carbohydrates monosaccharides d-glucosed-galactose

CarbohydratesCarbohydrates

• Monosaccharides

D-glucose D-galactose

Reducing substance: hydroxyl group near an aldehyde or ketone group can react with Cu2+, converting it to Cu+

D-glucose D-fructose

• Disaccharides

Lactose

reducingend

Maltose

reducingend

Sucrose not a reducingsubstance

• Polysaccharides

Cellulose

reducingend

Glycogenbranches every8-12 glucose units

Amylopectinbranches about every24-30 linear linkages

Intestinal absorption of carbohydrates

Jejunum villimicrovilli

Intestinal absorption of carbohydrates

Starch

Glycogen

-amylase

saliva andpancreaticjuice

maltose

sucrose

lactose

galactose

GUTMICROVILLIBRUSHBORDER

BLOOD

glucose

fructose

galactose

glucose

fructose

Monosaccharides

1

2

3

Glucose Production Glucose Consumption

Glycogen (75%)

Glucose

Bloodglucose

125g

50g

50g

brain

rbcwbc

muscle

fat cell

Glucose transportersName Tissue Function

GLUT1 (erythrocyte) wide distribution, esp. brain, kidney, colon, fetal tissues

Basal glucose transport

GLUT2 (liver) Liver, -cells of pancreas, small intestine, kidney

Non-rate-limiting glucose transport

GLUT3 (brain) Wide distribution, esp. neurons, placenta, testis

Glucose transport in neurons

GLUT4 (muscle) Skeletal muscle, cardiac muscle, adipose tissue

Insulin-stimulated glucose transport*

GLUT5 (small intestine) Small intestine, kidney, skelatal muscle, brain, adipose tissue

Fructose transport

*insulin low…GLUT4 in intracellular compartments; insulin high…GLUT4 translocates to membrane

Glucose Production Glucose Consumption

Glycogen (75%)

Glucose

Bloodglucose

125g

50g

50g

pyruvatelactate (10-15%)

certainamino acids (10-15%)

glycerol (2%)

CO2

CO2

brain

rbcwbc

muscle

fat cell

Regulation of blood glucose

• Glycogenesis: glucose glycogen

(liver, muscle)

• Glycogenolysis: glycogen glucose

• Gluconeogenosis:

non-CHO sources glucose

• Glycolysis: glucose CO2 + H2O + ATP

• Renal threshold: proximal convoluted tubule

Regulation of blood glucose

Glucose uptakeLipogenesis

Glucose uptakeGlycolysis

Insulin

Pancreatic IsletPancreatic Islet

Somatostatin

glucose

epinephrine

glucagon

CortisolGrowth hormone

GlycogenolysisGluconeogenesis

LiverLiver Adipose tissueAdipose tissue MuscleMuscle

stimulates

inhibits

Determination of glucose• Specimens used

– whole blood

» used with home glucose monitoring units

» cellular use of glucose gives 7% decrease/hour

» NaF preserves glucose 24 hr, RT

• cannot use such a specimen for enzyme assays, especially urease

» lithium iodoacetate preserves glucose; does not interfere with urease

» capillary blood = fasting venous level + 5 mg/dL

– plasma, serum

» 10-15% higher level than whole blood glucose

» RI: 70-105 mg/dL

– CSF

» RI: 60-70% plasma glucose = 40-70 mg/dL

– urine

» RI: <30 mg/dL random; <500 mg/24 hr

Glucose MethodsGlucose Methods• hexokinase

glucose + ATP gluc-6-PO4 + ADP

gluc-6-PO4 + NAD+ 6-phosphogluconate + NADH + H+

INT + NADH + H+ formazan + NAD+

– most widely used

– reference method against which others are compared

– serum, plasma and urine

– avoid hemolysis

HK

G6PD

PMS

Glucose MethodsGlucose Methods• glucose oxidase

glucose + O2 gluconic acid + H2O2

H2O2 + reduced dye oxidized dye + H2O• peroxidase reaction interference by uric acid, vitamin C, bilirubin

• suitable for spinal fluid

measure O2 consumption via pO2 electrode• suitable for all body fluids

measure H2O2 production via H2O2 electrode• suitable for plasma, serum, whole blood

GO

POD

• glucose dehydrogenase

glucose + NAD+ D-gluconolactone + NADH + H+ » highly specific as “GDH-NAD”, with little interference, EXCEPT…

• Pyrroloquinolinequinone (“GDH-PQQ”)

– 2005 FDA warning

– giving false increased glucose readings when patient is receiving maltose, icodextrin (dialysis), galactose, d-xylose

Glucose MethodsGlucose Methods

GDH

Manufacturer Proper Name Sugar

Octapharma Immune Globulin Intravenous (Human)

Maltose10%

Talecris Immune Globulin Intravenous (Human)

Maltose9-11%

Cangene Rho(D) Immune Globulin Intravenous (Human)

Maltose10%

Cangene) Vaccinia Immune Globulin (Human Maltose10%

NERL Diagnostic d-Xylose Usual dose 25g

Baxter Peritoneal dialysis soln (icodextrin) 7.5 mg/dL icodextrin

• glucose dehydrogenase

glucose + NAD+ D-gluconolactone + NADH + H+ » highly specific as “GDH-NAD”, with little interference, EXCEPT…

• Pyrroloquinolinequinone (“GDH-PQQ”)

– 2005 FDA warning

– giving false increased glucose readings when patient is receiving maltose, icodextrin (dialysis), galactose, d-xylose

» Coulometry• http://www.medisense.com/au

• FreeStyle glucometer, Abbott Laboratories

• Electrons released in the reaction are measured as a current

• Allows very small volume (0.3 uL) to be used, with results in 15 seconds


GDH

http://www.medisense.com/au



• oxidation-reduction reactions Fe3+ Fe2+ or Cu2+ Cu1+

• least specific for glucose


Clinical Significance

• Hyperglycemia– diabetes mellitus

– endocrine disorders

» acromegaly: incr. growth hormone

» Cushing’s syndrome: incr. cortisol

» thyrotoxicosis: incr. T4

» pheochromocytoma: incr. epinephrine

– drugs

» certain anesthetics

» steroids

• Hypoglycemia– insulin overdose

– drugs

» sulfonylureas

» antihistamines

– alcoholism (long term)

– insulinoma

– galactosemia

– glycogen storage diseases


• Diagnosis of Diabetes Mellitus– Symptoms of diabetes mellitus

» Polyuria

» Polydipsia

» Unexplained weight loss

– Any TWO of the following tests, on different days

» Casual plasma glucose > 200 mg/dL

» Fasting plasma glucose (FPG) > 126 mg/dL

» 2hr Post prandial glucose (PPG) > 200 mg/dL after a meal with 75g glucose load

Expert Committee on the Diagnosis Expert Committee on the Diagnosis and Classification of DM - 2005and Classification of DM - 2005

• Type 1– Type 1a

» characterized by beta cell destruction caused by an autoimmune process, usually leading to absolute insulin deficiency

» patients must take insulin to survive

» usually young, with acute onset (days to weeks)

» islet-cell antibodies usually present

– Type 1b

» idiopathic



• Type 2– insulin resistance in peripheral tissue and an insulin

secretory defect of the beta cell

– variable [insulin]

– highly associated with a family history of diabetes, older age (>40), obesity and lack of exercise

– more common in

» Women

» African American

» Hispanics

» Native Americans

• “Other specific types” – pancreatic, hormonal disease

– Pancreatitis, cystic fibrosis

– Acromegaly (GH), Cushing’s syndrome (cortisol)

– drug/chemical toxicity

– insulin receptor abnormalities

– no renal or retinal complications


• Gestational diabetes mellitus– pregnancy

– frequent but transitory glucose intolerance

– greater risk of perinatal complications

– placental lactogen?

– > 140 mg/dL one hour after 50-g glucose load screening

– TWO of four results abnormal in 100 g glucose load test:

– fasting plasma glucose > 105 mg/dL

– > 195 mg/dL at 1 hr

– > 165 mg/dL at 2 hrs

– > 145 mg/dL at 3 hrs


National Diabetes Association National Diabetes Association 20032003

• Pre-diabeteshttp://diabetes.niddk.nih.gov/dm/pubs/diagnosis/index.htm

http://www.diabetes.org/pre-diabetes.jsp

Fasting Plasma Glucose Diagnosis Result (mg/dL)

70 – 99 Normal

100 to 125 Pre-diabetes(impaired fasting

glucose)

126 and above Diabetes mellitus**Confirmed by repeating the test on a different day.

http://diabetes.niddk.nih.gov/dm/pubs/diagnosis/index.htm

http://www.diabetes.org/pre-diabetes.jsp

• Glucose tolerance test (still used for gestational diabetes diagnosis)– patient preparation

» normal diet three days prior to test

» no food after regular evening meal on day before test

» take fasting blood, urine specimen

» drink 100 g glucose load within 5 minutes

» allow water, but no food, chewing gum, smoking, exercise during test

» specimens taken 1, 2, 3 hours after ingestion


Plasma glucose (mg/dL)

Minutes after glucose ingestion60 120 180

100

200

Normal

Diabetic

• Other conditions, tests associated with diabetes mellitus– white cell antigens

» HLA types DR3, DR4, DQB1*0302

» Note that diabetes resistance genes = DR2, DQB1*0602

– lipid studies

» hyperlipoproteinemia type IV• increased TG

– microalbuminuria

– microangiopathies

» retinal, renal, neural


Management of diabetes mellitus

• Glycated hemoglobin (A1)

COOH

COOH

COOH COOH

COOH

COOH

H2N

H2N

H2N

H2N H2N H2N

N N

gluglu

non-enzymatic process conversion of HbA into HbA1 at N-terminal valine

• Glycated hemoglobin– irreversible reaction occurring throughout the 120-day

life span of rbc

– reflects timed average [glucose] over previous 4-8 weeks

– HbA1c = 80% total glycohemoglobin

– reference range: 3-6% total Hgb

– uncontrolled diabetes mellitus: 12-20% total Hgb

– controlled: 9-12% total Hgb

• Considerations when measuring HbA1c

– abnormal hemoglobins can also be glycated

– variability in levels of “labile fraction” (intermediates)


For every 1% decrease in HbA1c, risk of microvascular complications is reduced by 35%

Diabetes Care 2000, 23: S27-S31


• Manual Methods for HbA1c compared


Ion-exchangechromatography

AffinityChromatography

PrincipleHbA HbA1c

++

+ ++

++

+

+

-- - - - - -- - - - - - - - - -- -- - -

“Fast fraction” HbA1c

elutes firstpba

pba

pbapba

HbA--Val--N | CH2

| C=O | HOCH | HCOH | HCOH | CH2OH

HbA1c

elutes last

Phenylboronic acid

• Manual Methods for HbA1c compared– other non-glycated Hb measured

» IEC: HbF and any others with charge like A1

» AC: none– time

» IEC: 2-3 hrs» AC: 15 minutes

– glycated hemoglobins measured» IEC: A1a, 1b, 1c only» AC: any glycated hemoglobin, including

abnormals– temperature sensitive?

» IEC: yes» AC: no


• Automated Method for HbA1c

– High pressure liquid chromatography (HPLC)

– Cation exchange method

» the eluant must be __________________

» The first form of hemoglobin eluting from the column must be _________________________

» The last form of hemoglobin eluting from the column must be __________________________


• Attempts to convert HbA1c value to “mean blood glucose” value– Nathan et al, 1984 using linear regression on data

from 21 patients

– 33.3 (%HbA1c) – 86

– Examples: 6.0% = ~115 mg/dL 7.5% = ~165 mg/dL 9.0% = ~215 mg/dL


• Revised calculation, effective 3/21/05– Rohlfing et al, 2002 using comparison data from

1500 patients

– (35.6 x %HbA1c) – 77.3

– Examples: 6.0% = ~136 mg/dL 7.5% = ~190 mg/dL 12.0% = ~350 mg/dL

– Only valid for A1c values between 6 and 12%


• Glycated serum proteins– albumin (“fructoseamine”)

– turnover = 2-3 weeks

– rapid method using tetrazolium dye reduction

colored product


Carbohydrate inborn errors of metabolism

• Glycogen storage diseases– lack of enzymes of glycogen metabolism

– incr. tissue glycogen

– results in severely limited lifespan

– von Gierke’s disease

» liver cells lack glucose-6-phosphatase

pyruvate

triose phosphate

glucose-6-PO4 blood glucose

• Lactose intolerance– deficiency in intestinal mucosal lactase

– GTT done as baseline

– 2nd day, give lactose instead of glucose

– normal: normal GTT curve

– abnormal: flat curve ( and much pain!)


• Galactosemia(1) galactose galactose-1-PO4

galactilol

(2) galactose-1-PO4 UDP-galactose

(3) UDP-galactose UDP-glucose cataracts

(4) UDP-glucose glucose-1-PO4

– deficiency in uridyl transferase *

– results in galactosuria, retardation, cataracts, no conjugation of bilirubin

– urine tests


galactose + O2 galactose dialdehyde + H2O2

gal. oxidase

*

• Complication of uncontrolled diabetes mellitus– Acid-base imbalance

– Can be life-threatening

– Acetone, acetoacetate, -hydroxybutyrate

Ketones

fatty acids amino acidsacetyl CoA

TCA cycleCO2

H2O

ATP

Ketones acetoacetate B-hydroxybutyrate acetate

Bloodketones

• Complication of uncontrolled diabetes mellitus– Sodium nitroprusside

– B-hydroxybutyrate dehydrogenase

Ketones

fatty acids amino acidsacetyl CoA

TCA cycleCO2

H2O

ATP

Ketones acetoacetate B-hydroxybutyrate acetate

Bloodketones

Extra slides

Glycogenesis, glycogenolysis

• Hormones involved– fed: insulin from pancreatic beta cells (Islets of Langerhans)

» preproinsulin proinsulin (A, B and C peptides)

insulin + C-peptide

» anabolic (synthesis)

» promotes cellular uptake of glucose

» increased:

• lipogenesis

• protein synthesis

• glycogenesis

» decreased:

• lipolysis

• ketone formation

• gluconeogenesis

• glycogenolysis

• Hormones involved– fasting: glucagon from pancreatic alpha cells

» catabolic

» liver: glycogen converted to glucose, released into blood

» muscle: glycogen converted to glucose-6-PO4, remains in the muscle cell for its own energy needs

– “fight or flight” : epinephrine from adrenal medulla

» action similar to glucagon


• Stimulation of insulin release– glucose

– leucine, arginine, histidine, phenylalanine

– sulfonylureas (tolbutamides)

– ACTH, GH

• Inhibition of insulin release– thiazide diuretics

– dilantin (antiseizure)

– human placental lactogen (diabetes of pregnancy)

• Decreased tissue response to insulin– glucocorticoids obesity

– estrogens inactivity

– progestins low CHO diet


Gluconeogenesis

• cortisol (hydrocortisone)– from adrenal cortex

– inhibits glucose entry into muscle, connective tissue, lymphoid tissue

– stimulates release of gluconeogenic amino acids from muscle

– promotes conversion of amino acids into glucose by liver

– stimulates lipolysis in adipose cells, releasing glycerol for conversion to glucose by liver

• ACTH– from anterior pituitary

– stimulates production of cortisol

• NOTE!!– if serum/plasma is not separated from cells soon after

collection, cellular use of glucose will continue, causing a falsely decreased glucose result

• proximal convoluted tubule– reabsorbs all glucose if <180 mg/dL

– glycosuria results if blood glucose >180 mg/dL

Glycolysis

Renal Threshold

carbohydrates monosaccharides d-glucosed-galactose

Documents

glucose blood glucose

plasma glucose

blood glucose ri

glucose glycogen liver

testis glucose transport

fructose slide

galactose slide

end slide