Exam #2

How is urea synthesized?
Proteins are catabolized into amino acids via enzymatic proteolysis. The amino acids undergo transamination and oxidative deamination to be converted into ammonia, which enters the “urea cycle”. Urea is synthesized from the “urea cycle”
What are some of the modes of urea excretion?

90% of urea is excreted through its kidneys

 

There are some losses through the gastrointestinal tract and skin

Disease states and disorders associated with urea (BUN) measurement

Kidney disease shows accumulation of urea in blood

 

Uremic (azotemic) state: increase in [blood urea] 

Discuss the methodologies used for urea nitrogen (BUN) measurement (pages 369 – 370)

Fearon Reaction (chemical method): molecules of diacetyl condense with those of urea to form the chromogen diazine, which absorbs strongly at 540 nm.

 

Enzymatic Methods: based on preliminary hydrolysis of urea with urease to generate ammonia; use spectrophotometry (Berthelot reaction and enzymatic assays)

Chemical structure of creatinine
cyclic anhydride of creatine
Synthesis of Creatinine (see p. 365)
final product of decomposition of phosphocreatine as well as the spontaneous dehydration of creatine
Mode of creatinine excretion

Excreted in the urine

 

(note: although it is present in all body fluids and secretions and is freely filtered by the glomerulus)

Clinical significance of creatinine

marker of glomerular filtration rate

 

implicated in diabetes, hypertension, and congestive heart failure

principle of Jaffe reaction
creatinine reacts with picrate ion in an alkaline medium to yield an orange-red complex
Analytical problem of Jaffe raction

lack of specificity for creatinine

 

many compounds have been reported to produce a Jaffe-like chromagen 

The greatest success in common usage and specificity for the Jaffe reaction (chemical method)
use of a kinetic measurement approach in combination with careful choice of reactant concentrations
Advantages of kinetic assays for Jaffe Reaction
more specific, faster, and the analysis is automated
Maximum concentration of picrate that can be used in a Jaffe reaction. Also name the concentration range employed in most methods.

30 mmol/L maximum

 

3 – 16 mmol/L

What can high concentrations of hydroxide cause in the Jaffe reaction?
interference
Absorbance maximum of the Jaffe reaction
490 – 500 nm
chemical sructure of uric acid
nitrogenous compound (2,6,8-trihydroxypurine)
Synthesis of uric acid (p. 370 – 371)

major product of catabolism of the purine nucleosides adenosine and guanine.

 

Both endogenous and exogenous nucleic acids are degraded/catabolized into uric acid.

Mode of excretion of uric acid

Highly present in the urine and can be found in serum

 

 

Did you know that it can be crystallized in the serum and deposited in the joints?

Clinical significance of uric acid

there are more than 20 disorders of purine metabolism that cause hyperuricemia and hypouricemia

 

suspicious symptoms: kidney failure/stones in children or young adult, unexplained neurological problems in infant – adolescent, gout in man/woman <30 years old

Cause of gout
monosodium urate precipitates from supersaturated body fluids
What are the three methods to measure uric acid?
Phosphotungstic Acid Method, Uricase Method, and HPLC Method
Drawback of the Phosphotungstic Acid (PTA) Method
sunject to many interferences
Advantage of the Uricase Methods
more specific than the PTA methods
Carbohydrate
aldehyde or ketone derivatives of polyhydroxy alcohols composed of carbon, hydrogen, and oxygen in a ratio of 1:2:1
Monosaccharide
a simple sugar that consists of a single polyhydroxy aldehyde or ketone unit and is unable to hydrolyzed to a simple form
Disaccharide
two monosaccharides joined covalently by an O-glycosidic bond, with the loss of a water molecule.
Polysaccharide
the linkage of multiple monosaccharide units
glycosidic linkage
chemical bond between monosaccharides?
Aldose
 monosaccharide (a simple sugar) that contains only one aldehyde (-CH=O) group per molecule
Ketose
a monosaccharide containing one ketone group per molecule.
Hexose
any of the class of simple sugars whose molecules contain six carbon atoms, such as glucose and fructose.
Isomer
each of two or more compounds with the same formula but a different arrangement of atoms in the molecule and different properties
Stereoisomer
compounds that are identical in composition and differ only in spatial configuration
Glycogenesis
formation of glycogen from glucose
Glycogenolysis
the breakdown of glycogen to glucose
Gluconeogenesis
production of glucose from noncarbohydrate sources
Glycolysis
the breakdown of glucose by enzymes, releasing energy and pyruvic acid
Anaerobic Glycolysis
transformation of glucose to pyruvate when limited amounts of oxygen
Acetyl-coenzyme A
acetyl ester of coenzyme A, involved as an acetylating agent in many biochemical processes
Glycogen
an extensively branched polysaccharide containing many glucose residues and found particularly in muscle and liver cells for glucose storage
Lipogenesis
the metabolic formation of fat/lipids
Lipolysis
the breakdown of fats and other lipids by hydrolysis to release fatty acids
Components of Maltose
Glucose + Glucose
Components of Lactose
Glucose + Galactose
Components of Sucrose
Glucose + Fructose
Starch Composition
a mixture of amyloses and amylopectins
Glycogen Composition
similar to amylopectins, but branching is more extensive and is evident every 8 to 12 glucose residues
Clinical Significance of Glycogen
Glycogen Storage Disease (p. 382)
Glycoprotein composition
proteins that have carbohydrate groups attached to the polypeptide chain
proteoglycan composition
protein bonded to glycosaminoglycan groups
Ingestion and Absorption of Carbohydrate

Once eaten, amylase (salivary) breaks down starch into glucose and disaccharides into monosaccharides

 

Carbohydrates are absorbed in the intestines and liver

main physiological functions of glucose

structureal components in RNA and DNA

 

source of energy

pentose phosphate pathway
a biochemical pathway parallel to glycolysis that generates NADPH and pentoses (5-carbon sugars)
Embden-Meyerhof pathway
metabolic pathway that converts glucose to pyruvate
Insulin effect in regulating blood glucose level
It decreases blood glucose level via glycogenesis, glucose uptake (entering the cell), and lipogenesis
Effects of glucagon in reguling blood glucose levels
increases blood glucose via glycogenolysis and gluconeogenesis
Maintenance of blood glucose levels in “fasting state”

1) Decrease in blood [glucose] stimulate ?-cells of pancreas to secrete glucagon

 

2) Glucagon causes lipolysis, glycogenolysis, and gluconeogenesis to occur, yielding normal blood [glucose]

The maintenance of glucose levels in the “fed state”

1) An increase in blood [glucose] cause the ?-cells of the pancreas to release insulin

 

2) Insulin causes lipogenesis, glucose uptake, and glycogenesis to occur

Growth hormone effect on blood glucose levels
The growth hormone stimulates gluconeogenesis, enhances lipolysis, and antagonizes insulin-stimulated glucose uptake
Type 1 Diabetes Mellitus

Abrupt onset of symptoms: polyuria, polydipsia, and rapid weight loss

 

Insulinopenia (deficiency of insulin) due to loss of pancreatic ? cells; autoantibody may be involved

 

Peak incidence occurs in childhood and adolescence

 

 

Type 2 Diabetes Mellitus

Minimal symptoms, not prone to ketosis, and not dependent on insulin

 

[Insulin] may be normal, decreased, or increased; insulin action may be impaired

 

Obesity is commonly associated

 

 

 

 

Gestational Diabetes Mellitus
Carbohydrate intolerance of variable severity with onset or first recognition during pregnancy.
Risks for Gestational Diabetes Mellitus

1) women who have marked hyperglycemia during or soon after pregnancy

 

2) women who are obese

 

3) women whose GDM was diagnosed before 24 weeks’ gestation

Principle of glucose oxidase method

Glucose + H2O + O2 >(glucose oxidase)> Glucaronic Acid + H2O2

 

H2O2 + Reducing Dye (color #1) >(peroxidase)> Oxidized Dye + H2O

 

The more oxidied dye there is, the more H2O2 produced, thus, the more glucose initially present

Interferences of the Glucose Oxidase method
uric acid, ascorbic acid, and bilirubin
Principle of Hexokinase Method

Glucose is phosphorylated in presence of hexokinase and forms Glucose-6-phosphate. Glucose-6-phosphate is oxidized by G6PD to another product in the presence of NADP+ or NAD+

 

The amount of reduced NADP or NADH produced is directly proportional to the amount of glucose in sample (340 nm ABS)

Required Sample Types for Hexokinase Method

plasma or serum.

 

NaF, with an anticoagulant may be used

 

Sources of Interferences for Hexokinase Methods

Hemolyzed specimens containing more than 0.5g of Hgb/dL 

 

drugs, bilirubin, and lipemia

Specimens for Glucose Oxidase Method
CSF
Method used in lab to measure Glycohemoglobin A1C
Helena GLYCO-Tek Affinity Column Method
Principle of the Helena GLYCO-Tek Affinity Column Method

The dihydroxyboryl group in the cellulose reesin has an affinity to cis-diol groups on simple sugars, thus allowing separation of glycated hemoglobins from non-glycated.

 

Elution with a basic developer removes non-glycated Hb. The glycated forms are eluted using sorbitol buffer. Then spectrophotometry

Specimen for Glycohemoglobin methods
Fresh, whole blood containing EDTA, heparin, or citrate. Packed cells are the specimen of choice.
Interfering substances for the Glycohemoglobin method
hemolyzed, lipemic, and icteric specimens
Patient Preparation for Glucose Tolerance Test

1) Patient has 3 days of unrestricted diet

2) Collect blood after patient has fasted for at least 8 hrs

 

Procedure for Glucose Tolerance Test

1) Collect patient sample from 8-hour fast

2) Give 75g of glucose

3) Collect blood every half hour (up to 2 hours)

4) Patient is considered diabetic if the 2-hour test tube (and another test tube) is ;200 mg/dL

 

Normally, 7% of blood glucose is lost every hour (cellular glycolysis)

GTT normal glucose level
70 – 90 mg/dL
Criteria for diagnosing Diabetes Mellitus in fasting plasma glucose
?126 mg/dL (7.0 mmol/L)
Criteria for diagnosing Diabetes Mellitus in 2-hour plasma glucose
?200 mg/dL (11.1 mmol/L)
The two families of protein that modulate glucose transport into the cells

1) sodium-dependent glucose transporter (promote the uptake of glucose and galactose from the lumen of the small bowel and their reabsorption from urine in the kidney)

 

2) facilitative glucose transporter (located on the surface of all cells)

Methods used to measure ketones in body fluids

1) Acetest

 

2) Ketostix

 

3) Determination of ?-hydroxybutyrate

Most common analytical method of ketones
sodium nitroferricyanide + ketones yield a purple complex
Qualitative Method to measure Glucose in Urine

Benedict’s Test

 

Aldose + Cu(citrate)22- (blue) > carboxylate anion and Cu2O (brick red ppt)

 

If the color of the solution changes, glucose is present (spectrophotometry?)

Quantitative Methods to measure Glucose in Urine
Hexokinase and glucose dehydrogenase procedures are recommended for greatest accuracy
Quantitative Method to measure Glucose in CSF
Glucose oxidase
Three Methods for Determination of Glycated Hemoglobins

1) High-Performance Liquid Chromatography

2) Immunoassay

3) Affinity Chromatography (*done in lab)

Lipids
a class of compounds that are soluble in organic solvents but are nearly insoluble in water and that contain nonpolar carbon-hydrogen bonds
Simple lipid
compound that contains fatty acids and no prosthetic groups; simple fatty acids; monomers of complex lipids
Complex lipid
Lipid containing esters, fatty acids, alcohol groups, and other prosthetic groups; a macromolecule composed of simple lipids (e.g. triglycerides)
triglyceride
an organic compound made up of glycerol and three fatty acid chains. Fat is stored as triglycerides
phospholipid
a polar amphipathic lipid located on the surface of a lipoprotein; they are also found at the aqueous interface of biological membranes
Lecithin

any group of yellow-brownish fatty substances occurring in animal and plant tissues composed of phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids.


major surfactant found in the lung; used along with sphingomyelin to assess fetal lung maturity

Sphingomyelin

a lipid compound that coats lung cells

 

substance that occurs widely in brain and nervous tissue, consisting of complex phosphoryl derivatives of sphingosine and choline

Glycolipid
a lipid with a carbohydrate component
apoprotein

protein that together with a prosthetic group forms a particular biochemical molecule such as a hormone or enzyme


apolipoproteins are the major components of lipoproteins

Lipoprotein
spherical particles involved in the transport of lipids with nonpolar neutral lipids (triglycerides and cholesterol esters) in their core and more polar amphipathic lipids (phospholipids and free cholesterol) at their surface
lipase

  1. a pancreatic enzyme that catalyzes the breakdown of fats to fatty acids and glycerol or other alcohols

lipemia
the presence in the blood of an abnormally high concentration of emulsified fat
Saturated fatty acids vs. unsaturated types

Saturated fatty acids have no double bond between their carbons

 

Monounsaturated fatty acids contain one double bond

Polyunsaturated fatty acids contain multiple double bonds

Chylomicron

A microscopically visible particle appearing in the plasma after a fatty meal.

 

Over 90% of TGL make up chylomicrons

VLDL – Very Low Density Lipoprotein

Composition: 50-65% TGL, 15% cholesterol, +proteins

 

Function: carries endogenous cholesterol and other TGL from liver to tissues

LDL = Low Density Lipoprotein

Composition: primarily cholesterol about 50%

 

Function: primary chosterol transport from liver to tissues; transports most of the TGLs?

HDL = High Density Lipoprotein

Composition: AI & AII apolipoprotein and contains cholesterol ester

 

Function: “cholesterol scavenger”; esterification of cholesterol and transports cholesterol from tissues to liver, where it is converted to bile acids and excreted.

Emusification and absorption of dietary lipids
The digestive enzyme, lipase, is water soluble and can only work at the surface of fat globules.  Digestion is greatly aided by emulsification, the breaking up of fat globules into much smaller emulsion dropletsBile salts and phospholipids areamphipathic molecules that are present in the bile. Motility in the small intestine breaks fat globules apart into small droplets that are coated with bile salts and phospholipids, preventing the emulsion droplets from re-associating
Physiological Function of Lipids
hormones, energy source, assisting digestion, structural component of cellular membrane
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