Carbohydrates

1
Carbohydrates

• Monosaccharides

D-glucose
D-galactose
2

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

D-glucose
D-fructose
3

• Disaccharides

reducing end
Lactose
4
reducing end
Maltose
5
Sucrose
not a reducing substance
6

• Polysaccharides

reducing end
Cellulose
7
Glycogen
Amylopectin
branches every 8-12 glucose units
branches about every 24-30 linear linkages
8
Intestinal absorption of carbohydrates
microvilli
Jejunum villi
9
Intestinal absorption of carbohydrates
MICROVILLI BRUSH BORDER
GUT
BLOOD
1
maltose
-amylase
a
Starch
saliva and pancreatic juice
glucose
Glycogen
sucrose
2
fructose
3
lactose
galactose
galactose
glucose
Monosaccharides
fructose
10
Glucose Production
Glucose Consumption
125g
Blood glucose
brain
Glucose
50g
Glycogen (75)
50g
rbc wbc
muscle
fat cell
11
Glucose transporters
insulin lowGLUT4 in intracellular
compartments insulin highGLUT4 translocates to
membrane
12
Glucose Production
Glucose Consumption
CO2
125g
Blood glucose
brain
Glucose
50g
Glycogen (75)
50g
rbc wbc
pyruvate lactate (10-15)
certain amino acids (10-15)
CO2
glycerol (2)
muscle
fat cell
13
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

14
Regulation of blood glucose
stimulates
Somatostatin
inhibits
d
Pancreatic Islet
a
b
Cortisol Growth hormone
glucose
Insulin
glucagon
epinephrine
Glucose uptake Glycolysis
Glycogenolysis Gluconeogenesis
Glucose uptake Lipogenesis
Muscle
Liver
Adipose tissue
15
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 lt30 mg/dL random lt500 mg/24 hr

16
Glucose 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
17
Glucose 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
18
Glucose Methods

• 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

GDH
19
Glucose Methods

• 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
20
Glucose Methods

• oxidation-reduction reactions
• Fe3 Fe2 or
  Cu2 Cu1
• least specific for glucose

21
Clinical Significance

• Hyperglycemia
• diabetes mellitus
• endocrine disorders
• acromegaly incr. growth hormone
• Cushings syndrome incr. cortisol
• thyrotoxicosis incr. T4
• pheochromocytoma incr. epinephrine
• drugs
• certain anesthetics
• steroids

22
Clinical Significance

• Hypoglycemia
• insulin overdose
• drugs
• sulfonylureas
• antihistamines
• alcoholism (long term)
• insulinoma
• galactosemia
• glycogen storage diseases

23
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 gt 200 mg/dL
• Fasting plasma glucose (FPG) gt 126 mg/dL
• 2hr Post prandial glucose (PPG) gt 200 mg/dL after
  a meal with 75g glucose load

24
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

25
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 (gt40), obesity and lack of
  exercise
• more common in
• Women
• African American
• Hispanics
• Native Americans

26
Expert Committee on the Diagnosis and
Classification of DM - 2005

• Other specific types
• pancreatic, hormonal disease
• Pancreatitis, cystic fibrosis
• Acromegaly (GH), Cushings syndrome (cortisol)
• drug/chemical toxicity
• insulin receptor abnormalities
• no renal or retinal complications

27
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?
• gt 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 gt 105 mg/dL
• gt 195 mg/dL at 1 hr
• gt 165 mg/dL at 2 hrs
• gt 145 mg/dL at 3 hrs

28
National Diabetes Association 2003

• Pre-diabetes
• http//diabetes.niddk.nih.gov/dm/pubs/diagnosis/in
  dex.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.

29
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
180
Minutes after glucose ingestion
30
Clinical Significance

• Other conditions, tests associated with diabetes
  mellitus
• white cell antigens
• HLA types DR3, DR4, DQB10302
• Note that diabetes resistance genes DR2,
  DQB10602
• lipid studies
• hyperlipoproteinemia type IV
• increased TG
• microalbuminuria
• microangiopathies
• retinal, renal, neural

31
Management of diabetes mellitus

• Glycated hemoglobin (A1)

COOH
COOH
COOH
COOH
H2N
H2N
H2N
H2N
COOH
COOH
H2N
H2N
N
N
glu
glu
non-enzymatic process
conversion of HbA into HbA1 at N-terminal valine
32
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)

33
Management of diabetes mellitus

• For every 1 decrease in HbA1c, risk of
  microvascular complications is reduced by 35
• Diabetes Care 2000, 23 S27-S31

34
Management of diabetes mellitus

• Manual Methods for HbA1c compared

Ion-exchange chromatography
Affinity Chromatography

HbA--Val--N
CH2
CO HOCH
HCOH
HCOH
CH2OH
Principle




HbA
HbA1c




pba
-- - - - - - - - - - - - - - - --
-- - -
pba
pba
Fast fraction HbA1c elutes first
HbA1c elutes last
pba
Phenylboronic acid
35
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

36
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 __________________________

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38
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

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40
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

41
Management of diabetes mellitus

• Glycated serum proteins
• albumin (fructoseamine)
• turnover 2-3 weeks
• rapid method using tetrazolium dye reduction
• 
  colored product

42
Carbohydrate inborn errors of metabolism

• Glycogen storage diseases
• lack of enzymes of glycogen metabolism
• incr. tissue glycogen
• results in severely limited lifespan
• von Gierkes disease
• liver cells lack glucose-6-phosphatase

glucose-6-PO4
blood glucose
triose phosphate
pyruvate
43
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!)

44
Carbohydrate inborn errors of metabolism

• 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

gal. oxidase
galactose O2
galactose dialdehyde H2O2
45
Ketones

• Complication of uncontrolled diabetes mellitus
• Acid-base imbalance
• Can be life-threatening
• Acetone, acetoacetate, b-hydroxybutyrate

Blood ketones
amino acids
acetyl CoA
fatty acids
Ketones acetoacetate B-hydroxybutyrate
acetate
CO2
TCA cycle
H2O
ATP
46
Ketones

• Complication of uncontrolled diabetes mellitus
• Sodium nitroprusside
• B-hydroxybutyrate dehydrogenase

Blood ketones
amino acids
acetyl CoA
fatty acids
Ketones acetoacetate B-hydroxybutyrate
acetate
CO2
TCA cycle
H2O
ATP
47
Extra slides
48
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

49
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

50
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 obesity
• estrogens inactivity
• progestins low CHO diet

51
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

52
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
• proximal convoluted tubule
• reabsorbs all glucose if lt180 mg/dL
• glycosuria results if blood glucose gt180 mg/dL

Renal Threshold