Physiology Labs Protocols Block 3 - fyziologie.lf1.cuni.cz · INSIPIDUS). Polyuria can be osmotic...

Physiology Labs v 0.2 (2016) 1

Physiology Labs

Protocols

Block 3 v. 0.2

physiology.lf1.cuni.cz

Labs aim: Explore biology in context through brain and hands


Metabolic Rate Measurement Basal Metabolism (Basal Metabolic Rate)

AIM of the lab

Get ideas about energetic needs and consumption REQUIRED KNOWLEDGE

TASKS 1. Measure resting metabolic rate (in details described here) 2. Measure BMR of experimental animals (principles same as in #1) 3. Find expected value of Basal Metabolism from Tables of norms or Harris-

Benedict Formula (use calculator available on PCs in the lab) 4. Estimate your typical daily energy needs 5. Estimate Fuel Value of your typical meal

WHAT

1. Basal Metabolism definitions PubMed: Heat production, or its measurement, of an organism at the lowest level of cell chemistry in an

o inactive, o awake o fasting state.

It may be determined: o directly by means of a calorimeter o OR indirectly by calculating the heat production from an analysis of

the end products of oxidation within the organism OR from the amount of oxygen utilized.

WHY …

1. … is MR or O2 consumption estimated? a. BMR can be altered in some diseases, typically endocrine (T-

hormones) b. O2 consumption is regularly tested in sports medicine

2. … do we perform the lab

a. Demonstration of measurement b. Training of use air ways c. Practical example of Energy consumption, approx. estimation of daily

E needs and intake (estimation of caloric value of meals) d. Get ideas obesity

HOW

1. Basal /resting metabolism measurement. o BM is determined through indirect calorimetry from oxygen

consumption. The method assumes, that during aerobic oxidation E – energy (and also P – product) is/are released from S – substrate:

S + O2 – > P + E


This means that energy released is proportional to O2 consumption:

E ~ O2

Aerobically, E is linearly proportional to O2 consumption.

E = O2 . EE

EE – energetic equivalent, i.e. amount of E released if 1lt of O2 is consumed. For average food EE ≈ 20 kJ/lO2

o O2 consumption. (O2 cons) It is calculated as difference between inspired O2 and expired O2:

O2 cons = O2 IN – O2 OUT

O2 IN

Amount of inspired O2 is cca 21% of inspired air since there is cca 21% of O2 in air we breathe in (atmospheric air - ATM)

O2 IN ~ 21% of inspired air O2 IN = pO2 ATM * ventilation

O2 OUT

Amount of expired O2 is unknown fraction of enspired air since this fraction (pO2 EXP – O2 partial pressure in expired air) is not constant and it must be measured!

O2 IN = pO2 EXP * ventilation

Ventilation, minute ventilation (MV) can be easily measured by spirometer. Similar method is used as Spirometry lab (1st semester) As air flows through turbine, propeller rotates. Rotations are counted and thus provide air flow = ventilation data.

Taken together: O2 cons = O2 IN – O2 OUT

O2 cons = pO2 ATM * ventilation – pO2 EXP * ventilation O2 cons = (pO2 ATM– pO2 EXP) * ventilation

Items in blue need to be measured. Since they can vary with each breath, we perform 10-minutes measurement for an averaging.

Temperature and pressure correction since the volume of air depends on temperature and pressure, the measured volume of O2 should adjusted to standard conditions, i.e. T= 0°C and p = 760 mm Hg:


Torr760TC273

C27310

BTVV

V0 = standard volume, V1 = O2 consumption measured during experiment, T = temperature during experiment, BT = barometric pressure during experiment

Final calculation EEconsOMR Vo 2

MR – metabolic rate. O2consVo – O2 consumption converted to standard volume, EE – energetic equivalent.

SETUP

1. Principle. Expired air is collected in reservoir and (with permanent flow of expired air →). In this averaged sample of expiratory air O2 concentration and air flow is measured.

2. Detailed scheme of interconnection of all setup components. In all cases, connectors are designed so that they fit in suitable sockets only. O2 sensor, barometer and thermometer can be connected into any of 4 analogue ports (so not only as shown on the picture)

AIR IN

AIR OUT

Flow of air


Blood Glucose Measurement Glucose tolerance test, Glycemia profile curve

AIM of the lab

Practical demonstration of some blood glucose regulatory mechanism. REQUIRED KNOWLEDGE

Blood Glucose (def., norms) Blood glucose regulation (insulin, glucagon, epinephrine, corticoids, …) Glucose estimation Diabetes Spectrophotometry (principle, absorbance, standard, blank, calibration curve,

Beer-Lambert law) TASKS

1. Perform Intravenous Glucose Tolerance Test (animal model) 2. Administer insulin, monitor glycemia, respiration and ECG. 3. Estimate blood glucose in all collected samples

WHAT

1. Glucose tolerance test [MeSH UID D005951] PubMed: A test to determine the ability of an individual to maintain HOMEOSTASIS of BLOOD GLUCOSE. It includes measuring blood glucose levels in a fasting state, and at prescribed intervals before and after oral glucose intake (75 or 100 g) or intravenous infusion (0.5 g/kg).

2. Blood Glucose (Glycaemia) [MeSH UID D001786]. PubMed: Glucose in blood. Normal fasting values around 5 mmol/l (90 mg/dL)

3. Diabetes Mellitus [MeSH UID D003920] PubMed:A heterogeneous group of disorders characterized by HYPERGLYCEMIA and GLUCOSE INTOLERANCE.

WHY …

1. … is glucose tolerance test performed clinically

a. To help in diagnosing diabetes or impaired glucose tolerance – IGT (“prediabetes”)

2. … do we perform the lab? a. To discuss diabetes b. To see the methods of blood

glucose estimation c. To practice vital functions

monitoring and acute care

Sample glucose profile curve during oGTT (oral glucose tolerance test):


HOW In Principle:

Blood glucose is estimated in 10 minutes intervals (8 samples) in two animals After 1st sample animals are treated with Insulin or glucose. Blood glucose is estimated enzymatically (spectrophotometry and personal

glucometer) and glucose profile is constructed Complex monitoring and care must be provided to both animals all the time

while in the lab. Experiment timeline

Glycemia estimation: 1. In Principle:

Glucose is estimated spectrophotometrically Glc being clear thus needs to be converted to coloured product. Set of enzymatic reactions (assuring HI specificity) result in colour change

of an indicator Glc concentration is proportional to optical density of processed sample.

More exactly, it is linearly proportional to absorbance of sample

2. Principle of enzymatic reactions:


Working solution thus contains: Glucose-oxidaze Peroxidaze Indicator in leuko (uncolored) state

3. Lab Procedure (principle) Dilution. Sample is diluted by 5% TCA. Higher volume is obtained, while

low volume of blood (< 100 µl) is withdrawn. It is much easier to work with >>100 µl than <50 µl.

Centrifugation. Hemoglobin would interfere with the spectrophotometry of glucose, thus it must be removed by centrifugation.

Enzymatic reaction. Glucose oxidation and subsequent peroxidation start immediately once supernatant and working solution are mixed

incubation. me. Under room temperature it

4. Photometry

(Wikipedia): In spectroscopy, the absorbance A is defined as:

Aλ = − log(I / I0),

where I is the intensity of light at a specified wavelength λ that has passed through a sample (transmitted light intensity) and I0 is the intensity of the light before it enters the sample (or incident light intensity).

5. NOTE!

During the whole experiment, monitor vital functions (respiration and ECG)

In case of emergency, provide proper care including but not limited to: Glucose administration in case of insulin treated animal CPR Pharmacological support (epinephrine or atropine)

Absorbance Aλ = − log(I / I0)

I0 I

I0 – intensity cuvette I - intensity of light IN of light OUT


Initially, insulin should be administered to the animal with higher starting

blood glucose level (at time 0). RESULTS weight

[g] Treat-ment

Animal 1

Animal 2

conc.

mmol/l A

standartd 1

standartd 2

standartd 3

Time [min] 0 10 20 30 40 50 60 70 A (Isulin treated)

A (Glc treated)

Glc (Ins) mmol/l

Glc (Ins) mmol/l

A – absorbance, Glc – blood glucose

10 20 30 40 50 60 70 Time [min]

Glc [ ]


Polyuria, clearence

Name Date

AIMS of the lab

Practical demonstration of osmotic polyuria, i.v. administration Detailed understanding the principles of clearance estimation

REQUIRED KNOWLEDGE

Diuresis Polyuria, osmotic/water Renal functions, principles Concentration of urine – mechanism Renal threshold

WHAT

1. Polyuria [MeSH UID D011141] PubMed: Urination of a large volume of urine with an increase in urinary frequency, commonly seen in diabetes (DIABETES MELLITUS; DIABETES INSIPIDUS).

Polyuria can be osmotic (driven by excess of solutes to be eliminated) or water polyuria (driven by excess of water to be eliminated) Osmotic polyuria results from increased amount of particles (substances) in renal tubules, (relative to norm) that cannot be dissolved in usual volume of urine Renal concentration capability is 600-1200 mOsmol/l. Roughly 600 mOsmols of solutes need to be eliminated daily (depending on diet, metabolism, extrarenal losses)

2. Glomerular Filtration rate (GFR) [MeSH UID D005919] PubMed: The volume of water filtered out of plasma through glomerular capillary walls into Bowman's capsules per unit of time. It is considered to be equivalent to INULIN clearance. Year introduced: 1966(1965)

TASKS

1. Osmotic polyuria.

a. In two anesthetized subject check anesthesia depth, all vitals and body weight.

b. Record all data into Anesthesia protocol and keep monitoring the subject continuously. Record the observation at least every 10 minutes

c. Empty urinary bladder (careful compression), record urine volume and check for glycosuria


d. In case sedation and vitals are satisfactory, administer: Substance: GLC 20% or Manitol 20% Route: i.v. Dose: 1 ml /100 g. Rate: 1 ml/min

e. Keep monitoring the subject. Correct any abmormalities f. Collect all urine produced (empty the bladder), record volume and

check for glycosuria Subject 1 Subject 2

Time relative to administration

Urine volume [ml] GLC

Glc in urine (approx.) GLC

Urine volume [ml] MANITOL

Glc in urine (approx.) MANITOL

Time 0 min

Time 5 min

Time 10 min Time 15 min Time 30 min Results Inerpretation:

. 2. Understand the principles of GFR measurement

a. Which substances can be used for GFR measurement b. Which parameters need to be measured and how in order to calculate

GFR? c. What is the relationship between GFR and plasma creatinine

concentration? d. Calculate clearance of creatinine based on sample values (Do search).

QUESTIONS

1. Why does osmotic polyuria occur? 2. What are the differences between mannitol and glucose? (Biochemistry, renal

filtration, renal absorption, clinical effect when administered, etc.) 3. Why do we use G20% rather than G5%? 4. What are necessary precautions when administering G20? 5. In clinical setup G20 would be administered together with insulin. Discuss

why. 6. Does glycosuria implicate polyuria and vice versa. 7. Comment on urinary Glc concentration in glycosuria in the lab.


Vision Name Date

AIM

Understand common refractive disorders, principles of evaluation and correction

Understand principles and use of ophthalmoscopy. Practice ohthalmoscopy Demonstrate some other tests for vision

REQUIRED KNOWLEDGE

Snell’s law, optical rays refraction (converging spojka, convex, concave lens)

Principles of ophthalmoscopy. Direct and indirect.

Purkyne images

Eye Anatomy. Eyeball, muscles, visual pathway, retina, aqueus humor

Accomodation – principles, significance, presbyopia

Weber-Fechner psychophysical law

(Optical transduction) TASKS

1. Visual acuity, visus. Visus checks the resolution of visual system (i.e. optical system, transduction, , pathway and image processing.) It is very sensiteive but low specific test.

a. Estimate visus by means of Snellen optotypes. (see biophysics) b. Explain the significance of Visual acuity test c. Write your results

Visus RIGHT LEFT

Inerpretation:

2. Near point (NP), Far point (FP) a. Estimate NP and FP by means of Snellen’s optometer b. Explain the principle of optometry, why is additional lens used c. Explain myopia, hyperopia. d. Write and comment the results of yourself and at least 5 other students:

RIGHT LEFT PF

NP FP NP

Inerpretation:


3. Astigmatism

Astigmatism is irregular curvature of the cornea and/or lens. It often results in reduced visual acuity.

a. Estimate astigmatism by means of ceratoscope, ophthalmometer and optometer

b. Understand differences between astigmatism of cornea, lens, and total. c. Understand differences between regular and irregular astigmatism d. Write and comment your results:

Astigmatism RIGHT LEFT

Horizontal

Vertical Horizontal vertical

Cornea (D) (Ophthalmometer)

FP (D) (optometer)

Inerpretation:

4. Ophthalmoscopy Ophthalmoscopy is observation of the inside the eye, mainly the posterior.

a. Draw the scheme explaining the principle of direct and indirect ophthalmoscopy

b. See and explain the operation of a real ohthalmoscope c. Perform ophthtalmoscopy on one of your friends. Try hard to see at

least some vessels. d. Which simple pathologies can be identified by ophthalmoscopy? Fill in

Structure

Observation Implication

Papilla

Retina

Retinal artery

…


5. Perimetry Perimetry tests the area of peripheral vision. Drops in peripheral (even central) vision are called scotomas and these reflect ongoing pathology of the eye or visual pathway.

a. Perform perimetry in at least one eye. Plot the results

b. Explain what is scotomoa.

c. Can scotoma be physiological? d. What is bitemporal hemianopsia?

6. Binocular vision or 3D vision, stereoscopic vision, perception of depth.

a. See the stereoscopic test available in the lab b. Can we perceive depth with monocular vision?

7. Colour Vision

a. See the chromatic charts and understand the core principle QUESTIONS Which refractive disorder/s cannot be corrected by regular lenses? Please explain. Which refractive disorders you know? Which parameter best reflects myopia/hyperopia? FP, NP, accommodation range, Visus? What does Accommodation range reflect? Introduce several indications for performing ophthalmoscopy What are the advantages of direct/indirect phthalmoscopy Why is it vital to perform ophthalmoscopy prior to lumbar puncture?

Physiology Labs Protocols Block 3 - fyziologie.lf1.cuni.cz · INSIPIDUS). Polyuria can be osmotic...

Documents

Transcript of Physiology Labs Protocols Block 3 - fyziologie.lf1.cuni.cz · INSIPIDUS). Polyuria can be osmotic...