The evolution and application of enzymes in the animal feed industry: the role of data interpretation.

1. Enzymes have been used commercially for nearly 40 years and save significant costs through sparing of expensive nutrients but the mechanism by which this is achieved is still debated. 2. The research focused on non-starch polysaccharidase (NSPase) enzymes is used as an example of where greater progress could have been made if the details of the work had been described more fully and the analysis of the data generated had been broader in scope and more critical. 3. Lack of standardisation of the details presented in the materials and methods has been identified as a significant barrier to meaningful retrospective analysis and thus limits advances in the understanding of the mode of action of these enzymes. 4. The identity of the enzyme employed and its activity is often lacking, and more importantly the purity is rarely disclosed. Contaminant activities which are neither listed nor assayed could play a significant role in the responses observed. 5. The dose optimum of most enzymes is often considerably higher than that employed in most studies. Thus studies claiming synergy between two 'activities' should ensure that the response is not related to each enzyme simply augmenting the dose of just one activity in the finished feed. This is a common problem, and coupled with the lack of factorial experiments to justify the presence of each enzyme in a multi-enzyme product, it is not surprising that there is still debate as to whether single or multi-enzymes are best suited poultry rations. 6. The three proposed mechanisms for NSPases (viscosity, cell wall and prebiotic) are discussed, and along with their strengths and weaknesses it is suggested that a re-evaluation of each is needed. Viscosity may have to be re-evaluated as being a function not only of the cereal being fed, but of the age of the animal as well. The cell wall theory as described is poorly modelled in vitro and hence the validity of these data is questioned. The prebiotic theory may need significant modification as it appears that the quantities of oligomers produced are insufficient to generate the additional volatile fatty acids (VFA)'s reported. It is likely that all three mechanisms play a role in the responses observed, but the prebiotic mechanism probably plays by far the most important part in low viscosity diets. 7. Future research would be improved if it considered all potential mechanisms when designing a trial. Significant failings are apparent as a result of adherence to tenets in explanation of the results. Most importantly, it should be emphasised that a hypothesis is there to be tested, not defended.

Sensitising effects of genetically modified enzymes used in flavour, fragrance, detergence and pharmaceutical production: cross-sectional study.

OBJECTIVES: The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. METHODS: Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. RESULTS: Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). CONCLUSIONS: Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays.

Prevalence of sensitization to 'improver' enzymes in UK supermarket bakers.

BACKGROUND: Supermarket bakers are exposed not only to flour and alpha-amylase but also to other 'improver' enzymes, the nature of which is usually shrouded by commercial sensitivity. We aimed to determine the prevalence of sensitization to 'improver' enzymes in UK supermarket bakers. METHODS: We examined the prevalence of sensitization to enzymes in 300 bakers, employed by one of two large supermarket bakeries, who had declared work-related respiratory symptoms during routine health surveillance. Sensitization was determined using radioallergosorbent assay to eight individual enzymes contained in the specific 'improver' mix used by each supermarket. RESULTS: The prevalence of sensitization to 'improver' enzymes ranged from 5% to 15%. Sensitization was far more likely if the baker was sensitized also to either flour or alpha-amylase. The prevalence of sensitization to an 'improver' enzyme did not appear to be related to the concentration of that enzyme in the mix. CONCLUSIONS: We report substantial rates of sensitization to enzymes other than alpha-amylase in UK supermarket bakers; in only a small proportion of bakers was there evidence of sensitization to 'improver mix' enzymes without sensitization to either alpha-amylase or flour. The clinical significance of these findings needs further investigation, but our findings indicate that specific sensitization in symptomatic bakers may not be identified without consideration of a wide range of workplace antigens.

IgE-Mediated Hypersensitivity and Desensitisation with Recombinant Enzymes in Pompe Disease and Type I and Type VI Mucopolysaccharidosis.

Enzyme replacement therapy (ERT) is important for the treatment of lysosomal storage disorders. Hypersensitivity reactions with ERT have been reported, and in these cases, desensitisation with the enzyme is necessary. Here we report the cases of 3 patients with lysosomal storage disorders, including Pompe disease and mucopolysaccharidosis type I and VI, who had IgE-mediated hypersensitivity reactions and positive skin tests. Successful desensitisation protocols with the culprit enzyme solution were used for these patients. All 3 patients were able to safely receive ERT with the desensitisation protocol.

Occupational risk factors associated with work-exacerbated asthma in Quebec.

BACKGROUND: There is limited information regarding the occupational exposures of subjects with a diagnosis of work-exacerbated asthma (WEA). OBJECTIVES: To: (1) identify potential specific occupational, chemical, biological and physical agents associated with incident cases of WEA and (2) compare these agents with occupational exposures of occupational asthma (OA) and non-work-related asthma (NWRA) cases. METHODS: Subjects were workers with work-related asthma (WRA) or NWRA referred between 2005 and 2008 to two Quebec clinics specialised in the field of WRA. Specific inhalation challenges were performed to differentiate OA from WEA. Work exposures were assessed using a detailed occupational questionnaire. Exposures to 41 chemical and biological agents were coded in a semiquantitative way according to a combination of indices for concentration in workplace air, frequency and confidence of exposure by an occupational hygienist expert in occupational exposure coding. This expert was blind to the medical status of WEA, OA or NWRA. Five physical agents were coded on a yes/no scale. RESULTS: 153 subjects were enrolled (53 WEA, 67 OA and 33 NWRA). WEA cases were significantly more exposed to ammonia, engine exhaust fumes, silica, mineral fibres, aerosol propellants and solvents, and significantly less exposed to animal derived dust and enzymes than were OA cases. Exposure to physical conditions did not differ between WEA and OA. CONCLUSIONS: Exposures associated with WEA differ from those associated with OA in this study. A proportion of subjects with WEA may suffer from low-dose irritant asthma, which remains a hypothesis to be tested.

Occupational asthma and rhinitis due to detergent enzymes in healthcare.

BACKGROUND: The use of proteolytic enzymes to improve the cleaning efficacy of washing powders was introduced in the mid 1960s. Many microbial enzymes are known to be potent respiratory sensitizers but previously there has been only one case of occupational asthma associated with workplace exposure in a healthcare worker. AIMS: To report two cases of occupational asthma associated with exposure to biological enzymes in health-care workers and related occupational cases. METHODS: Reporting of clinical case reports from three different work places. RESULTS: One case of occupational asthma and three other cases with work-related asthma or rhinitis occurred in one workplace. A single case of probable occupational asthma presented at a second workplace with another case of work-related asthma at a third workplace. Exposures occurred in areas used for cleaning medical instruments and endoscopy suites. Hygiene measurements confirmed the potential for exposure. Control measures were not in place and recognition of the hazard was missing in these workplaces. CONCLUSIONS: Detergent enzymes when used in healthcare settings should be recognized as potential respiratory sensitizers. Healthcare institutions and professional bodies that recommend the use of detergent enzymes should review their risk assessments to ensure that the most appropriate methods for preventing or reducing exposure are in place.

Enzymes, trophoblasts, and cancer: the afterlife of an idea (1924-2008).

In the early 20th century, advocacy of the enzyme therapy of cancer was primarily the work of one man, John Beard, DSc (1858-1924). He and his collaborators made a determined effort to establish this mode of therapy, especially in the years 1905 to 1911. Despite a brief flowering of international interest, Beard's efforts came to naught. During the 20th century, there was a succession of American researchers who continued to investigate this topic. This included Marshall William McDuffie, MD (1882-1945), Frank LeForest Morse, MD (1876-1953), Franklin Lloyd Shively, MD (1887-1971), and William Donald Kelley (1926-2005). In central Europe, India, and other parts of the globe, the use of pancreatic enzymes as an adjuvant treatment for cancer has become a fairly routine practice, at least among those doctors who utilize complementary and alternative medicine (CAM). It is also a well-established method for reducing inflammation and mitigating the adverse effects of cytotoxic treatment.

Evaluation of the Safety and Efficacy of a Multienzyme Complex in Patients with Functional Dyspepsia: A Randomized, Double-Blind, Placebo-Controlled Study.

Functional dyspepsia (FD) is a highly prevalent disorder having nonspecific symptoms and varied pathophysiology. Its treatment remains a challenge as therapeutic options are limited, unsatisfactory, and elusive. Thus, safety and efficacy of DigeZyme®, a proprietary multienzyme complex (MEC), was evaluated as a dietary supplement in FD patients. In this randomized, double-blind, placebo-controlled, parallel-group study, 40 patients were randomly assigned (1:1 ratio) to receive either MEC (50 mg, TID; n = 20) or placebo (n = 20) for 60 days. Reports of adverse or serious adverse events (AEs), abnormal results of vital signs, abnormal findings during physical examination, and abnormal laboratory investigations were monitored closely. Efficacy measures were change in Short-Form Leeds Dyspepsia Questionnaire (SF-LDQ), Nepean Dyspepsia Index-Short Form (NDI-SF), Visual Analog Scale (VAS), Clinical Global Impression Severity Rating Scale (CGI-S), and Glasgow Dyspepsia Severity Score (GDSS) at baseline and follow-up visits on day 15, 30, and 60. Supplementation with MEC was associated with statistically significant differences (P value ranging from .0401 to .0033) in all efficacy parameters compared with placebo. The between-group comparison also revealed that MEC supplement had a significantly greater effect (P < .001) versus placebo. No investigation product-related AEs were reported. There were no clinically significant abnormalities in physical findings and no statistically significant changes in biochemical and hematological parameters, vital signs, body weight, and body mass index observed between the two groups at baseline and follow-up visits. MEC supplementation represents an effective and safe alternative to manage dyspepsia symptoms in FD patients.

Amino acid deprivation using enzymes as a targeted therapy for cancer and viral infections.

INTRODUCTION: Amino acid depletion in the blood serum is currently being exploited and explored for therapies in tumors or viral infections that are auxotrophic for a certain amino acid or have a metabolic defect and cannot produce it. The success of these treatments is because normal cells remain unaltered since they are less demanding and/or can synthesize these compounds in sufficient amounts for their needs by other mechanisms. Areas covered: This review is focused on amino acid depriving enzymes and their formulations that have been successfully used in the treatment of several types of cancer and viral infections. Particular attention will be given to the enzymes L-asparaginase, L-arginase, L-arginine deiminase, and L-methionine-γ-lyase. Expert opinion: The immunogenicity and other toxic effects are perhaps the major limitations of these therapies, but they have been successfully decreased either through the expression of these enzymes from other organisms, recombination processes, pegylation of the selected enzymes or by specific mutations in the proteins. In 2006, FDA has already approved the use of L-asparaginase in the treatment of acute lymphoblastic leukemia. Other enzymes and in particular L-arginase, L-arginine deiminase, and L-methioninase have been showing promising results in vitro and in vivo studies.

Investigation on possible allergenicity of 19 different commercial enzymes used in the food industry.

The aim of the study was to investigate the safety to allergic patients of 19 commercially available and authority-approved enzymes used in the food industry. Enzymes produced by genetically modified organisms were included. Four hundred consecutive adult patients with a diagnosed allergy to inhalation allergens, food allergens, bee or wasp were included. All had at least one positive skin prick test to the above allergens. Skin prick testing with the 19 enzymes was performed on the forearm and if positive (in 13 patients), in vitro histamine release from blood basophils were performed. Patients with positive results in skin prick test were subsequently reinvestigated with further purified enzymes and finally challenged orally with the enzymes in a double-blind, placebo-controlled protocol. Only one reaction to a placebo challenge was seen. In some instances a positive skin prick test result or a positive histamine release was seen elicited by the enzymes, but since none of the patients were positive to any of the commercial enzymes in the subsequent oral challenges using exaggerated dosages of the enzymes compared to normal daily intake, the findings are without clinical relevance. A wide variety of enzyme classes and origins was included in the study. Because there were no allergenic findings of clinical relevance it is concluded that ingestion of food enzymes in general is not considered to be a concern with regard to food allergy.

Enzymes approved for human therapy: indications, mechanisms and adverse effects.

Research and drug developments fostered under orphan drug product development programs have greatly assisted the introduction of efficient and safe enzyme-based therapies for a range of rare disorders. The introduction and regulatory approval of 20 different recombinant enzymes has enabled, often for the first time, effective enzyme-replacement therapy for some lysosomal storage disorders, including Gaucher (imiglucerase, taliglucerase, and velaglucerase), Fabry (agalsidase alfa and beta), and Pompe (alglucosidase alfa) diseases and mucopolysaccharidoses I (laronidase), II (idursulfase), IVA (elosulfase), and VI (galsulfase). Approved recombinant enzymes are also now used as therapy for myocardial infarction (alteplase, reteplase, and tenecteplase), cystic fibrosis (dornase alfa), chronic gout (pegloticase), tumor lysis syndrome (rasburicase), leukemia (L-asparaginase), some collagen-based disorders such as Dupuytren's contracture (collagenase), severe combined immunodeficiency disease (pegademase bovine), detoxification of methotrexate (glucarpidase), and vitreomacular adhesion (ocriplasmin). The development of these efficacious and safe enzyme-based therapies has occurred hand in hand with some remarkable advances in the preparation of the often specifically designed recombinant enzymes; the manufacturing expertise necessary for commercial production; our understanding of underlying mechanisms operative in the different diseases; and the mechanisms of action of the relevant recombinant enzymes. Together with information on these mechanisms, safety findings recorded so far on the various adverse events and problems of immunogenicity of the recombinant enzymes used for therapy are presented.

Efficacy of chelation therapy to remove aluminium intoxication.

There is a distinct correlation between aluminium (Al) intoxication and neurodegenerative diseases (ND). We demonstrated how patients affected by ND showing Al intoxication benefit from short-term treatment with calcium disodium ethylene diamine tetraacetic acid (EDTA) (chelation therapy). Such therapy further improved through daily treatment with the antioxidant Cellfood. In the present study we examined the efficacy of long-term treatment, using both EDTA and Cellfood. Slow intravenous treatment with the chelating agent EDTA (2 g/10 mL diluted in 500 mL physiological saline administered in 2 h) (chelation test) removed Al, which was detected (using inductively coupled plasma mass spectrometry) in urine samples collected from patients over 12 h. Patients that revealed Al intoxication (expressed in µg per g creatinine) underwent EDTA chelation therapy once a week for ten weeks, then once every two weeks for a further six or twelve months. At the end of treatment (a total of 22 or 34 chelation therapies, respectively), associated with daily assumption of Cellfood, Al levels in the urine samples were analysed. In addition, the following blood parameters were determined: homocysteine, vitamin B12, and folate, as well as the oxidative status e.g. reactive oxygen species (ROS), total antioxidant capacity (TAC), oxidized LDL (oxLDL), and glutathione. Our results showed that Al intoxication reduced significantly following EDTA and Cellfood treatment, and clinical symptoms improved. After treatment, ROS, oxLDL, and homocysteine decreased significantly, whereas vitamin B12, folate and TAC improved significantly. In conclusion, our data show the efficacy of chelation therapy associated with Cellfood in subjects affected by Al intoxication who have developed ND.

ATTEMPTS: a heparin/protamine-based triggered release system for the delivery of enzyme drugs without associated side-effects.

A prodrug type delivery system based on competitive ionic binding for the conversion of the prodrug to an active drug has been developed for delivery of enzyme drugs without their associated toxic side-effects. This approach, termed "ATTEMPTS" (antibody targeted, triggered, electrically modified prodrug-type strategy), would permit the administration of an inactive drug and then subsequently triggered release of the active drug at the target site. The underlying principle was to modify the enzyme with small cationic species so that it could bind a negatively charged heparin-linked antibody, and the latter would block the activity of the enzyme drug until it reached the target. To provide the enzyme drug with appropriate binding strength to heparin, a cationic poly(Arg)(7) peptide was incorporated onto the enzyme either by the chemical conjugation method using a bifunctional crosslinker or by the biological conjugation method using the recombinant methodology. Methods for drug modification, heparin-antibody conjugation, and the prodrug and triggered release features of the "ATTEMPTS" approach are described in detail in this review article.

Occupational asthma and allergy in the detergent industry: new developments.

This review highlights the latest developments in the control of enzyme-induced occupational asthma and allergy (rhinitis and conjunctivitis) in the detergent industry. The industry has developed guidelines for the safe handling of enzymes in order to reduce the risk of occupational allergy and asthma. Those manufacturing facilities that follow all of the guidelines enjoy very low or no cases of asthma and allergy among workers exposed to enzymes. The key to the success of the management of enzyme-induced allergy and asthma is prospective surveillance for the development of enzyme-specific IgE antibody before the onset of allergic symptoms. This allows for continuing interventions to reduce exposures, so as to minimize or eliminate those associated with symptoms. Workers with IgE to enzymes can still continue to work in the industry symptom-free for their entire career. This indicates that exposures needed to induce sensitization are different and probably lower than exposures needed to elicit enzyme allergic symptoms. The experience of the detergent enzyme industry in controlling occupational allergens can be applied to other industries. The detergent enzyme story can be viewed as a model for the control of type 1 protein allergens in the workplace.

Efficacy of enzyme replacement therapy in Fabry disease.

Enzyme replacement therapy has recently been introduced to treat Fabry disease, a rare X-linked lysosomal storage disorder. The disease occurs due to deficient activity of alpha-galactosidase A, leading to progressive accumulation of globotriaosylceramide in multiple organs and tissues. Renal, cardiac and cerebrovascular manifestations of the disease result in premature death in both hemizygous males and heterozygous females. This paper outlines the clinical signs, symptoms and diagnosis of Fabry disease, and the development of the two available enzyme replacement therapies -- agalsidase alfa and agalsidase beta. Agalsidase alfa and agalsidase beta are produced in a human cell line and in Chinese hamster ovary cells, respectively, resulting in products with the same amino acid sequence as the native human enzyme, but with different patterns of glycosylation. Correct post-translational glycosylation is important in terms of the pharmacokinetics, biodistribution, clinical efficacy and tolerability of genetically engineered protein therapeutics. Differences in glycosylation, which may affect immunogenicity and mannose-6-phosphate receptor-mediated cellular internalisation of administered enzyme, possibly account for the differences in dosing, clinical effects and safety profiles reported for agalsidase alfa and agalsidase beta.

Control of malabsorption in cystic fibrosis.

Intestinal malabsorption is severe and of early onset in virtually all people who have cystic fibrosis. The main cause is deficiency of pancreatic enzymes, but bicarbonate deficiency, abnormalities of bile salts, mucosal transport and motility, and anatomical structural changes are other contributory factors. Appropriate pancreatic replacement therapy will achieve normal or near normal absorption in many patients. It is important to identify both malabsorption and evidence of a pancreatic lesion in all patients who are to receive pancreatic enzymes. All who have evidence of fat malabsorption are deemed pancreatic insufficient and candidates for enzyme replacement therapy. Effective treatment should allow a normal diet to be taken, control symptoms, correct malabsorption and achieve a normal nutritional state and growth. The occurrence of fibrosing colonopathy in some patients receiving very high doses of those enzymes that have the copolymer Eudragit L30 D55 in their covering has resulted in guidelines in the UK to avoid dosages greater than the equivalent of 10,000 IU lipase/kg/day for all patients and also to avoid preparations containing this copolymer in children and adolescents. For patients not responding to 10,000 IU lipase/kg/day, review of adherence to treatment, change of enzyme preparation, variation of the time of administration and reduction in gastric acid may improve absorption. The importance of excluding other gastrointestinal disorders as a cause of the patient's symptoms and the need for early investigations, rather than merely increasing the dosage of enzymes, is stressed. With modern enzymes, adequate control of gastrointestinal symptoms and absorption can be achieved at dosages of 10,000 IU lipase/kg/day or only slightly more, and a normal nutritional state and growth rate maintained in most patients with cystic fibrosis.

Asthma on the job: work-related factors in new-onset asthma and in exacerbations of pre-existing asthma.

Occupational asthma (OA) can be defined as variable airways narrowing causally related to exposure in the working environment to airborne dusts, gases, vapours or fumes. There are many agents in the work-place that can induce asthma or cause substantial deterioration in pre-existing asthma. It has been estimated that 5-15% of adult-onset asthma can be attributed to occupational exposures. Hence adult patients, especially those with new-onset asthma, must be investigated with regard to occupational risk factors for disease. The prognosis for OA is improved if the causal exposure is controlled either by controlling the exposure at the workplace or by moving the patient out of the workplace.

Study on OELs for enzyme-containing detergent in China.

This study is aimed at setting occupational exposure levels for total detergent dust and enzymes in detergent industries. The study population consisted of 795 workers from four enzyme-containing detergent manufacturing plants (A1, A2, B1 and B2), and 156 control workers from an electronic assembly factory. Work environment monitoring was conducted using high volume of air sampler fro measuring the concentration of total dust (mg/m3), and analyzing the level of enzyme (ng/m3) by ELISA method. A standard questionnaires, pulmonary function test, and skin prick test are used to assess health effects. The levels of detergent total dust varied from 0.2 mg/m3 to 12.54 mg/m3. For enzyme levels, in A1, B1 and B2, the concentration ranged from non-detectable to 9.92 ng/m3 and in A2, the concentration was analyzed by enzyme activity methods and was expressed as Gu/m3 (1 Gu/m3 = 16 ng/m3). The concentration is between 0.16-31.36 ng/m3. Non-specific irritation rates in exposed workers were significantly higher than that in controls. Based on the data collected from A1, B1 and control plants, 95% benchmark dose lower bound were calculated as 1.17 mg/m3. The difference of pulmonary function between exposed workers and controls is not significant. The results of SPT showed that neither Savinase- nor Alcalase-induced sensitization was found in controls. The prevalence rates of sensitization for Savinase and Alcalase were ranged between 3.2% and 31% in all enzyme-containing detergent manufacturers investigated. No case of occupational asthma was observed. For total dust, 1 mg/m3 is suggested as permissible concentration-time weighted average (PC-TWA), and 2 mg/m3 as permissible concentration-short term exposure limit (PC-STEL). For the enzyme Subtilisins, 15 ng/m3 is suggested as PC-TWA, and 30 ng/m3 as PC-STEL.