Names and abbreviations of 20 Amino Acids

1. Pakistan Society Of Chemical PathologistsDistance Learning Programme In Chemical Pathology(DLP-2)Lesson No 4Plasma ProteinsBy Brig Aamir IjazMCPS, FCPS, FRCP (Edin)Professor Of Pathology / Consultant Chemical PathologistAFIP Rawalpindi

2. leucine - leu - L lysine - lys - K methionine - met - M phenylalanine - phe - F proline - pro - P serine - ser - S threonine - thr - T tryptophan - trp - W tyrosine - tyr - Y valine - val - V alanine - ala - A arginine - arg - R asparagine - asn - N aspartic acid - asp - D cysteine - cys - C glutamine - gln - Q glutamic acid - glu - E glycine - gly - G histidine - his - H isoleucine - ile - I Names and abbreviations of 20 Amino Acids

3. b. Sixty one codons Q 1.In the process of protein synthesis, 20 amino acids are coded by:a. Sixty four codons b. Sixty one codonsc. Sixty one tRNA speciesd. Three nucleotides in DNAe. Two purines and one pyrimidine in mRNA

4. For biosynthesis of a protein (chain of upto 20 amino acids) one gene is required • A gene is a part of DNA molecule that codes for the synthesis of one specific protein (polypeptide chain) through its sequence of nucleotides. • Nucleotides of DNA are four i.e. Thymine, Cytosine, Guanine and Adenine DNA codes

5. T-A-C-A-A-G-C-A-G-T-T-G-G-T-C-G-T-G Example of DNA codes

6. Through transcription DNA code is copied into a single stranded messenger RNA (mRNA) • mRNA is coded as mirror image of DNA code with the exception of Thymine which is replaced by Uracil. Messenger RNA

7. DNA CODE T-A-C-A-A-G-C-A-G-T-T-G-G-T-C-G-T-G corresponding mRNA code A-U-G-U-U-C-G-U-A-A-C-C-A-G-C-A-C Example of mRNA codes

8. A codon is part of mRNA which comprises three nucleotides • Each codon has three positions: • First position (5\ end) • Second position (middle) • Third position (3\ end) e.g. in codon GAC, G is at 5\ end , A at 2nd position while C is at 3\ end A codon

9. Four nucleotides of mRNA makes 64 codons • Why? 64 Combinations of Nucleotide Triplets (codons)

10. The order of nucleotide in the triplet (i.e. codon) is important e.g. • U-U-U codes phenylalanine • U-C-U codes serine • U-A-A codes tyrosine • U-G-G codes tryptophan • So 16 codons will result with Uracil at First Position. • Another 16 with Cytosine at First Position and so on…. • In this way all four nucleotides will result into 64 codons • Please note that one amino acid may be coded by one, two, three, four, five or six codons (Please see table on next slide) 64 Codons

11. The Genetic Code(slide courtesy of Dr SabihaWaseem, Canada)

12. Three codons do not code any amino acids but they are stop codons • That is why we have 61 codons for 20 amino acids !!! Why 61 Codons for 20 Amino Acids

13. mRNA code A-U-G-U-U-C-G-U-C-A-A-C-C-A-G-C-A-C Name the Amino Acids coded in the above mRNA code (tip : Take help from table in Slide No 11) An Exercise

14. Biosynthesis of Proteins Slides courtesy of CaptSaima Bashir, AFIP Rawalpindi

15. A codon or triplet codeis determined by a base triple (three contiguous nucleotides). A codon provides information about a certain amino acid to be added into the peptide chain or for the chain to stop. Three codons in mRNA (UAA, UAG and UGA) are stop codons. The signals encoded by the codon form the genetic code. The end of the mRNA does not either code for amino acids, and makes up the 3'untranslated region The first nucleotides on the mRNA form a regulatory sequence, and do not code for amino acids. This sequence is also called the 5'untranslated region

16. Ribosomal RNA (rRNA) molecules diffuse from the nucleus to the cytoplasm and into the ribosomes, where it plays an essential role in the protein synthesis • The amino acids are carried on small RNA molecules called transfer RNA (tRNA). Each of these tRNA molecules contains an anticodon consisting of 3 unpaired nucleotides Every anticodon or base tripletrecognizes its complementary codon in the mRNA. The tRNA deposits its amino acid in the peptide chain.(Translation )

17. Following ribosomal translation, proteins are modified by addition of carbohydrates, cleavage of bonds within the new protein, shortening or folding. These processes are called post-translational modification Post-translational modifications

18. b. Cysteine Q 2.A 62 year male patient has following liver function tests:• Serum bilirubin: 123 μmol/L (<17)• Serum ALT: 92 U/L (<42)• Serum Albumin: 22 g/L (35-50)• PT: Failure to clotWhich of the following amino acids you will like to add in his diet:a. Alanineb. Cysteinec. Cystined. Glutamic acide. Glutathione

19. Essential amino acids (Slides Courtesy of Dr SobiaIrum, AFIP Rwp)

20. What are Essential Amino Acid An essential amino acid is an amino acid that cannot be synthesized by the organism and therefore must be supplied in its diet. Essential amino acids are "essential" not because they are more important, but because the body does not synthesize them. If they are not taken through diet, they will not be available for protein synthesis.

21. List of Essential Amino Acids phenylalanine valine threonine tryptophan methionine leucine isoleucine lysine histidine Mnemonic memorizing the essential amino acids : "Any Help In Learning These Little Molecules Proves Truly Valuable”

22. Nonessential Amino Acids *Are conditionally essential amino acids Alanine Arginine* Aspartic acid Cysteine* Glutamic acid Glutamine* Glycine* Proline* Serine* Tyrosine* Asparagine* Selenocysteine

23. Conditionally Essential Amino Acids The amino acids arginine, cysteine, glycine, glutamine, proline, serine and tyrosine are considered conditionally essential, meaning they are not normally required in the diet, but must be supplied exogenously to specific populations that do not synthesize them in adequate amounts For example individuals living with PKU cannot synthesize tyrosine from phenylalanine, so tyrosine becomes essential in the diet of PKU patients.

24. Cysteine L-cysteine is an important protein and a deficiency could cause serious health problems. It is used to help prevent major organ damage that can be caused by taking acetaminophen. In certain dietary supplement form, L-cysteine counteracts the mild pain reliever and its effects on the liver

25. c. Ornithine Q 3: A newborn with neurological deterioration with moderate hepatocellular disturbance, hypotonia, seizures and coma. His biochemical findings are:• Alkalosis: +• Ketones : +• NH3: ++++• Lactate: Normal• Glucose: NormalWhich of the following amino acid metabolism is most likely affected in this baby?a. Leucineb. Methioninec. Ornithined. Phenylalaninee. Tyrosine

26. Urea Cycle Defect

27. b. Glutamine Q 4: A multi-centre clinical trial carried out in UK examined the effects of an amino acid in the nutrition of infants undergoing gastrointestinal surgery.Which of the following amino acids was studied?a. Alanineb. Glutaminec. Homocysteined. Methioninee. Tryptophan

28. Surgical Infant Glutamine Nutritional (SIGN) Trial

29. e. β2 microglobulin Q 5 : A 59 year male has developed CKD with following biochemical picture:• Urea: 25.5 (3.3-6.7 mmol/l)• Creatinine: 656 (55-100 μmol/l)• pH : 7.23 (7.35-7.45)• PCO2 : 25.0 (35 - 45 mm of Hg)• HCO3 : 16.7 (22 - 28 mmol/l)• Serum Sodium 141 (136-149 mmol/l)• Serum Potassium 4.8 (3.5-5.0 mmol/l)Which of the following proteins of this patient will be markedly raised?a. Albuminb. Alpha 1 Antitrypsinc. Apolipoprotein Bd. Fibrinogene. β2 microglobulin

30. d. Pro-calcitonin c. Lipopolysaccharide Binding Protein (Marks will be given to both) Q 6 : A 28 year female patient is being treated in an ICU for suspected gram-negative septicaemia following a complicated delivery. Which of the following proteins will be helpful for the diagnosis or exclusion of a gram-negative etiology? :a. C-Reactive proteinb. Haptoglobinc. Lipopolysaccharide Binding Proteind. Pro-calcitonine. Serum amyloid A

31. Both are markers of bacterial septicaemia. • LPBP has been claimed to be a differentiating marker for gram negative bacteria • But recent studies have shown doubts as its role in differentiating gram positive from gram negative septicaemia. • PCN has been shown to be a differentiating marker. Lipopolysaccharide Binding Protein (LPBP) and Pro-calcitonin (PCN)

32. c. Multiple sclerosis Q 7 : A 31 year female has undergone CSF examination for the exclusion of a neurological disorder lasting for the last a few weeks. Her CSF findings are: CSF• WBCs : 29 x 106/L• Lymphocytes: 80%• Glucose : 4.1 mmol/L • Total Protein: 420 mg/L • Lactate: 1.7 mmol/L• Serum/CSF Albumin Index: 7• IgG: Albumin Ratio: 0.35• Oligoclonal band seen in CSF by isoelectric focusingSerum• Glucose: 5.5 mmol/L• No oligoclonal band seen by isoelectric focusingThese findings are most probably suggestive of:a. Clinically isolated syndromeb. Monoclonal gammopathyc. Multiple sclerosisd. No neurological disease e. Tuberculous meningitis

33. A neurological deficit which lasts for < 24 h is called clinically isolated syndrome (CIS) while in MS the deficit(s) may last for > 24 h. • Most important biochemical finding in MS is demonstration of local synthesis of oligoclonal band (OCB) in CSF. • Detection of OCB in CSF by Isoelectric Focusing is the gold standard in the lab diagnosis of MS.. Multiple Sclerosis (MS)

34. Matching the options:(Extended Matching Questions)

35. Options • Biuret method • Bromocresol green • Chemiluminescence • Chromatography on a dedicated HPLC • Chromatography on an HPLC used for Vitamin A estimation • Detection of Bence Jones proteins • Immunoturbidimetry • Isoelectric focusing • Protein electrophoresis • Pyrogelol dye binding method • Radial immunodiffusion • Radioimmunoassay

36. Generally proteins present in serum in large quantities e.g. Total Proteins and Albumin require relatively simple methods e.g. Biuret and bromocresol green. • More sophisticated techniques are required for estimation of smaller quantities e.g. albumin in urine and CSF. • Protein electrophoresis requires an expertized interpretation which a qualified pathologist can provide. • Quantitation of individual amino acids require highly specialized equipment e.g. dedicated HPLC (e.g. Biochrome) Selection of Method for Proteins and Amino Acids – Some Guidelines

37. a.Biuret Method Q 8 :DrZahid is working in a District Headquarters Hospital and wants to analyse Total Protein in patient samples on a semi-automatic analyser. He expects a range of protein of 65 – 80 g/L (6.5 – 8.0 g/dl). Which method / technique he should select?

38. b. Bromocresol Green Q 9 :DrAlam has started his private lab in a neighbourhood of many GP and specialist clinics. He wants to include Albumin analysis in his LFT profile. He has already procured a Chemical Pathology Autoanalyser. He expects a range of albumin of 35 – 50 g/L (3.5 – 5.0 g/dl). Which method / technique he should select?

39. d. Chromatography on a dedicated HPLC Q 10 : Prof Mahjabeen is planning to start service for the diagnosis of Inborn Errors of metabolism in her lab. She is especially interested to estimate branched chain amino acids for the diagnosis of Maple Syrup Urine Disease. Which method / technique she should select?

40. f. Detection of Bence Jones proteins Q 11 :Mr Zain is in-charge of a side lab in a cancer outpatient clinic. He has been requested by the clinicians to start a preliminary urine test for the screening of Multiple Myeloma. Which method / technique he should select?

41. g. Immunoturbidimetry Q 12 :DrZoya, a Resident of Chemical Pathology at a teaching hospital, has been given an assignment to establish a service of Albumin: Creatinine ratio. There is already a method for urinary creatinine in the lab and she is now in search of a method for albumin. Which method / technique she should select?

42. g. Immunoturbidimetry Q 13 :DrFakharUddin is planning a research project on biochemical analysis of CSF. He wants to estimate albumin in CSF for the determination of integrity of blood brain barrier. Which method / technique he should select?

43. g. Immunoturbidimetry Q 14 :Dr Ali, a consultant Chemical Pathologist of a tertiary-care hospital lab, wants to start ceruloplasmin levels for the monitoring of Wilson`s disease. Which method / technique he should select?

44. Q.15: Several thousand proteins have been described in the plasma. Please write THREE lines each regarding clinical significance of following plasma proteins : Factor H Gc-Globulin Gelsolin Haptoglobin Inter-α-trypsin inhibitor (Slides courtesy of: DrUzma Ansari and DrSafia Anwar, AFIP Rwp)

45. It is soluble complement control glycoprotein that principally regulates the alternative complement pathway and prevents complement mediated injury to healthy host tissue. • Overactive factor H may result in reduced complement activity on pathogenic cells, increasing susceptibility to microbial infections. • Underactive factor H may result in increased complement activity on healthy host cells resulting in autoimmune diseases. Factor H

46. Predisposition to : • Age related macular degeneration. • Atypical haemolytic uremic syndrome. • meningioproliferative Glomerulonephritis type II with 50% chances of developing ESRD. Mutation or SNPs in FH gene

47. Group specific component globulin ( Gc-globulin ) is a multifunction glycoprotein. • Its main function is to bind with vitamin D and procoagulantactin released from damaged tissue thus also called vitamin D binding protein (DBP). • Gc-globulin has over 124 variants worldwide and these are related to variable susceptibility and prevalence of diseases like COPD, DM, and multiple sclerosis. Gc-Globulin

48. Low levels are bad prognostic sign in • Fulminant hepatic failure • Sepsis • Paracetamol overdose • Massive trauma (as it can lead to a vicious circle of actin release, organ damage and further actin release) • Macrophage stimulating activities of Gc-globulin are utilized in inhibition of the growth of various types of cancers. Clinical significance of Gc-Globulin

49. Gelsolin-like actin-capping protein (CapG) is a ubiquitous gelsolin-family actin-modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis, and motility • It is a tumor marker in Oral squamous cell carcinoma & premalignant condition, overexpression is detected either immunohistochemistry or qRT-PCR • Reduced levels of plasma Gelsolin are used as prognostic marker in sepsis, malaria, prolong hypoxia and respiratory distress syndrome Gelsolin (CapG)

50. It is a plasma protein (α2-globulin) produced by liver. As an antioxidant, it binds free haemoglobin in blood thereby preventing Hb-induced oxidative damage especially renal injury. • Haptoglobin assay is used to screen and monitor for intravascular hemolysis. • It is a positive acute phase protein and its levels are increased in any inflammatory, infectious or malignant condition. Haptoglobin