© Dept.

of Medical and Clinical Biochemistry UPJŠ in Košice,

Medical Faculty
Eva Ďurovcová, MD. PhD.
5 GM CLB
2
 GLUCOSE HOMEOSTASIS
Influx blood-glucose Clearence by
GLYCEMIA peripheral tissues

3.9 – 5.6 mmol/L

3 sources of blood-glucose: 3 ways of glucose removal:
1. Food: gut → liver → glycolysis/conversion of 1. Utilisation for energy
other ingested carbohydrates
2. Storage (glycogen + TAG !)
2. Glycogen stores – liver + other (glycogenolysis)
3. Excretion by kidney
3. Gluconeogenesis: liver (60 – 80%, kidney 20 –
40%)

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HORMONAL REGULATION OF GLYCEMIA
Tissue and hormone Insulin Glucagon Adrenalin Cortisol GH
LIVER
Glycogenolysis ↓↓ ↑↑ ↑↑↑ None ↑
Gluconeogenesis ↓↓ ↑↑↑ ↑ ↑↑↑ ↑
Ketogenesis ↓ ↑↑ ↑ ↑ ↑
MUSCLE = INS dependent
Glucose uptake, glycolysis ↑↑↑ None ↓ ↓↓ ↓
Glycogenogenesis ↑↑ ? ↓ ? ?
ADIPOCYTES = INS dependent
Glucose uptake ↑↑↑ ↓ ↓ ↓↓ ↓
Lipolysis ↓↓↓ ↑ ↑↑↑ ↑↑ ↑

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GLUCOSE TRANSPORTERS
 SGLTs – sodium coupled glucose co-transporters - active transport of GLU against
a concentration gradient
 GLUTs – glucose transporting proteins - passive transport of GLU („downhill“
movement of GLU across cell membrane)
Type Distribution
SGLT1 Kidney – PT, intestine, brain, heart
SGLT2 Kidney – PT, brain, liver, muscle, heart, thyroid....
SGLT 3-6 Ubiquituous
GLUT1 Kidney, intestine, liver, brain, uterus, stomach, pancreas....
GLUT2 Liver, pancreas, intestine, renal PT
GLUT4 Adipose tissue (white, brown), muscle (skeletal, heart)
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INSULIN SECRETION IS STIMULATED BY
 Glucose – increased plasma concentration

 Insulin secretagogues –free fatty acids and amino acids can augment
glucose-induced insulin secretion

 Incretin hormones: „amplify“ effect of GLU on β-cells;

 produced in small intestine in response to oral glucose or mixed food intake
(≈ modern antidiabetic drugs)
 GLP-1 glucagon-like peptide 1
 GIP gastric inhibitory polypeptide
 various hormones, such as melatonin, estrogen, leptin, growth hormone
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 DM – PATHOGENETIC FACTORS
 DM = chronic hyperglycaemia + disturbances of CH, F and P metabolism

 Generally DM arises from a combination of :

1. -cell dysfunction
2. insulin resistance
 ? increased GLU renal reabsorption
Genetic factors,
Western lifestyle,
obesity,


Insulin b-cell
resistance IR dysfunction

T2DM 9
WHY DOES THE -CELL FAIL?
EXHAUSTING OF -CELLS
AUTOIMMUNITY, GENETICS Oversecretion of insulin
to compensate for
insulin resistance

Chronic Toxic compounds,

Pancreas ROS High circulating
hyperglycemia free fatty acids
GLUCOTOXICITY LIPOTOXICITY

-cell dysfunction/ apoptosis

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INSULIN RESISTANCE – REDUCED RESPONSE TO CIRCULATING INSULIN

Insulin
resistance
IR

Liver Muscle Adipose

tissue

 Glucose output  Glucose uptake  Glucose uptake

HYPERGLYCEMIA
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METABOLIC DISORDERS IN DM
Feature Cause
Insulin resistance + dysfunction of β-cells
1. Fasting and postprandial
Hepatic overproduction of glucose
hyperglycemia
Adipocte dysfunction in T2DM
2. Atherogenic dyslipidemia*
↑TAG, ↓HDL-CH, ↑ sdLDL Insulin resistance, ↓activity and clearance of LPL

Metabolic syndrome: accumulation of several metabolic RFs with high risk of CVD

central obesity + hyperglycemia + dyslipidemia* + hypertension

laboratory markers of
insulin resistance
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Rationale for screening:
1. The onset of type T2DM is estimated to occur 4 – 7 years
before clinical diagnosis
2. 25% of diabetic patients manifest complications at
diagnosis
3. We have well-established treatments for T2DM
4. Early diagnosis = prevention or reduction of
cardiovascular complications.
SCREENING OF TYPE 2 DM
 recommended for asymptomatic people ≥ 45 yrs at risk of DM

Risk factors:
 First degree relatives with diabetes

 Central obesity, increased BMI

 Hypertension or cholesterol abnormality

 Women diagnosed with GDM or having a baby > 4.5 kg

 Other clinical conditions associated with insulin resistance (PCOS)

 Members of a high risk population groups (e.g. race)

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CRITERIA FOR DIAGNOSIS OF DIABETES
Any one of the following:
1 FPG ≥ 7.0 mmol/L (126 mg/dL)
OR
2 2h-PG ≥ 11.1 mmol/L (200 mg/dL) during an OGTT
OR

3 Symptoms of hyperglycemia and casual P-glucose ≥ 11.1 mmol/L (200 mg/mL)

OR
4 HbA1c ≥ 6.5% (48 nmol/mol Hb)
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SCREENING OF TYPE 2 DM IN CHILDREN
Recommended for children at risk of DM every 3 years:
 Overweight, obesity

 2 other risk factors

 Family history

 Race/ethnicity recognized to increased risk

 Signs of INS resistance

 Maternal history of DM or gestational DM

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 GLUCOSE: PREANALYTICAL CONSIDERATIONS

 Blood should be drawn:

 in the morning (FPG is lower afternoon → misdiagnosis of DM) WHY?

 after an overnight fasting (no caloric intake at least 10-12 hrs)

 glucose measured in venous plasma (whole blood, serum )

 GLU concentration in plasma is app. 10 – 15% higher than in whole

blood (at normal HTK) due to different water content

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PREVENTION OF GLYCOLYSIS IN THE TUBE

 Decrease of GLU concentration in whole blood due to

glycolysis in vitro
 ↓ 0.5 mmol/L (10 mg/dL)/hour – depends on glucose
concentration, temperature, WBC count...

Preventing actions:
 Immediate separation of plasma from blood cells

 Cooling of tube in ice-water (crushed ice) immediately after blood sampling

 Use of glycolysis inhibitors to prevent short-term glycolysis (NaF and citrate buffer +
anticoagulant)
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ACTIONS FOLLOWING FASTING P-GLUCOSE
Normal Diabetes
FPG result in
mmol/L < 5.6 5.6 – 6.9 ≥ 7.0
Interpretation Normal Prediabetes Diabetes
Perform OGTT
Two results
Re-test annually in >7 mmol/L on two
Retest in 3 – 5 years
persons: different days are
Action for those at risk.
 at increased risk of diagnostic of diabetes.
DM
 with IFG or IGT OGTT is not required.

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 OGTT – standard load of 75 g glucose
How to prepare patient:
 3 days before test obvious Test is not performed if patient :
physical activity + diet without
is not fasting
CH reduction (min. 150g/D)
has an acute illness (fever, diarrhoea,
 10 – 12 hrs fasting vomiting....)
 Adequate hydration has impaired intestinal resorption
(➩ i.v. GTT ?)
 No physical activity and smoking
has vomited after ingestion of glucose
during test solution
FPG > 7.0 mmol/L repateadly - depends
on instruction of clinician 22
INTERPRETATION OF OGTT
Fasting 2 hours post load
P-GLUCOSE mmol/L mmol/L
Normal < 5.6 and < 7.8
IFG 5.6 – 6.9 and < 7.8
IGT < 6.9 and 7.8 – 11.1

DM ≥ 7.0 and/or ≥ 11.1

 IFG – increased fasting plasma glucose
PREDIABETES (R73, ICD)
 IGT – impaired glucose tolerance

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HBA1C – PREANALYTICAL, ANALYTICAL
PROS
 Patient does not need to be
fasting
 Nonsignificant effects of age,
 Sampling at any day time
 More stabile than blood glucose
CONS
 Higher price (sampling tube, method)
 Interferences and limitations:
1. Any conditions that shorten RBC survival
race or decrease mean RBC age falsely lower
HbA1c test results
2. Hb variants (S,C,D,E) and chemically
modified Hb derivates interfere with
some methods ( F+ or F-)
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OTHER TESTS FOR DIAGNOSIS OF DM
1. INSULIN/C-PEPTIDE

2. Autoimmune markers

3. Genetic testing

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 INSULIN /C-PEPTIDE – CLINICAL UTILITY
Purpose
 Distinguish between T1DM and T2DM
 Select the best hypoglycemic
agent for initial TH in T2DM
 Differential dg of hypoglycaemia
 Investigation of factitious hypoglycemia
 Monitoring a patient after the removal of
an insulinoma or pancreas or islet cell
transplantation1
Comments
Normal - high INS/CPEPT in T2DM, 0 – low in
T1DM
The lower pre-treatment INS concentration the
more appropriate might be INS or INS
secretagogues as the first choice drugs
The diagnosis of an islet cell tumour: ↑INS
↑CPEPT
After administration of exogeneous INS: ↑INS
↓CPEPT
Assessing the effectiveness of the procedure
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 AUTOIMMUNE MARKERS
In type 1 DM beta cell are destroyed and lost
in majority of pts by
 autoimmune attack
 multiple genetic predisposition
 undefined enviromental influences

Autoantibodies against various

structures of islet cells are present
• Anti-GAD: glutamic acid decarboxylase Ab
• ICA: islet cell cytoplasm Ab Positive in 75-90% of T1DM
• IA2A: thyrosine phosphatase Ab
• IAA: insulin autoanibodies

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 CLINICAL SIGNIFICANCE OF AUTOAb TESTING

Autoantibodies Clinical condition

Positive (antiGAD) Differentiation of adults with T2DM phenotype = diagnosis of LADA.

Positive autoantibodies identify progression to insulin dependence

Positive Differentiation of T1DM and MODY in unclear cases (especially in children)

Positive Risk of T1DM in patients with other autoimmune disease

Negative Neonatal DM - genetic cause

Negative Search for donors suitable for pancreatic transplantation

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MONITORING OF BLOOD GLUCOSE
 Glycemic profile (4-10-times daily) – capillary blood
 Self-momitoring – glucometers
 less accurate results
 Continuous real-time glycemic monitoring – interstitial fluid – less accurate
 physiological lag of the balance of glucose concentration between the interstitial fluid and
blood
 Calibration several times a day

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 Glycated hemoglobin HbA1c
 an indirect indicator of glycemias
over the life of erythrocytes
(~ 120 days)
 ’view from perspective’ - the
recent glycemias influence HbA1c
more then ancient glycemias
 50% of glycated hemoglobin is
formed during the last month of
RBC's life

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HBA1C – TESTING FREQUENCY
 Routine component of clinical management of patients with DM
 No consensus on the optimal frequency of HbA1c testing – national differences
 At least one test/year in all DM patients
 4 x/year in patients treated by insulin
 4 x/year in pts whose therapy has changed or who are not meeting treatment
goals
 2 x in stabile patients treated by PAD
 Always in case of admission to a hospital, if results of previous testing (2-3 Ms)
are not available

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 HBA1C – INTERPRETATION
EXAMPLE OF TREATMENT
stringent goals GOALS:
in pts without tendency to hypoglycemia
Each 10% reduction of HbA1c ( e.g. 12 vs 10.8%) - associated with 45%
lower risk of progression
HBA1c NGSP/DCCT retinopathy
% (DCCT, UKPDS)
IFCC mmol/mol
 higher treatment goals are aceptable for children and adolescents, pts
excellent
with limited life expectancy, comorbide illnesses, advanced
< 6.5 < 48complications
control
 Recent studies have not demonstrated benefits of extremly low HbA1c with
regard to cardiovascular disease
satisfying
6.6 – 7.5 50 – 58
control
 Each 1% (11 mmol/mol)change in HbA1c is related to app. 2 mmol/L
poor control
change > 7.5
in mean plasma glucose > 60

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URINARY GLUCOSE - DISADVANTAGES
 Glucosuria corelates with glycemia only if GFR is normal
 gives no information about glycemia below the variable renal glucose
threshold;
 does not distinguish between normo- and hypoglycemia;
 in pts treated with SGLT2 inhibitors (gliflozins) – ↑glucosuria;

 Semiquantitative urine glucose testing

is not recommended for routine care –
only if SMBG is refused or impossible

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 KETONES, KETONE BODIES
 acetoacetic acid (AcAc), β-hydroxybutyric acid (βHBA) = catabolic
product of FA oxidation
• present in blood and urine in very low concentrations
 2 major mechanisms increasing ketones in blood and urine:
• Increased degradation of TAG
• Decreased utilisation in the liver

 Both are due to an absolute or relative insulin deficiency and excess of

counter-regulatory hormones (cortisol, epinephrine, glucagon, GH)
 Use: diagnosis of ongoing of DKA (routinely performed in office/hospital
setting, at home)
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INTERPRETATION OF POSITIVE KETONES
Possibility of DKA:
 in patient with known DM
 the first presentation in patient not previously diagnosed with DM
Other:
 lack of carbohydrate in diet of diabetic patient
 alcoholic ketoacidosis
 starvation in non-diabetic person
 30% of pregnant women + KETONES in the
1st morning urine

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 ALBUMINURIA
1. DM is the leading cause of CKD in Western world
2. DM is associated with very high rate of CVD

 Albuminuria = early marker of both risks (diabetic nephropathy, premature

atherosclerosis)
 Albuminuria is a marker of generalized endothelial dysfunction
 2 from 3 urine samples during 6 months should be positive for confirmation
(40% day-to-day variability)

Parameter Normal Increased albuminuria Pathological proteinuria

ALB mg/mmol creat <3 3 – 30 >30

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DIABETIC HYPERGLYCEMIC EMERGENCIES

GESTATIONAL DIABETES MELLITUS

HYPOGLYCEMIAS
 HYPERGLYCEMIC EMERGENCIES
1. Diabetic ketoacidosis - DKA
2. Hyperosmolar hyperglycemic state - HHS

(= two extremes in the spectrum of decompensated DM

 DKA = absolute lack of insulin

 HHS = relative lack of insulin
 there is enough INS to prevent lipolysis and ketogenesis, but not adequate to cause
glucose utilization
 + increase in counter-regulatory hormones

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 DKA – BIOCHEMICAL BACKGROUND
 Severe alteration of carbohydrate, protein and lipid metabolism
 Body is shifted into a major catabolic state with :
1. breakdown of glycogen stores
2. hydrolysis of TAG from adipose tissue
3. mobilisation of AA from muscles

 Product of β-oxidation and AA from peripheral tissues are substrates for

gluconeogenesis and ketone production
 DKA – typical for T1DM, but almost 50% of newly diagnosed pts with DKA have T2DM
(ketose-prone subtyp)
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 CLINICAL FINDINGS
Precipitating factors:
• Inadequate or inappropriate INS therapy = INSULIN DEFICIENCY
• Infection
• Fasting
• Other acute conditions: AMI, stroke, pulmonary embolism, alacohol abuse,
pancreatitis, drugs

Signs and symptoms (> in DKA): polyuria, polydispsia, weight loss, weakness, signs of
dehydration, Kussmaul breathing, acetone breath, nausea, vomiting, abdominal pain
(may be confused with acute abdomen)
Mental status: in DKA varies from alertness to profound lethargy or coma, in HHS coma
more frequent
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 DIFFERENCES BETWEEN DKA AND HHS
Diabetic ketoacidosis Hyperosmolar hyperglycemic syndrome
Short onset (few days) Long onset (many days)

Osmolality rarely > 320 mmol/kg Osmolality frequently > 320 mmol/kg

Significant hyperketonemia and acidosis None or low ketonemia and acidosis

Hyperglycemia relatively modest (up to 35 mmol/L Hyperglycemia severe (frequently above 50
in average), rarely euglycemic DKA !! mmol/L)
Occurs mostly in T1DM Occurs mostly in T2DM

Mortality < 6% Mortality up to 30%

Hyperglycemia, hyperosmolality,
Acidosis, ketonemia, hyperglycemia
dehydration 47
 TYPICAL FINDINGS
DKA HHS
mild moderate severe
P - glucose > 15 > 25 > 35 > 50 – 60
pH 7.25 – 7.30 7.00 – 7.25 <7.00 > 7.30
HCO3 15 – 18 10 – 15 <10 > 15
U-ketone positive positive weak posit +-
P-βOH-butyrate >2 > 5 – 10 > 15 +-
(Variable: depends on glycaemia, uraemia, dehydration
S-OSM > 325
etc.) <320
AG ↑ ↑ ↑ normal
Mental status alert drowsy/stupor stupor/coma stupor
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 OTHER LABORATORY FINDINGS
Water and ions deficit (K, Na, Mg) due to osmotic diuresis - results in next laboratory
findings:
1. Hyperkalemia in presense of MAC – redistribution from ICT, low or normal S-K (initial,
or after treatment) is a sign of possible dificit ↑urea ( ≈ 15 mmol/L) – dehydration,
prerenal kidney failure
2. ↑creatinin (≈ 150 μmol/L) – result of dehydration (prerenal uremia) or interference
of ketones
3. ↑AMS + lipase: are not reliable tools for diagnosis od pancreatitis in the setting of
DKA
4. Hyperlipidemia (lipolysis > utilization)
5. Leukocytosis – due to ↑counter-regulatory hormones
- but > 25 x109/L possibly signalizes ongoing infection !
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GESTATIONAL DIABETES
(seminar)
 CONCLUSION

 During normal pregnancy, insulin sensitivity declines with advancing gestation.

In a physiological situation, the compensatory increase in insulin secretion
maintains a normal glucose homeostasis.
 Because of the physiological insulin resistance, pregnancy is a suitable period to
reveal undiagnosed diabetes.
 GDM is most commonly a forerunner of type 2 diabetes (T2DM).
 High risk patients should be screened as early as possible using fasting plasma
glucose, and if normal, rescreen ed between 24–28 WG using 75 g OGTT.

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HYPOGLYCEMIA
 HYPOGLYCEMIA
 Clinical state associated with abnormally low B-GLU and typical clinical
sings
 Definition is arbitrary – glycemic threshold at which symptoms occur differs
between individuals (< 2.8 – 3.3 mmol/L)
 Especially women and children may have B-GLU < 3.0 mmol/L without symptoms
(brain uses ketones for energy)
 Diabetics pts may manifest hypoglycemic symptoms at higher glycemia

Whipples triad:
 Symptoms consistent with hypoglycemia
 Low plasma glucose concentration
 Resolution of those symptoms after the plasma glucose levels raised
57
FASTING HYPOGLYCEMIA
Cause Examples
 Increased insulin
Endogenous overproduction of insulin Hyperinsulinisms of childhood, insulinoma, pancreatic
tumour (MEN 1)
Exogenous insulin Incidental or intentional overdosing
 Insulin normal or low
Adrenocortical insufficiency and hypothyroidism, GH
Endocrine disorders (↓gluconeogenesis)
deficiency
Failure of critical organs (↓gluconeogenesis +
Liver failure, kidney failure
↓degradation of insulin
Extra pancreatic tumours (↑glucose utilisation + leiomyosarcoma, fibrosarcoma, mesothelioma, hepatoma,
production of IGF-1 carcinoma (stomach, rectum, pancreas)
Low intake, increased demands of the organism malnutrition, starvation, sepsis,

Drugs and toxins (various mechanisms) sulphonyl urea, ethanol, salicylates, beta-blockers etc.
Inherited metabolic disorders of saccharides, fatty glycogen storage disease, galactosemia, fructose
acids and amino acids intolerance, carnitine deficiency, leucinosis, tyrosinemia,etc.
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 POSTPRANDIAL- REACTIVE HYPOGLYCEMIA
 occurs usually 1,5 - 3 hours after meal in people without
DM:

 due to a rapid increase in insulin concentration (high

sugar meal on empty stomach, alcohol)
 following rapid entry of food into the small intestine -
after gastric surgery (gastric bypass or surgery for the
management of ulcer disease).
 enzyme deficiency (imborn metabolic errors) -
galactosemia, congenital fructose intolerance

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 PSEUDOHYPOGLYCEMIA (IN VITRO)

 due to consumption of glucose in the collection tube while the sample

waits for testing, in case of:

 leukocytosis (leukemias)
 erythrocytosis (polycythemia vera)

 It may occur when the separation of plasma from the formed elements
of the blood is delayed.

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 LABORATORY INVESTIGATION
AIM: 1. TO DEMOSTRATE HYPOGLYCAEMIA
2. TO IDENTIFY THE CAUSE
 Collection of blood during an episode of hypoglycemia and before administration
of glucose
 in asymptomatic patient – provocation tests (72-h controlled fasting or mixed
meal test)

LAB TESTS: basic metabolic panel, LFTs, EtOH, toxi screen, infectious diseases workout
Test for identification of the cause of hypoglycemia:
 INSULIN and C-PEPTIDE
 Ketones, β-OH butyrate
 Hormones: Cortisol, ACTH, GH, IGF-1, IGF-2, TSH... 62
Thank you for
attention

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