Semaglutide in Obesity and Non-Alcoholic Fatty Liver Disease

Introduction

Semaglutide injection was introduced in the US as an antidiabetic medication for type 2 diabetes in December 2017 by Novo Nordisk. It is available in subcutaneous pen injector and oral tablets.
Solution pen injector, subcutaneous: Ozempic 0.25mg, 0.5 mg, 1 mg
Tablet, oral: Rybelsus 3 mg, 7 mg, 14 mg
Solution pen injector, subcutaneous: Wegovy 2.4 mg (approved 2021)
Semaglutide is a selective glucagon-like peptide -1 (GLP-1) receptor agonist that activates the receptor for incretin, increasing glucose dependent insulin secretion, decreases inappropriate glucagon situation and slows gastric emptying. As we know, the first GLP-1R agonist approved for clinical use was exenatide (synthetic exendin-4), a peptide originally isolated from Heloderma suspectum lizard (Gila monster) venom by John Eng in 1992. It was approved in the US by the FDA in 2005.

Semaglutide and Obesity

Recent research results from the STEP1 study group showed significant improvements with once weekly semaglutide at 2.4 mg subcutaneous in overweight or obese adults who did not have type 2 diabetes. The study was a double-blind trial which enrolled 1961 adults with a BMI of 30 or greater who did not have diabetes and randomly assigned them in 2:1 ratio for 68-weeks trail of once-a-week treatment with subcutaneous semaglutide at a dose of 2.4 mg or placebo plus lifestyle intervention. Results were very encouraging at the end of 68 weeks. It was noted that in the semaglutide group mean body weight from baseline to weeks 68 was -14.9% compared to -2.4% with placebo, a change in weight of -15.3 kg in the semaglutide group as compared to -2.6 kg in the placebo group [1].

Participants who received semaglutide were more likely to lose 5% or more, 10% or more, 15% or more and 20% or more of baseline body weight at 68 weeks. 1047 participants in the semaglutide group achieved a coprimary endpoint of weight reduction of 5% or more compared to 182 adults in the placebo group. 838 adults had a weight reduction of 10% or more in the semaglutide group compared to 69 in the placebo group and 612 adults had 15% or more compared to 28 adults in the placebo group at week 68. Other supportive secondary endpoints that were achieved during the study were greater reduction from baseline than placebo with semaglutide in waist circumference, BMI, systolic and diastolic blood pressures, glycated hemoglobin, fasting plasma glucose, C-reactive protein and fasting lipid levels. SF–36 physical functioning score also improved significantly more with the semaglutide than the placebo at week 68. In the DEXA subpopulation, total fat mass and regional visceral fat mass are reduced from baseline with semaglutide.

Main adverse events noted during the study were gastrointestinal disorders typically nausea diarrhea vomiting and constipation which were reported and 74.2% of the semaglutide group versus 49.9% in the placebo group. Serious adverse events were noted to be serious gastrointestinal disorders in 1.4% of the semaglutide participants compared to 0% in the placebo group and hepatobiliary disorder in 1.3% with semaglutide compared to 0.2% with placebo. More participants in the semaglutide group than the placebo group discontinue treatment owing to gastrointestinal events 4.5% versus 0.8%. Overall 94.3% of the participants completed the trial, 91.2% had a body weight assessment at week 38 and 81.1% adherent to treatment. With obesity being a global health challenge and limited pharmacotherapeutic options, once weekly semaglutide injection in addition to lifestyle modifications might be a great option for people looking for nonsurgical treatment.

Semaglutanide and NAFLD/ NASH

Nonalcoholic fatty liver disease (NAFLD) is admitted because of liver disease worldwide and among the top indications for liver transplant in developed countries. NAFLD encompasses a spectrum of liver disease ranging from simple nonalcoholic steatosis/nonalcoholic fatty liver to Nonalcoholic Steatohepatitis (NASH) which is progressive and can lead to cirrhosis as well as hepatocellular carcinoma. Prevalence of NASH among NAFLD patients is estimated to be close to 60%. Based on the original article published in New England Journal of Medicine on March 25, 2021 [2]. It is encouraging to see that subcutaneous semaglutide has significant morbidity and mortality benefits for patients with hepatic fibrosis/advanced NASH. This was a double-blind phase 2 trial involving biopsy confirmed NASH and liver fibrosis F1–F3 patient’s who received once daily subcutaneous semaglutide at a dose of 0.1, 0.2 or 0.4 mg or corresponding placebo. The primary endpoint was resolution of NASH. Secondary endpoint was improvement of at least 1 fibrosis stage. He exclusion criteria were hemoglobin A 1C of greater than 9.5 at 6 screening, causes of chronic liver disease other than NASH, excessive alcohol consumption and confounding concomitant drug use.

After 72 weeks of following these patients, the percentage of patients and home Nash resolution was achieved with no worsening of fibrosis was significantly higher in the semaglutide group than in the placebo group (59% in the 0.4 mg group versus 17% in the placebo group; odds ratio 6.87; 95% confidence interval 2.60-17.63; P<0.001). The difference between the semaglutide 0.4 mg group in the placebo group and the percentage of patients who had an improvement of at least 1 fibrosis stage without worsening of NASH after 72 weeks was not significant. An improvement of at least 2 fibrosis stages is according to 25% of the patients in the semaglutide 0.1 mg who, 19% in the 0.2 mg group, 20% in the 0.4 mg group and 17% in the placebo group. Among all the patient’s underwent randomization, worsening of fibrosis according to 10%, 8% and 5% of the patients in the semaglutide 0.1 mg, 0.2 mg and 0.4 mg groups respectively and 19% of the patients in the placebo group. Gastrointestinal disorders were the most common adverse events reported which included nausea, constipation, decreased appetite, vomiting and abdominal pain. These seem to be dose dependent. The percentage of patients will discontinue treatment because of adverse events per 7% and semaglutide and 5% in placebo.

Semaglutide was associated with an increase from baseline to weeks 72 and amylase and lipase levels that were greater than those in the placebo group. Based on the discussion provided by the researchers, we still do not understand the mechanism of action of GLP-1 receptor agonists–. They are speculating if this could be an indirect benefit from weight loss and improvement in insulin resistance which would reduce metabolic dysfunction, likely toxic effect and inflammation and Nash patients. The safety profile of subcutaneous hemoglobin eyes was consistent with that observed in patients with type 2 diabetes and other trials.

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Expanded Carrier Screening for Neuromuscular Disorders

Introduction

Genetic disorders are characterised by deleterious changes in nucleic acid sequence called mutation. This alteration in genetic makeup could be inherited from ancestors or acquired during the course of life [1] and can be potentially life threatening depending on various factors like age, family history, [2] ethnicity [3,4], gender, comorbidities etc. However, mutations are also proven to be beneficial [5] or neutral. The latter is considered and proved to play an important role in evolution by Kimura in his Neutral theory of Molecular Evolution where he held random genetic drift to be the reason for molecular level evolutionary changes [6]. Most genetic disorders that are passed from parents to their offspring are carried by determining units of genome called genes by the concept of inheritance. Hence, it is important that we understand its pattern of flow, phenotypic and genotypic expression and variation and the probability of passing to next potential generation. Genes have two alternate forms of themselves called alleles, each from both parents, forms the basis of inheritance. Expression of gene can be influenced by genetics and environment [7]. Concept of inheritance first described by Gregor Johann Mendel laid the foundation for single gene disorders where one gene is affected by mutation hampering its normal function [1,7]. Effect of mutation is scrutinized up to the level of loci of the mutated gene, autosome or sex chromosome, number of mutated alleles and phenotypic expression of inherent genotype [1].

Disease phenotype can be distinguished as dominant where presence of one mutant allele will suffice the requirement to express the condition or recessive where both alleles are to be mutated to present the condition [1,8]. Autosomal dominant as in Huntington’s disease refers to expression of one mutated allele in autosomes, with no carrier states, heterozygous affected or homozygous unaffected [1,7,8]. Autosomal recessive like Phenylketonuria (PKU) refers to the requirement of two mutated alleles to present a condition, homozygous affected, unaffected or heterozygous carriers [1,7,8]. Hypophosphatemic rickets is X – linked dominant identified by inheritance of one mutated allele on X chromosome with gender bias towards females [1,8]. Hemophilia A is X – linked recessive, which has inheritance of two mutated alleles with only females as carriers [1,7,8]. Heterozygous female carriers can express clinical symptoms called as lyonization due to random inactivation of either of X chromosomes [7]. Y – linked inheritance is in Y chromosome in males and transmission only between them like in non-obstructive spermatogenic failure [1]. Mitochondrial inheritance is possible in females only with all offsprings at equal risk [7].

Carrier states in inheritance can be understood as individuals with one mutated allele in the place of two, with no phenotypic expression of concerned traits, who carry the risk through generations without awareness. Identifying such hidden secrets can help us foresee and be prepared for unplanned expressions [8]. Carrier screening is a specialised program to detect carriers for inherited recessive genetic disorders [2-4,9-11] that focuses on particular age groups with a family history. It is available for premarital or pre-relationship, preconception and prenatal screening [4]. It can be for the couple or individuals who are considered to be at high risk [3], which happens when there is enough awareness about the prevailing condition in the family, else identified in preconception time, which gives more reliable data about risks and chances. It was once referred as single-disease [2], ancestry-based screening [2,9] and now widely accepted as expanded carrier screening (ECS) or pan ethnic or universal screening [2,3,9]. The traditional screening explained by Wick and Rose was typically for single disease on particular ethnic group that were considered to impact normal living [4], which lacked focus on other prevailing genetic disorders, and this emphasized the improvisation to ECS, which has multiple addressing points for various population and disorders [10,12]. It was in 1970s when Tay-Sachs’s disease was first taken for carrier screening in Ashkenazi Jewish ethnic group [3,9,12].

Necessity

ECS is necessary to throw light on common and uncommon genetic disorders that run through generations, indicating high risk for acquiring the recessive condition without prior precaution. It gives possible options for alternative reproductive interventions [4], spouse or partner selection, a part of newborn screening not as a replacement [13], fall in birth rate of affected offsprings and gives equal importance to all ethnic groups. The main source of information in the genetic front is the frequency of sequence variants, which determine the effects of carrier status [2]. On the public health perspective, there is better planning of treatment strategies for the improvisation of quality of life. On gaining good reach there is observable data accumulated for rare inherited diseases. It has contributed to reduced morbidity and mortality rates [3]. In the economical perspective, this universal screening has been considered cost effective for resources invested and turnover of reliable information [2,3,9,14].

Evolution of Carrier Screening

Detection of single gene disorders was first implemented in 1963 as newborn screening (NBS) for phenylketonuria, a metabolic genetic disorder caused by mutation in phenylalanine hydroxylase (PAH) gene. [15] NBS is mandatory, non-ethnic based procedure followed to identify expressing clinical conditions and not asymptomatic carriers. First carrier screening began with Tay- Sachs disease in Ashkenazi Jewish ethnic group in 1970s. Around 1990s to 2000s the same was recommended for Cystic Fibrosis (CF) in Northern European Caucasian and Ashkenazi Jewish populations. A breakthrough ethnic barrier occurred in 2001 when American College of Medical Genetics and Genomics (ACMG) [16] and American College of Obstetricians and Gynaecologists (ACOG) established initially the guidelines for CF screening for Northern European and Ashkenazi Jewish descents, which was later improvised in 2017 to recommend CF screening to high risk categorised people (pregnant women, strong familial predisposition) and no ethnic considerations [14]. It was further refined for other conditions like Spinal Muscular Atrophy (SMA), Hemoglobinopathies, Fragile X Syndrome for the Eastern and Central European Jewish descents. However, in 2009, it took the shape of pan-ethnic screening for many genetic disorders despite of commonness and ethnic considerations [3,12]. ACMG in 2013 defined certain criteria for carrier screening which helped in identifying at risk patients who can be directed for prenatal diagnosis and prior consent and counselling for adult-onset disorders. It also emphasizes in understanding the role of causative gene and mutation in population level screening and establishing a direct association between mutation and severity of the disorder. Quality control and proficiency tests maintains the integrity of the process [16].

Spinal Muscular Atrophy (SMA)

It is an autosomal recessive disorder caused by degeneration of motor neurons in spinal cord, leading to weakness, hypotonia and paralysis. The incidence and carrier frequencies were found to be 1 in 10,000 [17,18] and 1 in 40 respectively [19]. Its prevalence was estimated to be 1 – 2 in 100,000. Caucasian and Asian population are found to have higher frequency of heterozygous carriers [18]. Carrier status of this disorder is indicated by one copy of SMN1 gene that is involved in pathogenesis. It is clinically classified as Type – I, II, III and IV. Type I or Werdnig-Hoffmann disease can be characterised as severe form in 6 months of age by hypotonia, unable to control head movement, paradoxical breathing, bulbar denervation, aspiration pneumonia finally leading to death within 2 years of onset. Type II can be distinguished by intermediate level of impact in 7 to 18 months of age, unaided sitting, kyphoscoliosis, deep tendon reflexes, fine tremors, weak swallowing, poor bulbar and intercostal muscles leading to respiratory failure and death. Type III or Kugelberg-Welander disease can be identified as heterogeneous with ability to walk without assistance but reduced significantly before or after 3 years of age, minor muscle weakness and scoliosis [20-22]. Type IV can be found in 2nd or 3rd decade of life with mild motor impairment and walking ability in later years [20,21]. The SMA gene was mapped on chromosome 5q11.2 - q13.3 in 1990 [21].

Five years down the line, survival motor neuron (SMN) gene was found to be responsible. The SMN exist as the telomeric SMN 1 gene (SMA – determining gene) and the centromeric SMN 2 gene. They differ by a few nucleotides which causes alternative splicing of exon 7 but no alteration in amino acid sequence [20,21] SMA is supposedly diagnosed by homozygous disruption of SMN1, leaving back a copy of SMN2, which when undergoes alternative splicing produces a truncated mRNA with exon 5 and/or 7 being lost. Further, a nucleotide transition produces a non-functional protein that is degraded. This when observed in diagnosis confirms the genetic condition. [20,21]. Some of the clinical features include floppy baby, proximal weakness, swallowing and breathing difficulty in extreme cases. Nocturnal hypoventilation is also seen. Assays that help assess type of SMA include creatine kinase dose, electromyography (EMG), nerve conduction study, multiplex ligation dependent probe amplification (MLPA), quantitative PCR (qPCR) and semiquantitative assays [21]. Genetic diagnosis is initiated for homozygous deletion of SMN1 particularly exon [7], which is observed in 95% of SMA cases [21]. Single deletion, frameshift, nonsense and missense mutation of SMN1 indicate other types of SMA. SMN1 dosage analysis is conducted for identifying 95% of carrier status of individuals who may have deletion or mutation in one SMN1 copy. SMN2 copy number can be used to determine severity of the disorder however, precise prediction when used stand-alone is not reliable [21,23].

Carrier status determination apart from SMN1 dosage testing is based on remaining 5% categorised for deletion followed by its duplication onto the other SMN1 or mutation sequencing of the remaining undeleted copy [23]. Single copy of SMN1 is more indicative of type I with very poor prognosis [21]. Single strand conformation polymorphism is widely used for understanding heterozygous deletion as in cases of carriers and point mutation in undeleted allele [24,25]. Genetic counselling is a challenging profession where the professionals are trained and evaluated for their knowledge in the field of genetics and counselling skills [26]. It is offered to people categorised for risk of having a child with SMA like parents or siblings of SMA affected infant, presymptomatic individuals [23], which is assessed by SMA screening on voluntary basis, with proper informed consent and confidentiality maintained. Prenatal diagnosis is available by chorionic villus sampling in 11th to 13th week of gestation and 14th to 16th week of gestation by amniotic fluid sampling [21]. Preimplantation Genetic Testing (PGT) can be facilitated for In Vitro Fertilization (IVF) to identify couples at risk for pregnancy [27]. Follow up coordination plays a crucial role in management of SMA. Complications in pulmonary function makes the situation difficult to tackle. Movement to muscles like walking is necessary. Surgical intervention benefits younger age group in prevent curve progression.

Drugs are being developed for cure of SMA, which have molecular level targets. Salbutamol, a β2-adrenergic agonist is proved to increase SMN levels [28]. Riluzole’s neuroprotective and neurotrophic activities is proved to promote neuron survival and expression of brain-derived neurotrophic factor [29]. Spinzara is an approved modified anti-sense oligonucleotide acting by including exon 7 for production of full-length SMN protein [30]. Adenoassociated virus serotype 9 (scAAV9) is employed for SMN1 gene replacement therapy. (21, 30) AAV9-mediated IGHMBP2 gene therapy for spinal muscular atrophy with respiratory distress type 1 (SMARD1) is under clinical trials, whose wild type has shown to increase IGHMBP2 protein levels, protect motor neurons and axons, resolve defects in neuromuscular junctions, increase myofibre calibre and causes extended life period [31].

Duchenne Muscular Dystrophy (DMD)

This X-linked recessive disorder [32,33] is reflected by weakness and loss of muscle mass at a very early stage of life [34] with limited life expectancy up to late teens [33]. The incidence rate is about 1 in 5000 boys [35,36] and global prevalence in general population was estimated to be 2.8 cases per 1,00,000 [37]. Mortality rates of nonhispanic whites was found to be statistically high on comparison with non-hispanic blacks and hispanics. [33] Mutation in DMD gene on X chromosome Xp21.2 [32,34,37] produces dysfunctional dystrophin protein [37,38], which is essential for cytoskeletal support of sarcolemma in skeletal muscle along with glycoprotein complex. Intragenic deletions hold higher responsibility followed by duplications and small insertions. The open reading frame of DMD gene when disrupted causes DMD with nonsense protein production [32,36]. Hotspots for deletion in the largest gene of the human genome include exons 45 to 55 for deletions (36) and exons 2 to 10 duplications [39]. Proportion of affected males is higher due to recessive inheritance; however, there are chances for female asymptomatic carriers who can be potential carriers in their family [32,36,37]. Almost fully absent (32, 36, 39) or higher deficiency of functional dystrophin protein [37] hampers muscle functions and they can be characterised by loss of regenerative potential of muscle mass, increase in serum creatine kinase (CK) [36,38], inflammatory response resulting in fibrosis and altered metabolism, deficiency of dystrophin associated proteins (DAP) that ultimately distorts muscle function.

Physical observation include abnormal gait [36], difficulty or inability to walk is a crucial milestone at 18 months hinting for diagnosis of DMD [38]. Since this can be indicative at a very young age around 3 to 5 years [36], where they are supposed to be on their feet always, it becomes a point of concern. Slow and stiff movement of muscles, scoliosis [36,38], pseudohypertrophy [36], lumbar lordosis and gowers maneuver [36,37] does not enable performing normal activities. Untreated can be wheelchair bound by 11 to 12 years of age [33,36]. Motor skills impairment or delay can also be seen in a proportion [36,37]. Cardiac concerns [34] like dilated cardiomyopathy [38], arrhythmias can be seen but mostly asymptomatic [37]. Respiratory insufficiency leads to fatal end of life [34,37,38]. Biochemical aspects include elevated serum CK levels around 5,000 to 15,000 units/L and hepatic transaminases [36]. However, these symptoms in a milder version can be seen in Becker Muscular Dystrophy (BMD). Hence, a sequencing analysis can clearly reveal the exact morbidity [32]. Diagnosis relies on muscle biopsy testing [38,40] by immunostaining and western blot for detecting absence of dystrophin [32], elevated enzyme levels [38] and cardiac examinations by electrocardiogram (ECG), echocardiography and chest X-ray are performed to check for development of cardiac symptoms [34]. Genetic testing [38] by MLPA which detects deletion of 25 exons [34], comparative genomic hybridization (CGH) [41], high resolution chromosome microarray, further to which direct sequence analysis can be availed for analysis of presence of small deletions, insertions and point mutations [42] apart from deletion and duplication [36].

Carrier testing is recommended for women who are closely associated with men genetically confirmed to have DMD. This can be well appreciated in a family tree. Female relatives of those suspected carrier women of a family are considered for carrier testing. However, asymptomatic nature poses a challenge in timely identification [40]. Creatine kinase test is most reliable for this context [43]. It can also be tested by Restriction Fragment Length Polymorphism (RFLP) [44] and Polymerase Chain Reaction (PCR) of short tandem repeat (STR) loci [45]. qPCR with STR markers and for dystrophin gene was also performed in Eastern Indian population for carrier status counselling also proving statistically significant difference in frequency in different parts of population [46]. As a developed diagnostic technique, MLPA holds great value that allows reading of 79 exons of the dystrophin gene than Quantitative Multiplex Fluorescence PCR (qmfPCR) where 51 exons are examined and STR-(CA) segregation analysis for 11 markers of the gene. Carrier frequency is observed to lower than theoretical values specially for deletion mutations. MLPA gives a reliable assessment before proceeding towards any invasive procedure and is termed to be first choice in carrier screening even in female relatives who are in families of affected males to confirm their status [42]. Prenatal diagnosis [36] by chorionic villus sampling or amniocentesis can be offered for females who have tested positive for carrier status and encourage family members to take up the testing to be well informed prior to family planning.

With all the results correlated, genetic counselling essentially prepares the susceptible individuals and parents or couples likely to plan, as it gives them the opportunity to receive prior information about their next step or to take the final call [36]. Linkage studies can also aid in locating the maternal X chromosome in the fetus [32]. DMD has no definite cure but symptom management is possible. Corticosteroid therapy with prednisolone, prednisone and deflazacort can be adopted to improve muscle strength with an eye on the side effects of hormone-induced actions [32,36]. AAV mediated transfer of miniature dystrophin genes for gene therapy [47] is an upcoming development [48]. Human trials are yet to be done for dystrophin producing mesenchymal-cell transplantation [32]. Aminoglycoside inhibitors used to interrupt encountering premature stop codons [49]. Ataluren is employed to target nonsense mutations and a possible drug therapy for DMD [37,50]. Eteplirsen is an anti-sense therapy used for skipping exon 51 in defective gene to produce a shortened yet functional dystrophin protein [36,37,51] which received FDA approval in 2016 [52].

Discussion

Genetic diagnosis when offered to a larger group and a wider spectrum of genetic disorders assists in taking well-informed decisions in all facets of suspicion. There is no ambiguity with presence or absence or carrying of disease, which can break barriers of discrimination. It covers major susceptible disease carrying aspects cost effectively with minimal risks involved. Some of the challenges for ECS include lack of interest in general public, not being considered as priority but as choice and social pressure on individuals or couples especially in reproductive decisions. This necessitates appropriate genetic counselling at the right time from a valid perspective and fine-tuning to get a deeper understanding of technical background. [9] Awareness about carrier status in genetic disorders begins from understanding own family history and current scenario in their lives. Focusing attention on highrisk classified individuals with strong familial background and immediate relations who are affected gives a major cue to diagnosis. With increasing knowledge and application of principles in viable technology, diagnosis has hastened multitude with maximum sensitivity and specificity. Availability of resources, reliability on testing and reproducibility of results are exceptionally crucial for validation of findings.

Conclusion

Expanded carrier screening is an upcoming initiative that involves identifying prospective symptomatic or asymptomatic carriers of genetic disorders which when left unnoticed poses great challenges in the later part of their live(s). The shape taken by ECS in today’s time will make a huge turnover in the upcoming times, provided there is support from both common public and healthcare experts. Role of a geneticist and a genetic counsellor takes a huge contribution to appropriate and timely diagnosis and planning their next step of action. The future prospects would be towards normalizing such testing and spreading awareness about keenly observing any inherited conditions in their family or acquaintances and report to concerned professionals to seek their observation and interpretation.

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Transient Zinc Deficiency Dermatitis in a Very Low Birth Weight Preterm Infant with Necrotizing Enterocolitis - A Case Report & Review of Literature

Introduction

Zinc plays a vital role in growth and development, cell proliferation, tissue repair, and cellular immunity. Zinc is considered very essential for oligodendrogenesis, neurogenesis, neuronal differentiation, white matter growth, and multiple physiological and biological roles in neurobiology [1]. Preterm infant is said to have high zinc dietary requirements because 60% fetal zinc is acquired during the third trimester of pregnancy which is not stored because of early delivery [2]. Therefore, premature infants are especially susceptible to zinc deficiency. It is the aim of this report to point out that a clinical suspicion of Zinc deficiency is warranted in any preterm who present with dermatologic manifestation that cannot be explained otherwise.

Case

A very low birth weight preterm delivered at 29 weeks of gestation with a birth weight of 1,180 grams born w/ dyspnea, intubated for congenital pneumonia treated with parenteral antibiotic. Moderate patent ductus arteriosus was closed with 3 doses of parenteral ibuprofen. Grade II B necrotizing enterocolitis (NEC) was diagnosed at 26th day of life which required treatment with parenteral antibiotics & prolonged total parenteral nutrition. She then presented with a 2 weeks of progressively extensive, erosive, eczematous crusted eschar looking like blistering skin lesions over the perioral region, right side of neck, hands, left axillary fossa, left thigh, and gluteal region (Figures 1-3). Laboratory investigations revealed WBC: 10,430/μl , CRP:1.73 mg/dl. Gentamycin cream was added for topical skin lesion use. IV antibiotics with Cefazolin and Gentamicin were given for suspected bacterial infection-associated skin lesions. Gram stains & cultures from skin lesions and blood were negative. For the condition of prolonged parenteral nutrition used, a clinical diagnosis of zinc deficiency was considered. The infant’s serum zinc level was decreased (511 mcg/L; normal range: 700-1200 mcg/L). Zinc deficiency was confirmed by the finding of low plasma zinc concentration. Parenteral zinc supplementation at a dose of 500 mcg/kg/day was started again after almost 1.5 months of shortage in supply of parenteral zinc in the country. After treatment with zinc supplementation, skin lesions dramatically improved within two-three days of treatment and recovered after one week of treatment. Complete resolution of the skin lesions is seen with no scar formation after zinc deficiency treatment.

Figure 1: Erythematous acral lesions with crusting on right hand.

Figure 2: Left thigh escharosis.

Figure 3: Erythematous erosive lesion all around left axillary fossa with desquamations.

Discussion

Severe zinc deficiency causes dermatitis, alopecia, diarrhea, neurologic symptoms such as irritability and altered behavior, and limitation of growth [3]. Zinc deficiency in breastfed mainly preterm infants is a condition with similar clinical findings as in acrodermatitis enteropathica, an autosomal recessive disorder of enteric zinc absorption [4]. Skin lesions are classically located in periorificial, intertriginous, and acral areas. Skin lesions usually appear as early small moist and erythema, progressing to eschar lesions [5]. These lesions may be the seat of numerous bacterial superinfections because of the impaired immune function induced by zinc deficiency. Premature infants are especially susceptible to a negative zinc balance, even under the conditions of adequate intake. Their zinc deficiency symptoms are associated with rapid growth and increased zinc requirements, with the immaturity of their digestive system combined with increased intestinal zinc elimination and decreased absorption, and with small zinc reserves in their muscles and bones [6].

However, premature and low-birthweight infants may also develop symptoms even when receiving milk with normal zinc levels. Infants with NEC were treated with long-term total parenteral nutrition, leading to the increased trace element lost from the inflamed gut mucosa and incapable intestinal absorption of zinc which make them especially susceptible to the development of zinc deficiency. The zinc deficiency in our case was caused by the transient shortage of supply of parenteral zinc in our country when she can not take oral zinc supplement because of her NEC. Administration of zinc preparation at a daily oral dosage of 1-2 mg/ kg/day is the treatment of choice in infants with zinc deficiency [7]. Rapid improvement in skin lesions is noted within days, and clearance occurs within 4–28 days [8]. Remission of other symptoms occurs within weeks to months. The differential diagnoses include chronic mucocutaneous candidiasis, atopic dermatitis, contact dermatitis, impetigo, and metabolic disorders such as biotin and other decarboxylase deficiencies, organic acidurias, and essential fatty disorders [9].

Conclusion

Premature infants have a negative zinc balance mainly secondary to inadequate stores because of their early delivery, and high requirements are at risk of developing zinc deficiency. Early recognition of the disorder and introduction of zinc supplementation rapidly reverses transient zinc deficiency. Thus, it is essential for a prompt diagnosis and effective treatment of zinc deficiency especially in premature to prevent a severe zinc deficiency resulting in neurologic sequelae and growth failure.

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Aluminium Induces Iron-Mediated Oxidative Stress in Brain Tissue

Introduction

Aluminium (Al) is an abundant element in the environment, but it does not perform any known physiological function in the human body (Exley, et al. [1]). Al is associated with toxic effects on various body systems (Pogue, et al. [2,3]), and especially with neurotoxicity (Exley, et al. [4,5]). Some studies have linked increased levels of Al in the brain to Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative disorders (Virk, et al. [6-9]). Al interferes with many physical and cellular processes by disturbing the redox status (Morris, et al. [5,10]). Although Al is a non-redox metal and occurs only in one oxidation state – Al3+, its strong pro-oxidant activity can cause oxidative damage through multiple mechanisms (Mujika, et al. [11]). As the brain is a target tissue for AI intoxication, a likely mechanism of its toxicity can be the generation of oxidative stress in the brain. To gain more knowledge about this complex mechanism, this study investigated Al-induced oxidative stress by analyzing biomarkers in the blood and brain tissue.

Materials and Methods

Experimental Animals and Exposure Protocol

Experiments were performed with 4 to 6-week-old male Balb/c mice weighing 20-25g. The mice were housed in a conventional area of animal housing facility at 22 °C and 50% humidity, with a 12-hour light/dark cycle. The mice were exposed to Al3+ by an intraperitoneal (i.p.) injection of AlCl3 solution daily for 14 days. AlCl3 was dissolved in deionized water. The dose was 5 mg (0.185 mmol) Al3+/kg body weight, corresponding to 0.1 LD50. Control animals were injected with the same volume of physiological solution (0.9% NaCl). Each group consisted of eight animals. After 14 days the mice were anesthetized and terminated. All procedures were performed according to the rules of the European Convention for the Protection of Vertebrates used for experimental and other scientific purposes (Republic of Lithuania, License of the State Veterinary Service for Work with Laboratory Animals No. 0221).

Preparation of Samples

The serum samples were prepared as follows. Whole blood was collected in Eppendorf test tubes after the decapitation of mice. The blood was centrifuged at 4000 rpm × 10 min (1600 × g) in a Savant centrifuge. The serum samples were immediately frozen at -80°C and shipped on dry ice from Kaunas to Bilthoven. The frozen samples were received and stored at -80°C until analysis. The brain was removed, chilled rapidly on ice, and homogenized in 3 volumes (e.g., 1g of tissue plus 3 ml of buffer) of homogenization buffer (50 mM Tris-HCl, pH 7.6; 5 mM MgCl2; 60 mM KCl; 25 mM sucrose). The tissue homogenate was centrifuged at 3000 rpm x 10 min (1000 × g) at 4°C in a Beckman J2-21 centrifuge. The first supernatant was poured into another reaction tube and the pellet was discarded. The supernatant was centrifuged at 10,000 rpm × 15 min (12,000 × g) at 4°C in a Beckman J2-21 centrifuge. The entire volume of the second (post-mitochondrial) supernatant was immediately frozen at -80°C. All the tissue data was adjusted and standardized for protein content.

Biochemical Analysis

The concentrations of Al and iron in the whole blood and brain were evaluated using an inductively coupled plasma mass spectrometer NexION 300 D (Perkin Elmer, USA). Blood and brain tissue samples were digested with 0.125 M NaOH at 90°C and diluted to an appropriate volume to be analyzed according to the manufacturer's recommendations. To ensure the accuracy of the analysis, we followed internal and external quality control procedures, including the use of analytically pure water, reagents, (Merck, Sigma-Aldrich) and certified reference materials ClinCheck® Whole Blood Controls Level (Recipe, Germany). Standard Reference Material®-1577c bovine liver (NIST-1577c, Gaithersburg, USA) was used as a standard reference material when determining iron in tissue samples. Also, we checked the laboratory equipment for trace element contamination.

ROS-derived hydroperoxides, as an indicator of ROS production, were measured using the Diacron test for reactive oxygen metabolites (d-ROMs kit, No. MC-003 from Diacron, Grosseto, Italy). The test is based on the estimation of the amount of organic hydroperoxides formed in blood serum caused by the presence of free radicals. In practice, a small amount of serum is diluted in an acidic solution (pH 4.8). In these conditions, iron ions become available to catalyze the breakdown of hydroperoxides to alkoxyl and peroxyl radicals. The chromogenic substrate is N,N-dimethylparaphenylenediamine transformed into a pink to red colored radical cation. Quantitation is possible with the use of a photometer (wavelength 505 or 546 nm). The concentration of colored complex is directly related to the levels of hydroperoxides in the tested biological sample. The levels of hydroperoxides were quantified in Carratelli Units (1CARR.U. = 0.08 mg H2O2 / 100 ml) (Schöttker, et al. [12]).

 

Total serum thiol levels (TTL) were measured with a kit from Rel Assay (Gyantzip, Turkey) (TTL kit No. RL0178) and expressed in µmol/l. Both ROM and TTL assays are adapted for automatic use on an LX-20 Pro autoanalyzer (Beckman-Coulter, Woerden, The Netherlands) (Leufkens, et al. [13,12]). Serum iron bound to transferrin (TrF-Fe) expressed in µmol/l was determined on the same autoanalyzer LX-20 Pro (kit No. 467910 from Beckman-Coulter). All the intra assay coefficients of variation for these assays were between 1.5 and 4.3%. Mouse transferrin (TrF) expressed in g/l (kit No. E-90TX), and mouse ferritin (FER) expressed in µg/L (kit No. E-90F), were determined by ELISA obtained from ICL (Portland, OR, USA). The intra-assay coefficients of variation were less than 5%, according to the manufacturer's data. Activity of alkaline phosphatase (ALP) (kit No. 476821 form Beckman-Coulter) and the concentration of total protein (TP) (kit No. 465986 from Beckman-Coulter) in the brain tissue were measured using the autoanalyzer LX-20 Pro (Beckman-Coulter, Mijdrecht, The Netherlands). The intra assay coefficients of variation were less than 5%.

Lipid peroxidation in the brain tissue was estimated by measuring the absorption of thiobarbituric acid reactants using UV/Vis spectrophotometer LAMBDA 35 (Perkin Elmer, USA) and expressed as malondialdehyde content in nmol/g wet organ weight. The mice brains were homogenized in cold 1.15% KCl solution to make 10% homogenate. To 0.5 ml of this homogenate, 3 ml of 1% H3PO4 and 1 ml of 0.6% thiobarbituric acid (Serva, Germany) aqueous solutions were added. The reaction mixture was placed in a boiling water bath for 45 min. After cooling, 4 ml of n-butanol was added and mixed vigorously. The butanol phase was separated by centrifugation and the light absorption measured at wavelength 535 and 520 nm (Uchiyama, et al. [14]).

The total brain glutathione concentration (totGSH) (expressed in µmol/g protein) was measured in the post-mitochondrial supernatant after deproteinization with an equal volume of 10% metaphosphoric acid at 20°C for 5 min. The mixture was centrifuged at 2000 x g for 2 min and the supernatant was incubated with glutathione reductase solution (2500 U/6 ml) at 25 °C for 60 min to convert oxidized glutathione (GSSG) to GSH. The GR kit No. G9297 was obtained from Sigma-Aldrich, Zwijdrecht, The Netherlands. The total GSH was determined after derivatization with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), kit No. D8130 from Sigma-Aldrich at 37°C for 5 min. The light absorption of reaction product was measured at 412 nm with the autoanalyzer LX-20 Pro from Beckman-Coulter, Woerden, The Netherlands. The intra assay variation was 6.7%.

Statistical Analysis

The results were expressed as the mean ± standard error of mean. The data was analyzed with ANOVA using Microsoft Excel. Statistical significance was set at p<0.05. In addition, the statistical significances in the serum biomarkers and brain biomarkers were checked separately to counteract the problem of multiple using the Bonferroni method.

Results

Systemic Biomarkers

Concentration of Al and iron after a 14-day intraperitoneal (i.p.) exposure of mice to Al3+ was determined in the whole blood. Al concentration increased sevenfold, from 74.0±13 µg/l to 535±77 µg/l (p <0.001), and the total iron concentration of decreased by 27%, from 208±18 mg/l to 153±28 mg/l (p <0.001). To evaluate the effect of Al on iron homeostasis, the biomarkers of iron status in the serum were measured: total content of iron bound to transferrin (TrF-Fe), transferrin (transport protein of Fe) and ferritin (indicating iron status in tissues). Several changes in iron status were observed after the exposure of mice to Al3+: an increased concentration of TrF from 2.10 to 2.52 g/l, and a decreased concentration of TrF-Fe from 68.2 to 36.5 µmol Fe/l. Ferritin concentration increased from 1.09 to 2.44 µg/l (Figure 1). Oxidative stress biomarkers were estimated in the blood serum of Al-treated mice. Al caused a statistically significant increase (almost 60%) in the concentration of lipid peroxides, measured by ROM assay from 83.7 to 133.7 CARR.U (p=0.0003) (Figure 1). Total redox status measured by TTL assay decreased statistically significantly from 371 to 280 µmol/l (24%) (p<0.004).

Biomarkers of Neurotoxicity

Exposure to Al3+ did not affect Al and iron levels in the mice brain. In the control and Al-treated groups, Al concentration was 11.3±2.6 and 11.7±1.6 µg/l, and Fe concentration was 18.6±1.6 and 18.5±1.6 mg/l, respectively. However, concentration of oxidative stress biomarker MDA, increased statistically significantly from 109±19.7 to 131±16.5 nmol/g (20.5%), (p=0.003). Also, concentration of totGSH, which reflects brain redox status, decreased from 15.0±3.3 to 8.3±2.7 μmol/g protein (64.7%) (p=0.0011) (Figure 2). In addition, we measured the enzymatic activity of ALP due to its possible relation with neurodegenerative, e.g. Alzheimer, diseases. ALP activity increased substantially in the brain of Al-treated mice from 6.64±1.07 to 9.97±1.69 U/g protein; ** p <0.0005 vs. control group.

Figure 1: Serum concentrations of oxidative stress biomarkers and iron status biomarkers: ROM (expressed in CARR.U), TTL (expressed in μmol/l), serum iron (TrF-Fe) (expressed in μmol Fe/l), ferritin (FER) (expressed in μg/l) and transferrin (TrF) (expressed in g/l) in control mice (C) and after Al exposure (Al). Statistics: * p<0.01; ** p<0.001 vs. control group.

Figure 2: Concentrations of MDA (expressed in nmol/g tissue), totGSH (expressed in μmol/g protein) and ALP (expressed in U/g protein*10) in brain post-mitochondrial supernatant in control and Al-treated mice. Statistics: * p<0.01 vs. control group; ** p<0.001 vs. control group.

Discussion

In this study, mice were exposed to Al for 14 days. It was determined that even low doses of Al3+ corresponding to 0.1 LD50 caused a sevenfold increase in Al concentration in the blood, while there were no changes in Al concentration in the brain. As Al is known to affect iron homeostasis (Peto, et al. [15,16]), we used this knowledge to explain the generation of oxidative stress in the blood and possibly in the brain. The serum iron status was measured because free iron is one of the major factors associated with the production of reactive oxygen species in vivo (Bresgen, et al. [17,18]), including the brain (Piloni, et al. [19,20]). Indeed, the data of this study showed that exposure to low Al3+ doses corresponding to 0.1 LD50 significantly affected serum iron status. It was demonstrated that Al3+ can displace iron ions from the iron-binding protein transferrin, which was reflected by a lower amount of bound iron. The transferrin saturation, which is the percentage of TrF that has been occupied by iron, decreased from 41.4% to 18.4% after exposure to Al. Our results are in agreement with the data of other authors (Jacobs, et al. [21]).

Figure 3: Schematic representation of Al-caused oxidative stress in brain tissue mediated by iron.

The release of iron from the serum transferrin was also confirmed by a higher level of serum ferritin, reflecting the increase in iron stores in tissues. The higher content of unbound iron in the serum and tissues may explain the development of oxidative stress (Figure 3). Exposure to Al3+ resulted in a significant decrease in total iron concentration in the blood by 27% but caused no changes in the iron levels of the brain. The blood-brain barrier is believed to protect the brain from the transport and accumulation of Al and iron. To assess the increased oxidative stress in serum, we measured ROM and TTL as biomarkers of lipid peroxidation and redox status. It was shown that ROM concentration increased, while TTL level decreased after exposure to Al3+. The importance of ROM and TTL as biomarkers of oxidative stress was proved in several large cohort studies on cancer (Leufkens, et al. [12,13,22]), cardiovascular diseases (Vassalle, et al. [23-25]), and aging (Schöttker, et al. [26,27]). The stability of serum samples for the ROM and TTL assays have been tested during short-term and long-term storage (Jansen, et al. [28,29]).

For the evaluation of oxidative stress in the brain tissue we used biomarkers MDA and totGSH. Due to technical reasons, the ROM test could not be performed on tissue extracts. The increased concentration of MDA and decreased concentration of totGSH indicated oxidative stress in brain tissue. In addition, the activity of ALP increased in the brain of Al-treated mice. Our results are in agreement with the data of other studies, in which higher activity of ALP in the brain of Alzheimer patients (Karabulut-Bulan, et al. [30,31]) was demonstrated. Increased enzymatic activity may be one of the indicators of increased oxidative stress in the brain. The observation that exposure to Al3+ did not change Al and iron levels in the brain implies the indirect effect of Al3+ on the brain via reactive oxygen species originating in the circulatory system. It was concluded that oxidative stress in the brain tissue is a result of systemic oxidative stress induced by aluminium through the iron mediated mechanism.

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Effect of Monosodium Glutamate Substitutes on Physiochemical, Microbiological and Sensory Properties of Fried Chicken Breast Strips

Introduction

Several major manufacturers have announced to move away from using artificial ingredients and flavors in their products. Monosodium glutamate (MSG) is one such ingredient that has been controversial for decades. It is one of the ingredients that some companies have committed to remove from products (Nguyen Thuy, et al. [1]) MSG is a flavor enhancer commonly added to processed food products like chicken to boost the palatability. Its remarkable effects on the sensory appeal have been proven in various studies (Baryłko Pikielna, et al. [2,3]) Removal of this ingredient is very likely to cause reduced consumer acceptability. Using MSG substitute is a promising approach to compensate for the sensory satisfaction loss caused by MSG elimination. The flavor enhancement effect of MSG is mainly from glutamate which contributes to umami or savory taste sensation. Besides glutamate, there are several other umami eliciting components such as aspartate and 5’-ribonicleotides.

Among nucleotides, inosinate (IMP) and guanylate (GMP) significantly contribute to flavor and taste enhancement (Wang et al. [4]) Theoretically, substances that are naturally rich in umami components have the potential to replace MSG in food products. Consumers preferred natural extracts such as yeast extract, mushroom extract, and tomato extract as MSG substitute in chicken products (Adhikari, et al. [5]). Sugars may also contribute to umami taste characters in the form of glutamate glycoconjugates (Hui, et al. [6]) Furthermore, salts of potassium are also responsible to enhance umami taste strength. However, during boiling process, significant levels of potassium leach out from potatoes (Bethke, et al. [7,8]). Sodium chloride is an important ingredient added to most of foods which contributes to flavor enhancement and food preservation (Chun, et al. [9]). MSG is a flavor enhancer that is found in some processed foods and Chinese cuisine. To avoid this sodium product there are some potential substitutes can be used as substitutes for MSG. Use 1:1 ratio mixture of sugar and salt as a substitute ingredient to your recipe instead of MSG.

This is safer to use, especially if you have children at home. MSG is a food additive used as a flavor enhancer The advantage of MSG goes to those who easily lose their appetite. This is a very common ingredient in fast foods and food seasonings. MSG is actually harmless but too much consumption would cause headaches and this is not good for people who have vertigo (a sensation of spinning) (Maluly, et al. [10]) Currently, there is limited research comparing the enhancement effects of MSG with these natural extracts in food products. Given the capability of salty taste enhancement, MSG substitute may also be able to increase the sensory appeal of meat products with reduced salt content. Previous study indicated that used of yeast extract successfully enhanced the taste of fermented sausage (Campagnol, et al. [11]) Ground mushroom has also been reported to improve the flavor of taco blend (Myrdal Miller, et al. [12]). To replace MSG, it is necessary to conduct more research to compare the performance between MSG and its alternatives in salt-reduced food matrix. The aim of the current study was to investigated the effect of replacement MSG with 1:1 ratio mixture of sugar and salt on physiochemical, microbiological and sensory properties deep fat fried chicken breast strips during frozen storage (-18°C).

Materials and Methods

Materials

Fresh boneless skinless chicken breast strips 74.33% moisture 20.72% protein, 2.26% fat, 1.18% carbohydrate, 1.18 Ash and pH 5.09, were obtained after 8 h of slaughtering from Cairo Poultry Processing Company, Sharkia, Egypt. The samples were transferred under cooling conditions to Food Technology Department Laboratory (Zagazig University, Egypt) and saved in freezer at -18°C for 3 months until processing.

Methods

Preparation of Chicken Breast Strips: After preparation of chicken breast strips as described (Tables 1 & 2), samples divided into two groups: control group containing MSG (C) and the other containing MSG substitution (T).

Table 1: Marinade formula of chicken breast strips.

Note: *Monosodium glutamate substitution (T): mixtures consist of table salt and sugar by ratio of 1:1.

Spices (onion powder 9gm., garlic powder 9gm., Celery powder 2.25gm., Ginger powder 2.7 gm.

Table 2: Coating formula of chicken breast strips.

Note: *Monosodium glutamate substitution (S) mixture consist of table salt and table sugar by ratio of 1:1 .

** Batter spices consist of (garlic powder 2.46 gm., Ginger powder 1.97 gm. And Black pepper powder 2.46 gm.

Preparation of Marinade Solution: The amount of water below5 °C was placed in a bag of high density polyethylene, after that the amount of food grade sodium tripolyphosphate (STPP) was dissolved in it, followed by dissolving the table salt and MSG in the case of control or MSG substitution (a mixture of table salt and table sugar in a ratio of 1: 1) in the case of treatment and then add spices, antioxidant, and stirring to homogenize the marinade solution. The amount of raw chicken fillet strips was added to previous brine after thawing it for 24 h in the refrigerator and reaching a temperature of 1- 4 °C. Finally the bags were closed and flipped for five minutes and placed in the refrigerator on a temperature of 1 – 4 °C. After 24 h, the bags were opened and the chicken breast strips were removed from the soaking solution and put on a stainless steel net for 5 min to drain excess brine solution, then the increase in the weight of the chicken breast strips acquired from the marinade solution was calculated according to the following formula (Smith, et al. [13]).

Deep Frying of Marinade Chicken Breast Strips: 1.5 liters of a mixture of sunflower and soybean oil 1: 1 were placed in an electric fryer and the oil temperature was raised to 186: 188 °C, then the marinated and covered chicken breast slices were placed in the oil at a rate of 4 pieces each time and the weight of the piece was approximately 40 g .When the temperature of the chicken breasts reached 74 - 76 °C, they were removed from the oil and placed on a stainless steel mesh to get rid of the excess oil from the throwing process in the control sample. In the treatment sample (without MSG), the same previous steps were repeated after getting rid of the frying oil used in the control sample and replacing it with a new oil of the same type of oil. Samples were preserved by freezing at -18 °C until the completion of the tests (Park, et al. [14]).

Chemical Analysis

Moisture, ash, crude protein, and crude lipids (%) were determined according to the methods recommended by (AOAC [15]) while total carbohydrate content was measured by difference.

Microbiological Examination

Preparation of Samples for Microbiological Examination

Ten g of each sample were homogenized with 90 mL of sterile saline solution (9 g NaCl/ L distilled water). The suspension was shocked by shaker for 5 min to give 0.1 dilutions. Then different dilutions (1: 10-1 to 1: 10-6) were prepared to be used for microbiological examination.

Aerobic Plate Count (APC)

The aerobic plate count (APC) was performed as described in (APHA [16]).

Molds and Yeasts

Potato dextrose agar was used for yeast and mold enumeration. Plates were incubated at 25°C for 5 days, according to (APHA [16]).

Total Coliform Bacteria Count

Violet red bile agar was used for the enumeration of coliforms. Plates were incubated at 37°C for 24 h, according to (APHA [16]).

Staphylococcus Aureus

Staphylococcus aureus test was performed as described in ISO, (ISO [17]).

Salmonella SPP

Salmonella SPP test was performed as described in ISO, (ISO [18]).

Freshness Tests

PH Value ( EOS [19])

In astomacher, approximately 10 g of the examined sample were homogenized with 25 mL of neutral distilled water, and left to stand for 10 min at room temperature with continuous shaking and filtered. The pH was determined by using electrical pH meter (ACTWA-AD1200-1034678) calibration of pH meter by using two buffer solutions of exactly known pH (alkaline pH 7.01, acidic pH 4.01). Therefore, pH electrode was washed with neutralized water and then introduced into the homogenate.

Determination of Total Volatile Basic Nitrogen (TVB/ N)”mg” % ( EOS [20])

Ten g of sample were minced in a stomacher for 1-2 min until homogenization. Then in a distillation flask add 2 g of magnesium oxide and 300 mL distilled water to the minced sample. Make distillation and receive 100 mL distillate within 30 min in a beaker contain 25 mL of 2% boric acid. Then titrate against H2SO4 0.1M until faint pink color.

TVN mg /100g = R×14

Where R is the volume of H2SO4 exhausted in titration.

Determination of Thiobarbituric Acid (TBA)”mg/Kg” ( EOS [21])

TBA number is expressed as milligrams of malondialdehyde equivalents per kilogram of sample. Ten g of sample were blended with 48 mLof distilled water, to which 2 mL of 4% of ammonium chloride (to bring’s the pH to 1.5) were added in astomacher for 2 min and left at room temperature for 10 min. The mixture was quantitatively transferred into Kjeldahl flasks by washing with additional 50 mL distilled water, followed by an anti-foaming preparation and few glass beads. The Kjeldahl distillation apparatus were assembled and the flask was heated to 50°C. 50 mL distillate was collected in 10 min from the time of boiling commences. The distillate was mixed, and then 5mL was pipette into a glassstoppard tube. 5mL of TBA reagent (0.2883g/100mL of 90% glacial acetic acid) were added. The tube was stoppered, shacked and immersed in boiling water bath for 35 min. A blank was similarly prepared using 5mL distilled water with 5ml TBA reagent and treated like the sample. After heating, the tube was cooled under tape water for 10 min. A portion was transferred to a curette and the optical density (D) of the sample was read against the blank by means of spectrophotometer (Perkin Elmer, 2380, USA) at a wave length of 538nm.

TBA value (mg malondialdehyde / kg of sample) = D×7.8

D: the read of sample against blank.

Physical Tests

Water Holding Capacity (WHC) and Plasticity

Water holding capacity (WHC) and plasticity were measured according to the method described by (Soloviev [22]). A weight of 0.3 g of ground meat was placed under ash less filter paper (Whatman, No. 41) between tow glass plates (20x20 cm) and pressed for 10 min., using 1 Kg weight. Two zones were measured using the planimeter, the water holding capacity was calculating by subtracting, the area of the internal zone from that of the outer zone. The internal zone represented the plasticity. Results were presented in cm2 per 0.3 g of raw sample.

Cooking Loss

Samples weighing 25-30 g (W1) were packed in plastic tubes. The tubes were then heated at 95°C, until the internal temperature of the samples reaches 75°C. The temperature was checked using thermocouples inserted in the center of the sample. The samples were considered cooked when the internal temperature reached 75°C after cooking, the meat was weighed again (W2) to determine the loss in weight during cooking as described by (Mamaghani [23]).

Cooking loss (%) = (W1− W2 / W1) x100

Sensory Evaluation

Ten experienced panelists made a sensory evaluation of full fried chicken strips.For the following attributes, each panelist was invited to give a numerical value from 0 to 10. Scores extended from 1 to 10 which illustrate dislike extremely to the like extremely. The sensory characteristics assessed were texture, color, odor and crispness (Ramadan, et al. [24]).

Statistical Analysis

All data of the present study were subjected to analyses of variance (AVOVA) using software (SAS institute [25]) Differences between means were collected by the least significant differences (LSD) at p< 0.05.All measurements were carried out in triplicate.

Results and Discussion

Physicochemical and Microbiological Properties of Raw Chicken Breast Strips

The chemical composition of raw chicken breast strips is presented in (Table 3) .Moisture, protein, fat, carbohydrate and ash contents of raw chicken breast strips were (73.66, 20.72, 2.4, 1.18 and 1.18 g/100g respectively. These results are in agreement with the data obtained by (Petracci, et al. [26]) who found that moisture, protein, fat and ash contents of raw chicken breast meat were 75.10, 22.90, 0. 78, and 1.30 g/100g respectively. Total volatile based nitrogen (TVBV) (mg/100g) and thiobarbituric acid milligrams of malonaldehyde (TBA) mg / kg of raw chicken breast strips were (11.34 mg/100g and 0.24 (MA) / kg), respectively. These results are in agreement with the data obtained by (Kim, et al. [27]). Data presented in (Table 3) showed that the color values (L*, a*, and b*) of raw chicken strips were 55.6, 3.2 and11.5 respectively. Whilewater holding capacity (WHC) and pH values of raw chicken strips were 44.2 and 5.09 respectively. These results are in agreement with the data obtained by (Qiao, et al. [13,28]). Total aerobic bacterial, coliform, E. coli, salmonella, staph. Positive coagulase psychrophilic bacteria and yeast and mold counts of raw chicken breast strips were presented in (Table 3). Total aerobic bacterial, coliform, salmonella, staph. aureus, psychrophilic bacteria and yeast and mold counts were 2.6× 105, 0.72× 102 , ND, ND, 0.67× 102 , 3.3× 106 and 6.4× 101 respectively. These results are in agreement with the data obtained by (Eglezo et al. [29-31]).

Table 3: Physicochemical and microbiological properties of raw chicken breast strips.

Chemical Composition of Deep Fat Fried Chicken Breast Strips During Frozen Storage (-18±1°c)

Chicken breast strips samples werechemically analyzed to determine the gross chemical composition and physical properties. The obtained data are shown in (Table 4). It could be noticed that moisture loss of deep fat fried chicken breast stripssignificantly decreased as a function of storage time for both samples. The control samples had statistically higher moisture contents than the treated fried samples. This could be due to water loss during frying. All coatings provided a beneficial barrier for moisture and preserved samples from moisture loss during storage. The lower water loss for the coated deep fat fried chicken breast stripsmight be due to controlling the loss of water and reducing dehydration. Similar results were observed by (Hwang, et al. [32] and Prejsnar, et al [33]). The crude protein content of deep fat fried chicken breast stripsdecreased by replacing MSG with a mixture of 1:1 sugar and salt this may be due to containing of MSG on amino acids. The crude protein content of all treatments slightly increased as storage period progresses.

Freezing storage has been shown to induce protein carboxylation, and the formation of Schiff bases in chicken meat (Utrera, et al. [34]) Freezing storage has impacts on the activities of endogenous proteolytic enzymes responsible for the degradation of meat protein as well as the relaxation of meat tissue structures (Farouk, et al. [35]) Study conducted by (Smiecinska, et al. [36]) revealed increased content of both total and soluble protein in breast meat after 6 weeks of freezing storage. Similar results were observed by (Hwang, et al. [32] and Prejsnar, et al. [33]) With regard to fat content of chicken breast strips died not affecting by replacing. Control samples had the highest fat content than treated samples. The fat content of all treatments slightly decreased as storage period progressed. This decrease of fat content may be explained by the autolysis of lipid (El-Nashi, et al. [37]). Carbohydrate and ash content were higher in sample containing sugar and salt mixture as alternative for MSG. The observed reduction in ash content was probably due to increased meat leakage during the fried process, hence the subsequent increased loss of mineral salts. (Chwastowska, et al. [38]) Also demonstrated the impact of thawing (in atmospheric air and microwave) methods on the ash content of pork meat.

Table 4: Effect of replacing monosodium glutamate with mix of sugar and salt in ratio of 1:1 on chemical composition of deep fat fried chicken breast strips during frozen storage (-18°C).

Note: Values (means ±SD) with different superscript letters are statistically significantly different (p≤ 0.05).

Physiochemical Quality of Deep Fat Fried Chicken Breast Strips During Frozen Storage (-18±1°c)

From data presented in (Table 5). It could be noticed that the pH value of deep fat fried chicken breast strips during frozen storage of both treatments increased as storage period progressed. Treatment contains mix of sugar and salt in ratio of 1:1 as MSD alternative had the higher pH values than treatment containing MSG (control sample). These results are in agreement with those obtained by (Hwang, et al. [32,33]). The slight increase in pH during storage may be due to inhibition of bacterial activity during frozen storage as (Bouacida, et al. [39]). The TVBN of both treatments increased as storage period progressed. Treatment containing mix of sugar and salt in ratio of 1:1 as MSD alternative had the lower TVBN values than treatment containing MSG (control sample) .The increasing in TVBN value due to the breakdown of nitrogenous substances by microbial activity as reported by (Prejsnar, et al. [33]). On the other hand, the TBA values of both treatments increased as storage period progressed. Treatment contains mix of sugar and salt in ratio of 1:1 as MSD alternative had the lower TBA values than treatment containing MSG control sample.

Table 5: Effect of replacing MSG with mix of sugar and salt in ratio of 1:1 on physiochemical quality of deep fat fried chicken breast strips during frozen storage (-18°C).

Note: Values (means ±SD) with different superscript letters are statistically significantly different (p≤ 0.05).

These results are in agreement with those obtained by (Hwang, et al. [32,33]) The increasing of TBA value taken place due to lipid oxidation as reported by (El Gharably, et al. [40]). However, a high degree of poly unsaturation accelerates oxidative processes leading to deterioration in meat flavor, color, texture and nutritional value (Mielnick, et al. [41]). Water holding capacity (WHC) of deep fat fried chicken breast strips during frozen storage of both treatments decreased as storage period progressed. Treatment contains mix of sugar and salt in ratio of 1:1 as MSD alternative had the higher WHC values than treatment containing MSG (control sample). These results are in agreement with those obtained by (Prejsnar, et al [33,39]) The cooking loss of deep fat fried chicken breast strips increased significantly as storage period progressed for all samples. Treatments containing MSG had the higher cooking loss percentage values than control sample. These results are in agreement with those obtained by (Aksu et al. [42-44,32,33])

Microbiological Examinations of Deep Fat Fried Chicken Breast Strips During Frozen Storage (-18°C)

The microbiology examinations of deep fat fried chicken breast strips during frozen storage (-18°C) were examined to determine some microbiological quality and shelf life validity throughout frozen storage. Microbial growth in meat and meat products can result in slime formation, structural components degradation, decrease in water holding capacity, off odors, and texture and appearance changes which reduce their quality, nutritional value and reduce the shelf life (Doulgeraki, et al [45]).

Total Bacterial Count

(Table 6) shows that there were significant differences in viable bacterial count between the control chicken breast strips and other chicken breast strips sample. The results indicated that total bacterial count decreased gradually throughout the storage period until the end of storage period. The obtained results also showed that control chicken breast strips had the highest counts of total bacterial count than other treatment. This might due to the antimicrobial activity of salt or sugar (Shee, et al [46]). Similar results were reported by (Aksu, et al. [42,47,32,33,39]).

Total Coliform Count

(Table 6) shows the differences in coliform counts. The results indicated that total coliform count decreased gradually throughout the storage period until the end of storage period. The obtained results also showed that control chicken breast strips had the lowest counts of total coliform count than other treatment. Similar results were reported by (Hwang, et al. [32,33,39,44]).

Table 6: Effect of replacing MSG with mix of sugar and salt in ratio of 1:1 on microbiological quality of deep fat fried chicken breast strips during frozen storage (-18°C).

E. Coli Count

The results presented in (Table 6) indicated that total E. coli count did not detect in both treatments until the end of storage period. Similar results were reported by (Hwang, et al. [32,33,39,44]).

Salmonella Count

The results presented in (Table 6) indicated that Salmonella did not detect in both treatments until the end of storage period. Similar results were reported by (Hwang, et al. [32,33,39,44]).

Staph. Aureus Count

(Table 6) shows the differences in Staph coagulase counts. The results indicated that total Staph aureus count decreased gradually throughout the storage period until the end of storage period. The obtained results also showed that control chicken breast strips had the lowest counts of total Staph coagulase count than other treatments. Similar results were reported by (Hwang, et al. [32,33,39,44]).

Psychrophilic Bacteria Count

(Table 6) shows the differences in psychrophilic bacteria counts. The results indicated that total psychrophilic bacteria count increased gradually throughout the storage period until the end of storage period. The obtained results also showed that control chicken breast strips had the lowest counts of total psychrophilic bacteria than other treatment.Similar results were reported by (Hwang, et al. [32,33,39,44]).

Yeast and Mold Count

The differences in yeast and mold counts of deep fat fried chicken breast strips during frozen storage are shown in (Table 6). The results indicated that total yeast and mold count decreased gradually as the storage period progressed until the end of storage period. The obtained results also showed that control chicken breast strips had the lowest counts of total yeast and mold than other treatment. Similar results were reported by (Hwang, et al. [32,33,39,44]).

Sensory Evaluation of Deep Fat Fried Chicken Breast Strips During Frozen Storage (-18°C)

Poultry meat is a nutritious food and it is consumed all over the world because of its relatively low cost and low fat content. However, it is highly perishable with a relatively short shelf life even when it is kept under refrigeration. Thus, developing more appropriate technologies for its preservation could be highly useful, in order to increase the shelf life of meat products (Mantilla, et al. [48]). Statistical analysis appears a significant difference in sensory evaluation between both samples. Treatment containing MSG (control) had the higher sensory proprieties (color,taste, crispness,odor and acceptability) than that treatment containing mix of sugar and salt as MSG replacer (Table 7). The overall acceptability of deep fat fried chicken breast strips during frozen storage (-18±1°c) were significantly higher in (C), while it was significantly lower in the sample treated with mix of sugar and salt as MSG replacer . Statistical analysis appears a significant difference in overall acceptability between both samples. These results are in agreement with those obtained by (Hwang, et al. [32,33,39,44,49- 51]).

Table 7: Effect of replacing MSG with mix of sugar and salt in ratio of 1:1 on sensory evaluation of deep fat fried chicken breast strips during frozen storage (-18°C).

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