Enzymes Part 2

Enzyme Concentraation

Substrate Concentration

Co-factors

Factors affecting Enzymatic Reaction

7 to 8

Most physiologic reaction of Enzymes occurs at a pH of

Low temperature (refrigeration, freezing)

renders an enzyme reversibly inactive

\-20C

ideal temperature for the preservation of enzymes (longer period of time)

2-8C

ideal storage temperature for substrates and coenzymes

22C or room temperature

ideal storage for LDH (LD4 and LD5)

Hemolysis

mostly increases enzyme concetration

Latescence or milky specimen

decreases enzyme concetration

Active site

a water free cavity, where substrate interacts with particular charged amino acid residue

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is a 3 dimensional protein structure

Allosteric site

a cavity other than the active site

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may bind regulator molecules

prosthetic group

When bound tightly to the enzyme, the coenzyme is called

proenzyme or zymogen

Digestive enzyme in its inactive form originally secreted from the organ of production is called

Fixed time

Continuous monitoring/Kinetic Assay

To measure the extent of enzymatic reactions, 2 general methods may be used

Alkaline Orthophosphoric Monoester Phosphohydroloase

Alkaline Phosphatase can also be called as

ALP

non specific enzyme capable of reacting with many different substrate

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functions to liberate inorganic phosphate from an organic phosphate ester with the concomitant production of an alcohol

ALP

no 1 indicator for obstructive jaundice

Liver ALP

Most anodal ALP isoenzyme

Bone ALP

Most heat labile ALP isoenzyme

Placental ALP

Most heat stable ALP isoenzyme

Intestinal ALP

Least anodal ALP isoenzyme

8\.6 – 10 (Rodriuez); 9.0 – 10 (Bishop)

ALP optimum pH

30-90 U/L

Alkaline Phosphatase reference value

Regan ALP

Found in lung, breast, ovarian, and gynecological cancers

Bone ALP co-migrator

Most heat stable  ALP (65C for 30 minutes)

phenylalanine 

Regan ALP is inhibited by

Nagao ALP

Found in adenocarcinoma of the pancreas and bile duct, pleural cancer

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Variant of Regan ALP

L-leucine and phenylalanine

Nagao ALP is inhibited

magnesium

activator of ALP

phosphorous

ALP is inhibited by

Placental ALP

us a useful tumor marker in serum and CSF for most germ cell tumors – CSF levels of ___ are of diagnostic value in differentiating whether a tumor in the pineal body is a pinealoma or a germ cell tumor.

Paget’s disease

For bone disorders the highest elevation of ALP occurs in

B1x

Bone ALP isoform, __ , was detected in the serum  of dialysis patient.

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__ isoform is used to study low bone mineral disease (BMD)  in patients with chronic kidney disease.

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___ isoform is also increased in the serum of the individuals with BMD of the hip, which is predominantly made up of trabecular bone.

Placental > Intestinal > Liver > Bone

Decreasing order of ALP heat stability:

phenylalanine

Placental and Intestinal ALP are inhibited by

Bone ALP

3M Urea inhibits

Bowers and Mc Comb (Szasz modification)

Is considered as the most specific method for ALP; IFCC recommended method.

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It is a continuous-monitoring technique which requires a pH environment of 10.15 and should be read at 405nm.

yellow

Bowers and Mc Comb (Szasz modification) color of product

1\.Electrophoresis

2\.Heat Fractionation/Stability Test

3\.Chemical Inhibition Test

4\.Bowers and Mc Comb (Szasz modification)

Alkaline Phosphatase methods of detection

Ingestion of food

leads to release of intestinal ALP into lymphatic fluid, and may transiently increase plasma levels of ALP.

zinc

an electrolye component of ALP

Acid Orthostatic Monoester Phosphohydrolase

ACP can also be called as

Acid Phosphatase (ACP)

For detection of Prostatic Carcinoma.

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It is also useful in forensic clinical chemistry, investigation of rape cases – vaginal washings are examined for seminal fluid ACP activity. (3 - 4 days)

Prostate (major)

RBC

Platelet

Liver

Bone (Osteoclasts)

Major sources of ACP

Thymolpthalein monophosphate

the specific substrate; substrate of choice for quantitative endpoint reaction of ACP.

Alpha-naphthyl PO4

upreferred for continuous monitoring methods of ACP

6\.5

uIf not assayed immediately, serum should be frozen or acidified to a pH lower than ___ With acidification, ACP is stable for 2 days at RT.

20mM L-tartrate ions

Prostatic ACP is inhibited by

1mM cupric sulfate and 2% formaldehyde ions

Red cell ACP is inhibited by

Serum glutamic-oxaloacetic transaminase (SGOT)

Aspartate Aminotransferase(AST) can also be called as

Cardiac tissue

Liver Tissue

Skeletal Tissue

Aspartate Aminotransferase(AST) major tissue sources

Kidney

Pancreas

RBC

Aspartate Aminotransferase(AST) other tissue sources

Cytoplasmic AST – predominant in Serum

Mitochondrial AST

2 Isoenzymes fractions of AST

Aspartate Aminotransferase(AST)

Diagnostic Significance:

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In the evaluation of :

Myocardial Infarction (AMI)

Hepatocellular disorder

Skeletal muscle involvement

6-8, 24, 5

In the AMI, AST level begins to rise __ hrs, peak at *hrs and normalize within* _days.

AST

used for monitoring therapy with potentially hepatotoxic drugs; 3x the upper border of normal signals cessation of therapy.

Karmen Method

Method currently used for AST identification

7\.5

Karmen Method pH

17 ± 5 hours

Half-life: AST

47 ± 10 hours

Half-life: AST

Serum glutamic-pyruvic transaminase (SGPT)

Alanine Aminotransferase(ALT) can also be called as

Alanine Aminotransferase(ALT)

It has enzymatic activity similar to AST.

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The highest concentration is in the liver; more specific than AST.

Liver

Alanine Aminotransferase(ALT) major tissue source

ALT

most specific marker for hepatocyte injury

ALT levels

used to screen blood donors.

plasma, bile, CSF and saliva

Aminotransferases are present in human

pyridoxal phosphate (vitamin B6)

Aminotransferases require ____ as coenzyme (prosthetic group).

Coupled Enzymatic reaction

Alanine Aminotransferase(ALT) reaction method

>1.0

In acute hepatitis, the De Ritis Ratio (ALT:AST) is

necrotic

ALT is slightly increase in obstructive jaundice but markedly increase in ___ jaundice.

Alpha-1-4 Glucan -4-Glucohydrolase

α-Amylase (AMS) can also be called as

α-Amylase (AMS)

It catalyzes the breakdown of starch and glycogen – an important enzyme in the physiologic digestion of starch.

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It is the smallest enzyme in size ( with a MW of 50,000 – 55, 000 daltons) – normally filtered in the renal glomerulus and also appears in the urine.

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It is the earliest pancreatic marker.

Acinar cells of the pancreas and salivary glands

Major Tissue Source of amylase

S-type (ptyalin)

α-Amylase isoenzyme anodal

P-type (amylopsin)

α-Amylase isoenzyme cathodal

P3

is the most predominant pancreatic amylase isoenzyme in acute pancreatitis. (There is no _ elevation in renal failure).

P2, S1 & S2

are the most commonly observed fractions of amylase

2 – 12, 24 hours, 3 – 5 days

α-Amylase In acute pancreatitis (AP) rises in after attack, peaks at _ normalizes in

NaCl

Samples with high activity AMS should be diluted with __ to prevent inactivation.

starch

Substrate for all the Methods in Amylase

Saccharogenic

Amylase Method

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It is the classic reference method expressed in Somogyi units.

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It measures the amount of reducing sugars produced by the hydrolysis of starch by the usual glucose methods.

Amyloclastic

It measures amylase activity by following the decrease in substrate concentration (degradation of starch).

Chromogenic

it measures amylase activity by the increase in color intensity of the soluble dye-substrate solution produced in the reaction.

Coupled - enzyme

It measures amylase activity by a continuous-monitoring technique.

wheat germ lectin

Salivary AMS is inhibited by

1% – 4% (0.01 -0.04)

Normal Amylase/Creatinine ratio

>4% (up to 15%)

α-Amylase (AMS)

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A:C Ratio (AP)

Triacylglycerol Acylhydrolase

Triacylglycerol Lipase (LPS) can also be called as

Triacylglycerol Lipase (LPS)

Is an enzyme that hydrolyzes the ester linkage of fats to produce alcohol and fatty acids.

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It is the most specific pancreatic marker – secreted exclusively in the pancreas; not affected by renal disorders.

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Plasma concentrations are normal in conditions of salivary gland involvement.

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Late pancreatic marker.

Pancreas

Major tissue source of Lipase

olive oil

Lipase uses __ as substrate

Hemoglobin

inhibits the activity of LPS leading to falsely low values.

Triolein

also as a substrate for LPS assay.

Cherry Crandal

reference method for Lipase

Cherry Crandal

Hydrolysis of olive oil after incubation for 24 hours at 37°C and titration of fatty acids using NaOH.

Fatty Acid

Cherry Crandal end product

Peroxidase coupling

most commonly used method for Lipase; does not use 50% olive oil.

LD-1 & LD-2

Heart, RBC, Kidneys

LD-3

Lungs, Pancreas, Spleen

LD-4 & LD-5

Skeletal muscles, Liver , Intestine

LD-5

cathodal isoenzyme of LD

LD-1

anodal isoenzyme of LD