Carbohydrates

Part 2
Risk Factors for Diabetes
 Obesity
 Family history in 1st degree relative
 History of GDM or > 9 lb baby
 Hypertension > 140/90
 Low HDL cholesterol (< 35mg/dl)
 Elevated triglycerides (> 250 mg/dl)

M. Zaharna Clin. Chem. 2009

Categories of Fasting plasma
glucose sugar
 Fasting Glucose measurements
 12-14 hours nothing but water
 Normal < 110 mg / dl
 Impaired 110 – 126 mg / dl
 Provisional Diabetes diagnosis > 126 mg / dl

M. Zaharna Clin. Chem. 2009

Criteria for Diagnosis
 Random plasma glucose of > 200mg /dl
 Fasting plasma glucose of > 126 mg / dl
 Two hour plasma glucose > 200mg /dl OGTT
 Any of the above plus symptoms

 Diagnostic = Fasting plasma glucose of > 140 mg/dl

M. Zaharna Clin. Chem. 2009

Oral glucose tolerance test
 The OGTT continues to be regarded as the
most robust means for establishing the
diagnosis of diabetes in equivocal cases.
 The WHO suggests that only when an OGTT
cannot be performed should the diagnosis
rely on FPG.
 OGTTs should be carried out under
controlled conditions after an overnight fast.

M. Zaharna Clin. Chem. 2009

 Collect blood sample while fasting.
 The patient is given 75 gm of glucose orally.
 Blood samples collected at 60, 120, and 180 minutes.
 Analyze the samples and draw a chart.
 In normal persons, a return to the fasting level occurs in 2
or at most 2½ h.
 In diabetics, the peak is higher and there is a delay in the
return of the blood glucose to a fasting level.
 Urine remains free from glucose throughout the test in
normal individuals and becomes positive in about 60
minutes in diabetics.

M. Zaharna Clin. Chem. 2009

 According to the American Diabetes Association the
corresponding categories when the OGTT is used
are the following:
 2-hour postload glucose <140 mg/dL = normal
glucose tolerance;
 2-hour postload glucose 140–199 mg/dL = impaired
glucose tolerance (IGT);
 2-hour postload glucose ≥200 mg/dL = provisional
diagnosis of diabetes (the diagnosis must be
confirmed).

M. Zaharna Clin. Chem. 2009

Pathophysiology of Diabetes
Mellitus
 Type 1 and Type 2 diabetes: there is
an increase in blood glucose levels
(hyperglycemic).
 There is also elevation of glucose in
urine (glucosuria) if glucose levels in
blood exceed 180 mg/dl.

M. Zaharna Clin. Chem. 2009

 Type 1: tend to produce ketones because of
the difference in glucagon and insulin
concentration
 Absence of insulin and with increased
glucagon which leads to gluconeogenesis
and lipolysis.

M. Zaharna Clin. Chem. 2009

 Type 2: have very little ketone
production, but have a greater
tendency to develop hyperosmolar
nonketonic states.
 There is increased insulin production
and less use of glucagon.

M. Zaharna Clin. Chem. 2009

Tests of Diabetes Control and
Disease Progression
 Laboratory testing for diabetes after
diagnosis of the disease is directed toward
the assessment of the progression of
disease.
 The laboratory offers analysis that helps the
physician determine the extent of glycemic
control and the risk for the severe
consequences of hyperglycemia.

M. Zaharna Clin. Chem. 2009

Laboratory tests
 Ketones
 Serum osmolality
 Electrolytes
 Glucose
 HbA1c

M. Zaharna Clin. Chem. 2009

Hypoglycemia
 Decreased glucose levels (< 50 mg\dl)
 If too low can be life threatening (< 30 mg/dl)
 Most effective on the CNS-
 there is shaking and tremors, heart rate increases-
dizziness, cold sweat, if not corrected can result in
unconsciousness-coma-death.
• Epinephrine act with glucagon to increase
plasma glucose.
• In addition cortisol and GH are released in
increased glucose metabolism.

M. Zaharna Clin. Chem. 2009

Reactive hypoglycemia
 Hypoglycemia that is caused by a stimulus
such as:
 excessive insulin administration,
 Reactive hypoglycemia is not usually related
to any underlying disease
 Spontaneous recovery of glucose level as
insulin levels return to normal.

M. Zaharna Clin. Chem. 2009

Fasting hypoglycemia
 Hypoglycemia that occurs after fasting is
rare.
 May occur as a response:
 to insulin-producing tumors of the pancreas
(insulinomas)
 hepatic dysfunction,
 glucocorticoid deficiency,
 sepsis,
 or low glycogen stores.

M. Zaharna Clin. Chem. 2009

Genetic defects
 Glycogen storage defect is due to a defect in
specific enzyme that cause an alternation of
glycogen metabolism.
 Most common form type 1 (von Gierke
disease) due to glucose-6-phosphatase
deficiency.
 Hypoglycemic state is due to the inability of
glycogen to be converted back to glucose by
hepatic glycogenolysis.
M. Zaharna Clin. Chem. 2009
Galactosemia
 Defect in enzyme needed to metabolize
Galactose- results in an increase in galactose
in plasma.
 Enzyme that is most commonly deficient :
galatose-1 phosphate uridyl transferase.
 Due to inhibition of glycogenolysis
 Must remove galactose from diet, if not, it will
build up in the system cause retardation and
cataracts.

M. Zaharna Clin. Chem. 2009

Glucose measurements
 Sample needs to refrigerated and separated
from cells with one hour of collection.
 Fluoride is the anticoagulant of choice.
 Glucose has the ability to function as a
reducing agent and aid in the detection of
and quatitation of carbohydrates .

M. Zaharna Clin. Chem. 2009

 Glucose and other carbohydrates are
capable of converting cupric ions in an
alkaline solution to form cuprous ions.
 Benedict reagent: uses cuprous /cupric
methodology forming a deep blue to red color
when cuprous ions are present.
 Reagent contains alkaline solution of cupric
ions stabilized by citrate or tartrate- which
detects the reducing substance.
M. Zaharna Clin. Chem. 2009
Methods
 Glucose oxidase method: converts beta-
glucose to gluconic acid. Mutarotase may be
added to facilitate to conversion to alpha-
glucose to beta-glucose.
 Oxygen is consumed and hydrogen peroxide
is produced.
 Horseradish perixidase is used as a catalyst.
Chromagens used for color change

M. Zaharna Clin. Chem. 2009

 Hexokinase: more accurate less interference
from uric acid, bilirubin and ascorbic acid.
 In the presence of ATP- hexokinas converts
glucose to glucose-6-phosphate.
 Glucose-6-phophate and NADP converted to
6-phosphogluconate and NADPH by glucose-
6-phosphate dehydrogenase- produces a red
color measured at 340 nm.

M. Zaharna Clin. Chem. 2009

Glucose monitoring and 2 hr
test
 2 hour test utilizes the knowledge that
normally a glucose level will return to normal
after 2 hrs if no disease or impairment
involved.
 GTT most sensitive, more accurate. Utilizes
fasting along with set time intervals.

M. Zaharna Clin. Chem. 2009

Glycosylated Hemoglobulin
(HbA1c)
 Is a term used to describe the formation of
Hb compound formed when glucose reacts
with the amino group of Hb.
 Used to monitor and manage diabetes,
monitors blood glucose levels over the last
60-90 days.
 Specimen of choice is EDTA whole blood

M. Zaharna Clin. Chem. 2009

Methods
 2 major categories
1. Based on charge difference between
glycosylated and nonglycosylated Hgb.
(cation-exchange chromatography,
electrophoresis, and isoelectric focusing)
2. Structural characteristics of glycogroups on
Hb. (affinity chromatography and
immunoassay)

M. Zaharna Clin. Chem. 2009

Ketones
 Ketone bodies are produced by the liver through the
metabolism of fatty acids to provide energy to
provide ready energy from stored lipids in low CHO
available.
 Acetone, Beta-hydroxybutyrate and acetoacetic acid
 Low levels present all the time, but when the body is
deprived of CHO (diet, vomiting, and glycogen
storage disease) ketones levels increase.
 Need fresh serum or urine – tightly stoppered
and analyzed immediately

M. Zaharna Clin. Chem. 2009

Microalbuminuria
 Because Diabetes mellitus cause
progressive disease in the kidneys
(nephropathy), the lab will monitor urinary
albumin through measuring microalbumin in
the urine.

M. Zaharna Clin. Chem. 2009

Self-Monitoring Glucose Meters
 At-home or near-patient monitoring by point of care testing
(POCT) with glucose meters provides information so that
therapeutic intervention may be initiated immediately.
 Glucose meters use the same chemical reactions that are
used in glucose analysis in the laboratory: glucose oxidase,
hexokinase, and dehydrogenase.
 Most systems use dehydrated reagents embedded in pads
on plastic strips.
 The strip is inserted in the meter, where the reaction is
measured.
 The reaction may be a color change that is measured by
reflectance spectrophotometry, or the reaction may
produce a change in current that can be measured by
electrochemistry.
M. Zaharna Clin. Chem. 2009
Self-Monitoring Glucose Meters
 A blood sample is applied to the surface layer,
which both acts as a spreading layer and is a semi-
permeable membrane that separates blood cells
from plasma.
 Plasma from the sample diffuses into the paper
analytical layer, which contains the buffered enzyme
reaction system, activated by plasma water.
 Within the analytical layer, glucose and atmospheric
oxygen are acted on by the glucose oxidase to
produce hydrogen peroxide and gluconic acid.
 In the presence of peroxidase, also contained within
the analytical layer, hydrogen peroxide oxidizes a
redox indicator to produce a visible colour change.

M. Zaharna Clin. Chem. 2009