Carbohydrates
• 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
reducing
end
Lactose
reducing
end
Maltose
Sucrose
not a reducing
substance
• Polysaccharides
reducing
end
Cellulose
Amylopectin
Glycogen
branches about every
24-30 linear linkages
branches every
8-12 glucose units
Intestinal absorption of
carbohydrates
Jejunum villi
microvilli
Intestinal absorption of
carbohydrates
GUT
Starch
Glycogen
a -amylase
maltose
1
saliva and
pancreatic
juice
sucrose
2
lactose
galactose
Monosaccharides
glucose
fructose
3
MICROVILLI
BRUSH
BORDER
glucose
fructose
galactose
BLOOD
Glucose Production
Glucose Consumption
Blood
glucose
125g
brain
Glucose
50g
Glycogen
(75%)
50g
rbc
wbc
muscle
fat cell
Glucose transporters
Name
Tissue
Function
GLUT1 (erythrocyte)
wide distribution, esp.
brain, kidney, colon, fetal
tissues
Basal glucose transport
GLUT2 (liver)
Liver, b-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
Blood
glucose
CO2
125g
brain
Glucose
50g
Glycogen
(75%)
50g
pyruvate
lactate (10-15%)
certain
amino acids (10-15%)
glycerol (2%)
fat cell
rbc
wbc
CO2
muscle
Regulation of blood glucose
• Glycogenesis: glucose
glycogen
(liver, muscle)
• Glycogenolysis: glycogen
• Gluconeogenosis:
non-CHO sources
• Glycolysis: glucose
glucose
glucose
CO2 + H2O + ATP
• Renal threshold: proximal convoluted tubule
Regulation of blood glucose
stimulates
Somatostatin
inhibits
d
a
b
Pancreatic Islet
Cortisol
Growth hormone
glucose
glucagon
Insulin
epinephrine
Glycogenolysis
Gluconeogenesis
Liver
Glucose uptake
Lipogenesis
Adipose tissue
Glucose uptake
Glycolysis
Muscle
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 Methods
• hexokinase
glucose + ATP
gluc-6-PO4 + NAD+
INT + NADH + H+
–
–
–
–
HK
G6PD
PMS
gluc-6-PO4 + ADP
6-phosphogluconate + NADH + H+
formazan + NAD+
most widely used
reference method against which others are compared
serum, plasma and urine
avoid hemolysis
Glucose Methods
• glucose oxidase
glucose + O2
GO
H2O2 + reduced dye
gluconic acid + H2O2
POD
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
Glucose Methods
• glucose dehydrogenase
GDH
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
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 Methods
• glucose dehydrogenase
GDH
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, dxylose
» 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
Glucose Methods
• oxidation-reduction reactions
Fe3+
Fe2+
or
Cu2+
• least specific for glucose
Cu1+
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
Clinical Significance
• Hypoglycemia
– insulin overdose
– drugs
» sulfonylureas
» antihistamines
– alcoholism (long term)
– insulinoma
– galactosemia
– glycogen storage diseases
Expert Committee on the Diagnosis
and Classification of DM - 2005
• 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
and 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
Expert Committee on the Diagnosis
and Classification of DM - 2005
• 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
Expert Committee on the Diagnosis
and Classification of DM - 2005
• “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
Expert Committee on the Diagnosis
and Classification of DM - 2005
• 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
2003
• Pre-diabetes
http://diabetes.niddk.nih.gov/dm/pubs/diagnosis/index.htm
http://www.diabetes.org/pre-diabetes.jsp
Fasting Plasma Glucose
Result (mg/dL)
70 – 99
100 to 125
126 and above
Diagnosis
Normal
Pre-diabetes
(impaired fasting glucose)
Diabetes mellitus*
*Confirmed by repeating the
test on a different day.
Clinical Significance
• 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
200
Plasma
glucose
(mg/dL)
Diabetic
Normal
100
60
120
Minutes after glucose ingestion
180
Clinical Significance
• 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
H2N
COOH
H2N
COOH
H2N
COOH
H2N
COOH
H2N
COOH
H2N
N
glu
N
glu
non-enzymatic process
conversion of HbA into HbA1 at N-terminal valine
Management of
diabetes mellitus
• 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)
Management of
diabetes mellitus
For every 1% decrease in
HbA1c, risk of microvascular
complications is reduced by
35%
Diabetes Care 2000, 23: S27-S31
Management of
diabetes mellitus
• Manual Methods for HbA1c compared
Ion-exchange
chromatography
Principle
“Fast fraction”
HbA1c
elutes first
+
+ +
HbA
+ + +
Affinity
Chromatography
+ +
HbA1c
+
-- - - - - - - ------ -- - -
HbA1c
elutes last
HbA--Val--N
|
CH2
|
C=O
|
HOCH
pba
|
pba
HCOH
|
pba
HCOH
|
CH2OH
pba
Phenylboronic acid
Management of
diabetes mellitus
• 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
Management of
diabetes mellitus
• 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 __________________________
Management of
diabetes mellitus
• 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
Management of
diabetes mellitus
• 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%
Management of
diabetes mellitus
• 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
glucose-6-PO4
triose phosphate
pyruvate
blood glucose
Carbohydrate inborn errors
of metabolism
• 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!)
Carbohydrate inborn errors
of metabolism
• Galactosemia
(1) galactose
galactose-1-PO4
galactilol
(2) galactose-1-PO4
*
UDP-galactose
(3) UDP-galactose
UDP-glucose
(4) UDP-glucose
glucose-1-PO4
cataracts
– deficiency in uridyl transferase *
– results in galactosuria, retardation, cataracts, no
conjugation of bilirubin
– urine tests
gal. oxidase
galactose + O2
galactose dialdehyde + H2O2
Ketones
• Complication of uncontrolled diabetes
mellitus
– Acid-base imbalance
– Can be life-threatening
– Acetone, acetoacetate, b-hydroxybutyrate
Blood
ketones
fatty acids
Ketones
acetoacetate
B-hydroxybutyrate
acetate
acetyl CoA
amino acids
CO2
TCA cycle
H2O
ATP
Ketones
• Complication of uncontrolled diabetes
mellitus
– Sodium nitroprusside
– B-hydroxybutyrate dehydrogenase
Blood
ketones
fatty acids
Ketones
acetoacetate
B-hydroxybutyrate
acetate
acetyl CoA
amino acids
CO2
TCA cycle
H2O
ATP
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
Glycogenesis, 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
Glycogenesis, glycogenolysis
• 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
– estrogens
– progestins
obesity
inactivity
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
Glycolysis
• NOTE!!
– if serum/plasma is not separated from cells soon after
collection, cellular use of glucose will continue, causing a
falsely decreased glucose result
Renal Threshold
• proximal convoluted tubule
– reabsorbs all glucose if <180 mg/dL
– glycosuria results if blood glucose >180 mg/dL