA content for six ligands [EGF, transforming growth factor alpha (TGF-alpha), amphiregulin (AR), betacellulin (betaCL), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EPI)] and two receptors [EGF receptor I Human EGF Receptor (HER1) and 2 (HER2)] was examined by a newly developed quantitative reverse transcription-PCR method. Five ligands and two receptors (HER1 and HER2) were present in median concentrations of (10(-21) mol/microg RNA) 0.39 (AR), 11 (betaCL), 2.4 (EPI), 40 (HB-EGF), 1.4 (TGF-alpha), 75 (HER1), and 39,000 (HER2). EGF was barely detectable. A significantly higher expression of EPI (P < 0.001), HB-EGF (P < 0.001), and TGF-alpha (P < 0.05) were observed in T2-T4 tumors as compared with Ta tumors. Especially the expression of EPI mRNA correlated strongly to survival (P < 0.0005), but increased expression of TGF-alpha (P < 0.005), AR, and HB-EGF (P < 0.02) was also associated with a reduced life span. For the first time, mRNA expression of six ligands and two receptors of the EGF family have been examined in bladder cancer tumors. Our data emphasize that members of the EGF family, especially EPI, may be potential bladder tumor markers.

Absorption and retention of free and milk protein-bound cyano- and hydroxocobalamins. An experimental study in rats.

INTRODUCTION: Cobalamin/Vitamin B12 (Cbl) is an essential vitamin, supplied mainly as hydroxocobalamin (OHCbl) by animal products, including cows' milk. Cyanocobalamin (CNCbl) is the usual form in vitamin pills. The aim was to explore absorption and tissue accumulation of two Cbl forms, administered alone or bound to milk protein. MATERIALS AND METHODS: We synthesized labeled OH[(57)Co]Cbl from commercially available CN[(57)Co]Cbl. Recombinant bovine transcobalamin (rbTC) was produced in yeast and skimmed milk obtained off the shelf. Male Wistar rats (250-300 g) received labeled Cbl by gastric gavage. First, we administered CN[(57)Co]Cbl, free or rbTC-bound (n = 15 in each group). Rats were sacrificed after two, 24, and 48 h. In the following studies, rats were sacrificed after 24 h. We compared absorption of free or rbTC-bound CN[(57)Co]Cbl added to cows' milk and analogous absorption of OH[(57)Co]Cbl, free or rbTC-bound, to absorption of free CN[(57)Co]Cbl, (n = 10 in each group). Blood, tissues, 24-h urine and feces were collected. Labeled Cbl was measured using a gamma counter. Results are expressed as percentage of administered dose. RESULTS: Absorptions of CNCbl and OHCbl were neither influenced by rbTC-binding nor administration in milk. Absorption increased in the first 24 h with no further tissue accumulation during the subsequent 24 h. Accumulation of free CNCbl and (OHCbl) was 1.4, (4.1) (liver); 20.2, (16.4) (kidney); and 0.05, (0.02) (plasma)% 24 h after administration. Total organ accumulations were 21.6, (20.5)%. While total accumulations of CNCbl and OHCbl were equal, distributions between liver, kidney, and plasma showed significant differences (p < 0.0001; p = 0.01; p < 0.0001). CONCLUSIONS: Cbl added to milk (spiked with rbTC) has high bioavailability matching that of free Cbl. OHCbl and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated.

A new principle in biospecific affinity chromatography used for purification of cobalamin-binding proteins.

To avoid using protein-denaturing agents for desorption, when purifying cobalamin-binding protein by biospecific affinity chromatography, an affinity column has been prepared where cobalamin is attached through a temperature-labile linkage to insolubilized 3, 3'-diaminodipropylamine. On desorption the protein is obtained in solution saturated with cobalamin. The method has been used for purification of intrinsic factor and transcobalamin I.

The interaction of human transcobalamin isopeptides in cerebrospinal fluid and plasma with cobalamin and the cellular acceptor.

By isoelectric focusing, transcobalamin from human cerebrospinal fluid was separated into the phenotypes X, M, MX, SX and MS. The corresponding plasma transcobalamins were of identical phenotypes. The unsaturated cobalamin-binding capacity in the cerebrospinal fluid was 0.12-0.54 nmol.1(-1), median 0.23 nmol.1(-1); no difference in binding capacity was found between the individual phenotypes. The isopeptides M, X and S bound cyano[57Co]cobalamin from pH 6 to 10. The apparent affinity constant was the same for all the isopeptides (0.4.10(12) l.mol-1, pH 7.4). The isopeptide-cobalamin complexes bound to acceptors on human placenta membranes with an apparent affinity constant of 11.10(9) l.mol-1, pH 7.4.

Changes in the ultraviolet and circular dichroism spectra of aquo-, hydroxy-, azido-, and cyanocobalamin when bound to human intrinsic factor or human transcobalamin I.

The ultraviolet and the circular dichroic spectra of aquo-, hydroxy-, azido-, and cyanocobalamin free or bound to purified human intrinsic factor or purified human transcobalamin I were recorded. Except for azidocobalamin-transcobalamin I, the molar absorption for the gamma1-band in the ultraviolet spectra increased with a factor between 1.2 and 1.4 when the cobalamins were bound to protein. The ultraviolet spectrum of azidocobalamin-transcobalamin I changed so that the gamma2-band became the most intense. Minor changes were observed in the other ultraviolet spectra. The most prominent change in the CD-spectra of protein bound cobalamins was the increase in negative elipticity above 400 nm. This increase was most intense for cyano- and azidocobalamin-transcobalamin I. The study emphasizes that the mechanism by which human intrinsic factor and human transcobalamin I bind cobalamins differ.

Purification and characterization of rabbit haptocorrin.

Rabbit haptocorrin (R-binder) has been purified from serum by affinity chromatography on cobalamin-Sepharose and Blue Sepharose. It proved to be a protein with a relative molecular mass of 60 000 and an amino acid content very similar to that of other haptocorrins (Nexø, E. and Olesen, H. (1981) in B12 (Dolphin, D., ed.), Wiley Interscience, New York/London, in the press). The pH optimum (pH 6-9) for binding of cyanocobalamin and the affinity to dicyanocobinamide were like those of human and hog haptocorrins. In spectral studies, the extinction coefficient of cyanocobalamin at 363 nm (gamma 1-band) increased by about 16% on binding to rabbit haptocorrin. Binding of azidocobalamin gave spectra changes similar to those for binding to rabbit transcobalamin.

Isoelectric focusing of apo- and holo-transcobalamin present in human blood. Identification of a protein complexing with transcobalamin.

By means of isoelectric focusing we studied the factors influencing the isoelectric point of transcobalamin. Upon binding of cobalamin, the transcobalamin isopeptides M, X and S increase their isoelectric points by 0.4 pH units. Serum protein different from gammaglobulin changes the isoelectric point of transcobalamin, but the attachment of the transcobalamin-cobalamin complex to its acceptor is unchanged. Transcobalamin bound to auto-antibodies shows a characteristic pattern upon isoelectric focusing.

Structural homologies of cobalamin-binding proteins. Tryptic peptide mapping of intrinsic factor, transcobalamin and haptocorrin from man, hog and rabbit.

We have explored the structural features of cobalamin binding proteins by peptide mapping. The present report is a comparison of the radioiodinated tryptic peptides of intrinsic factor, transcobalamin and haptocorrin from man, hog and rabbit. The results show that the homology between analogous proteins from different species is close for intrinsic factor and transcobalamin and weaker for haptocorrin. The results also suggest the existence of one or more regions which, with minor changes, are conserved among all proteins investigated. This implies a common evolutionary origin for all the cobalamin binding proteins studied.

Characterization of the particulate and soluble acceptor for transcobalamin II from human placenta and rabbit liver.

We describe in both human placenta and rabbit liver membranes specific acceptors which bind the human transcobalamin II-vitamin B-12 (cobalamin) complex with an affinity of 2.3 . 10(9) (placenta) and 6.7 . 10(9) (liver) M-1 and which bind the rabbit transcobalamin II-cobalamin complex with an affinity of 1.1 . 10(9) (placenta) and 1.9 . 10(9) (liver) M-1, respectively. The binding requires Ca2+ and is sensitive to both 1 M NaCl and acid pH. A new ligand binding assay, based on the ability of the acceptor, but not transcobalamin II, to bind to concanavalin A, is described and is used to characterize the solubilized acceptors. The solubilized acceptors bind human transcobalamin II-cobalamin with high affinity (about 2-9 . 10(9) M-1) but do not bind free cobalamin; unsaturated transcobalamin II is bound with an affinity approximately one-third of that for transcobalamin II saturated with cobalamin. On gel filtration, the human acceptor saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 6.7 nm, whereas the free acceptor has a Stokes radius of 5.1 nm. The rabbit liver acceptor either unsaturated or saturated with transcobalamin II-cobalamin exhibits a Stokes radius of 5.7 nm. Both acceptors bind to lectins such as concanavalin A, wheat germ agglutinin and phytohemagglutinin, indicating their glycoprotein nature, and both acceptors can be purified approximately 30-fold by affinity chromatography on wheat germ agglutinin-Sepharose columns. The concanavalin A assay, combined with lectin-Sepharose and transcobalamin II-cobalamin-Sepharose affinity chromatography will provide for the isolation and study of pure acceptors from a variety of tissue sources.

Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor.

Human, rat and mouse epidermal growth factors (EGF) bind to the same receptor on human placenta, but the binding characteristics differ. The apparent affinity constant (KA) for human EGF is higher (15 X 10(9) l/mol) than KA for rat EGF (10 X 10(9) l/mol). Mouse EGF binds with the lowest KA (5 X 10(9) l/mol). The pH optimum differs so that human and rat EGF bind with a pH optimum of 8.0, whereas mouse EGF binds with an optimum of pH 7.4. Half maximal dissociation is 130, 50 and 25 min for human, rat and mouse EGF, respectively. The structures of human, rat and mouse EGF differ somewhat. At least 11 of the first 24 residues differ. The N-terminal sequence of rat EGF is: Ala/Ser-Gly-X-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-X-Lys-Asp-Gly-Gly-Val-X-Met-Ty r-Val -Glu.

Transcobalamin from cow milk: isolation and physico-chemical properties.

The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, reacted with Cbl even in the presence of a B12-analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displace human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.

The membrane fraction of homogenized rat kidney contains an enzyme that releases epidermal growth factor from the kidney membranes.

High levels of epidermal growth factor (EGF) are excreted in the urine and high levels of mRNA for the EGF-precursor have been demonstrated in the kidney. The EGF-precursor is a membrane bound peptide in the kidney, but little is known about the renal processing of the precursor. The present study shows that the membrane fraction of homogenized rat kidney contains an enzyme that releases immuno and receptor reactive EGF from the kidney membranes when incubated at 37 degrees C. Gel filtration shows that the EGF reactivity released from the membranes is similar to the EGF reactivity in rat urine. The EGF releasing enzyme is inhibited by the serine proteinase inhibitor aprotinin and by low temperatures (4 degrees C). The pH optimum of the reaction is pH 7.5-8.0.