Physiology in Anaesthesia Flashcards
Name the afferent receptors of ventilation
- Chemoreceptors: central and peripheral;
- Lung stretch receptors: bronchial and smooth muscle;
- Irritant receptors: epithelial cells;
- Juxtacapillary receptors: unmyelinated;
- Other: bronchial c-fibres, nose and upper airway, joint and muscle receptors, airway baroreceptors, pain and temperature receptors
Foetal circulation: which organs get the best oxygenated blood
brain
liver
heart
Foetal circulation: what causes the fall in peripheral vascular resistance after birth
- raised PaO2
- decreased lung fluid
- alveolar surface tension
- shear stresses stimulating nitrous oxide production
- prostaglandins, endothelins, prostacyclin
Foetal myocardium: differences from adult
- fewer myofibrils
- greater water content
- fewer contractile elements
- poorly organised t-tubules
- immature sarcoplasmic reticulum
- under-developed calcium cycling and contraction-coupling
- less tension per gram
- impaired compliance
- downregulated adrenergic receptors
- incomplete sympathetic control
Blood volume in the human foetus
100-110 ml/kg
10-12% body weight
5 Stages of foetal lung development
- embryonic
- pseudoglandular
- canalicular
- sacular
- alveolar
What is the surface tension of:
1. water
2. surfactant (on functional film of alveoli)
- 70 mN/m
- 25 mN/m
Contents of surfactant
A. Lipids:
- phospholipids
- neutral lipids
- cholesterol
B. Proteins:
SP A to D
Functions of the various surfactant proteins
SP-A: regulates secretion and reuptake of phospholipids; part of the innate pulmonary host defence system.
SP-B and C: interact with lipids to promote formation and adsorption of surface tension.
SP-D: part of the innate pulmonary host defence system.
What triggers breathing (in humans)
- respiratory rhythm generator
- central respiratory in brainstem
- continuous respiratory pacemakers
Why do neonates have accelerated heat loss
- relatively larger body surface area
- thin layer of insulating subcutaneous fat
- limited capacity for thermogenesis
Foetus: what is the functional endocrine state
- raised plasma insulin
- reduced glucagon levels
Describe: Endocrine stress response after birth
Increased production of:
catecholamines, glucagon, glucagon receptors, cortisol
Decreased production of: insulin
Promotion of: glycogenolysis, gluconeogenesis, lipolysis
Neonatal fluid status characteristics
increased total body water,
raised extracellular fluid volume,
increased water turnover rate
Four phases of the left ventricular pressure-volume loop
- isovolumetric contraction
- ventricular ejection
- isovolumetric relaxation
- diastolic ventricular filling
Name and define
K1
K2
of the oxygen delivery equation
K1: 1.34
amount of oxygen that binds to Hb
K2: 0.003
solubility coefficient of oxygen in blood at 37C
With relevance to the oxygen delivery equation:
which parameters may cause deficient oxygen delivery
low PaO2
low Hb
low cardiac output
What are the values of PaO2 and SaO2 of the:
1. arterial point
2. mixed venous point
3. P50
- PaO2: 100mmHg, sats 97.5%
- PaO2: 40mmHg, sats 75%
- PaO2 26.6mmHg, sats 50%
What is meant by the term P50
The partial pressure of oxygen at which Hb is 50% saturated;
It is plotted on the x-axis only
What changes occur after smoking cessation at:
12-18 hours
6 weeks
6-8 weeks
After 8 weeks
- Increase of P50 from 22.9mmHg to 26.4mmHg,
decreased CO2 from 6.5% to 1.1% - Improved immunity and wound healing
- Hepatic enzyme activity restored to normal
- improved pulmonary function,
increased mucociliary transport,
decreased mucus secretion,
increased airway diameter
Disadvantage of pre-operative smoking cessation
Increased incidence of PONV
Thickness of the average human cell membrane
10nm
Venous drainage of the lower and upper rectum
Lower: inferior and middle rectal veins - drain directly into the IVC
Upper: superior rectal vein - drain into the portal system
Which organs regulate gastric emptying, and how
Stomach: promotes emptying
Duodenum: inhibits emptying
Name the stages of deglutition
- voluntary stage
- pharyngeal stage (involuntary)
- oesophageal stage (involuntary)
What are the motor functions of the stomach
- storage of large quantities of food
- mixing of food into chyme
- slow emptying of chyme into the duodenum
Factors that inhibit gastric emptying
- distension of the duodenum
- irritation of duodenal mucosa
- acidity of chyme in the duodenum
- osmolality of the chyme
- breakdown products of meat and fats in the chyme
What is the stomach mucosa made up of
- mucus-secreting cells
- oxyntic glads / gastric glands
- pyloric glands
What cell types are found in the oxyntic/gastric glands, and what do they secrete
- mucous neck cells: secrete mucus
- peptic/chief cells: secrete pepsinogen
- parietal/oxyntic cells: secrete HCl and intrinsic factor
- enterochromaffin-like cells: secrete histamine
What are the final secretions of a canaliculus of the stomach
water
HCl
KCl
NaCl
What chemical factors stimulate gastric secretion
acetylcholine
gastrin
histamine
In the stomach: what secretions do these chemical factors stimulate:
1. acetylcholine
2. gastrin
3. histamine
- pepsinogen, HCl, mucus
- HCl
- HCl
Define: transitional physiology
dynamic state of changes
that enable the foetus
to adapt to extrauterine life
What is the final outcome of transitional circulation
Reduction of the PVR to one-fifth of the SVR
Name the foetal shunts of circulation
- ductus venosus
- ductus arteriosus
- foramen ovale
Name causes of increased urea production
high protein diet
increased catabolism
corticosteroids
GIT bleeding
malignancy
tetracyclines
Name causes of decreased urea production
low protein diet
reduced catabolism e.g. elderly
liver failure
reduced absorption of nutrients
Name causes of increased urea elimination
elevated GFR e.g. pregnancy
Name causes of decreased urea elimination
glomerular disease
reduced renal blood flow
tubulo-interstitial disease
How is urea formed
Amino acids are deaminated;
NH2 groups form ammonia (NH3);
ammonia is converted to urea in the liver.
Is urea an accurate marker of glomerular filtration -
why/why not
No;
urea can also be reabsorbed by the tubules. Urea clearance is less than GFR.
What is cystatin C
protein secreted by all nucleated cells;
potential new marker for detecting kidney injury;
reflects a reduction in GFR;
min. influenced by weight, sex, race, age, muscle mass
True/False:
normal creatinine reflects normal renal function;
explain
False;
healthy individuals have a large reserve of renal excretory function. Serum creatinine does not rise above normal until the GFR drops by 50-60%.
Factors which affect differences in creatinine concentrations
body weight,
sex,
race,
age,
muscle metabolism,
protein intake
What is the Jaffe Reaction;
What factors may complicate interpretation
The most commonly used measurement of serum creatinine;
affected by ketones, glucose, proteins
Normal values:
Serum osmolality
Urine osmolality
282-295 mOsm/kg
500-800 mOsm/kg
Normal renal blood flow
1,200ml/min
Kidneys receive what percentage of cardiac output
20-25%
What is the renal filtration fraction
The fraction of renal plasma flow filtered by the glomerular membrane;
normally 0.2
The glomerulus is made of three types of cells, namely
- endothelial
- epithelial
- mesangial
What is the effect of mesangial cell action
Contraction reduces cross sectional area of the glomerular basement membrane and reduces GFR.
Relaxation increases GFR.
Factors that cause mesangial cell contraction
catecholamines
rening-angiotensin-aldosterone
pituitary hormones
endothelins
platelet factors
eicosanoids (leukotrienes, thromboxane A2, prostaglandin F2)
Factors that cause mesangial cell relaxation
natriuretic factors
Nitric oxide
dopamine
prostaglandin E2
What are the filtration mechanisms of the glomerular membrane
- size selectivity
- charge selectivity (albumin and negatively charged proteins are repelled)
Define osmosis
The net movement of water across a semi-permeable membrane. Water tends to move from an area of high concentration to an area of low concentration.
Define osmolarity cf. osmolality
Osmolarity is the osmolar concentration expressed as osmoles per litre of solution;
Osmolality is the osmolar concentration expressed as osmoles per kilogram of water.
Osmolality is responsible for osmotic pressure, but osmolarity is more practical.
Formula for calculating osmolarity
2Na + Glucose + Urea
What is the osmolar gap, and what is the normal value
The difference between the measured osmolality and the calculated osmolarity;
difference should be <10
Causes of a high osmolar gap
mannitol
methanol
ehtanol
ethylene glycol
sorbitol
polyethylene glycol
propylene glycol
glycine
maltose
Define Nernst Potential
The potential across the cell membrane
that exactly opposes net diffusion
of a particular ion
through that membrane
What percentage of body mass is water
60%
What percentage of body water is intracellular was extracellular
extracellular one third
intracellular two thirds
What fraction of the extracellular fluid is plasma volume vs interstitial volume?
Plasma volume one fourth
interstitial volume three fourths
What is heparan sulphate, and what is its function
a component of the fused basement membrane of the glomerulus;
it is negatively charged and prevents filtration of negatively charged substances
If renal clearance is less than the glomerular filtration rate of substance x, then there is net tubular _____ (reabsorption/secretion) of x.
Reabsorption
If renal clearance is greater than the glomerular filtration rate of substance x, then there is a net tubular _____ (reabsorption/secretion) of x.
Secretion
What is para-aminohippuric acid (PAH)
a derivative of hippuric acid;
a substance secreted by renal tubules;
almost all arterial blood entering the glomerulus is cleared of PAH;
venous blood contains almost no PAH;
the amount of PAH in urine is almost the same as that of plasma entering the glomerulus.
What is the formula for estimating the effective renal plasma flow using para-aminohippuric acid?
Effective renal plasma flow = urine concentration of para-aminohippuric acid times the urine flow rate divided by the plasma concentration of para-aminohippuric acid (UPAH × V/PPAH)
What is the formula for estimating renal blood flow if renal plasma flow is known?
Renal blood flow = renal plasma flow divided by (1 - the hematocrit), or RBF = RPF/(1 - Hct); in a normal individual, renal blood flow will be approximately double the renal plasma flow
Effective renal plasma flow _____ (over-/under-) estimates true renal plasma flow by approximately _____%.
Under; 10
it is an underestimate because 10% of renal blood flow perfuses the kidney parenchyma rather than being filtered through the glomerulus
How is the filtration fraction for a molecule determined?
By determining the ratio of the glomerular filtration rate to renal plasma flow
What are the effects of MOST prostaglandins on the glomerulus?
Prostaglandins cause dilation of the afferent arteriole and an increased glomerular filtration rate
What are the effects of angiotensin II on the glomerulus?
Angiotensin II causes constriction of the efferent arteriole and increased glomerular filtration rate
What type of drug blocks the effect of prostaglandins on the afferent arteriole?
NSAID
What type of drug blocks the effect of angiotensin II on the efferent arteriole?
ACE-inhibitor
What is free water clearance?
A measure of the kidney’s ability to dilute urine
What is the relevance of isotonic urine
In isotonic urine
free water clearance is equal to zero;
this is seen with the use of loop diuretics.
What is the formula for filtered load?
Filtered load = glomerular filtration rate × Px; where Px is the plasma concentration of x
What is the formula for excretion rate
Excretion rate = V × Ux; where V is the urine flow rate and Ux is the urine concentration of x
What is the formula for the amount of substance reabsorbed in the kidney
Reabsorbed = Filtered - Excreted
In the nephron, glucose at normal plasma concentrations is reabsorbed in which structure? By which transporter?
Proximal tubule,
sodium-glucose cotransporter
At normal plasma glucose concentrations, how does the proximal tubule handle glucose in the urine?
completely reabsorbed
How and where are amino acids reabsorbed in the kidney?
proximal tubule;
three distinct sodium-dependent transporters
What substance in the urine acts as a buffer for hydrogen ions
ammonia
In the thin descending loop of Henle, water is passively reabsorbed, because the tonicity of the medulla is _____.
hypertonic
The thin descending loop of Henle in a kidney nephron is permeable to _____ (sodium/water) but not to _____ (sodium/water).
water;
sodium
Does the thin descending loop of Henle reabsorb water passively or actively?
Passively
Which three ions are actively reabsorbed in the thick ascending loop of Henle of a kidney nephron?
sodium
potassium
chloride
In the thick ascending loop of Henle of a kidney nephron, which two ions are indirectly reabsorbed as a result of the active reabsorption of sodium, potassium, and chloride?
Magnesium
Calcium
The thin descending loop of Henle is _____ (impermeable/permeable) to water, whereas the thick ascending loop of Henle is _____ (impermeable/permeable) to water.
permeable;
impermeable
What two ions are actively reabsorbed in the early distal convoluted tubule of a kidney nephron?
sodium
chloride
Which hormone controls the reabsorption of calcium in the early distal convoluted tubule of a kidney nephron?
parathyroid hormone
In the collecting tubule of a kidney nephron, which ion is reabsorbed in exchange for secreting potassium or hydrogen ion?
sodium
Which segment of the kidney nephron does the hormone aldosterone act on?
collecting tubules
Which segment of the kidney nephron does the hormone antidiuretic hormone (vasopressin) act on?
collecting tubules
Sodium/potassium exchange and sodium/hydrogen exchange in the collecting tubule of a kidney nephron is regulated by which hormone?
aldosterone
The reabsorption of water in the collecting tubule of a kidney nephron is regulated by which hormone?
antidiuretic hormone
What type of cell in the collecting tubule of a kidney nephron is responsible for sodium/potassium exchange and the reabsorption of water?
principal cell
Define simple diffusion
Diffusion along a concentration gradient; i.e. solutes move from a higher concentration gradient to a lower concentration gradient. No external energy is required.
Define facilitated diffusion
Diffusion through channels, which allow only certain ions to pass through. No external energy is required.
Define active transport;
and primary vs secondary active transport
process that moves solutes against their concentration gradient, and required energy input.
Primary: relies on breakdown of ATP to power a pump;
Secondary: transport of one ion along its concentration gradient, which facilitates diffusion of another ion against its concentration gradient.
Substances reabsorbed in the proximal tubule
sodium
chloride
bicarbonate
urea
glucose
albumin
amino acids, phosphates, sulphates
Substances secreted into the proximal tubule
penicillins, thiazides
histamine, thiamine
EDTA
hydrogen ions
Which transporter initiates all reabsorption in the proximal tubule
Na-K-ATP-ase
Reabsorption of bicarbonate in the proximal convoluted tubule relies on which enzyme
carbonic anhydrase
Two methods of reabsorption of chloride in the proximal tubule
- antiport mechanism pumps organic anions into the lumen in exchange for chloride ions;
- concentration gradient for paracellular diffusion of chloride develops due to earlier reabsorption of sodium and bicarbonate.
Water is reabsorbed in the proximal tubule by which routes
transcellular
paracellular
What is responsible for glucose reabsorption in the proximal tubule
SGLT (sodium glucose cotransporters)
What is the main role of the long loops of henle
To produce a hypertonic environment in the renal medulla, in order to facilitate more osmosis and therefore water reabsorption
How much filtrate enters the distal tubule and collecting ducts per day
23l/day
How much filtrate:
enters the glomeruli;
is reabsorbed in the proximal tubules;
is reabsorbed in the loops of Henle
- 180l/day
- 127l/day
- 30l/day
What is reabsorbed in the distal tubule
sodium
chloride
Antidiuretic hormone controls:
ECF osmolality
ADH causes the appearance of what in the collecting ducts
Aquaporin-2
Aquaporins assist in the reabsorption of…
water and urea in the collecting ducts
Aldosterone controls:
ECF volume
How does angiotensin II cause increased blood pressure
- release of ADH by the hypothalamus
- vasoconstriction by contraction of smooth muscle walls of arterioles
- aldosterone release from the adrenal cortex (zona glomerulosa)
- increased sodium reabsorption in proximal tubule through Na-H antiporter
- increased sympathetic outflow from CNS
Where is renin produced
juxtaglomerular apparatus
What is the effect of thyrotropin-releasing hormone on pituitary hormone secretion?
TSH secretion
prolactin secretion
What is the effect of dopamine on pituitary hormone secretion?
inhibition of prolactin secretion
What is the effect of corticotropin-releasing hormone on pituitary hormone secretion?
stimulates adrenocorticotropic hormone secretion
What is the effect of growth hormone-releasing hormone on pituitary hormone secretion
stimulates growth hormone secretion
What is the effect of somatostatin on pituitary hormone secretion?
inhibits TSH secretion
inhibits growth hormone secretion
What is the effect of gonadotropin-releasing hormone on pituitary hormone secretion?
stimulates luteinising hormone secretion
stimulates follicle-stimulating hormone secretion
What is the effect of prolactin on pituitary hormone secretion?
inhibits gonadotrophin-releasting hormone;
therefore inhibits LH and FSH secretion
How does prolactin provide negative feedback to its own secretion?
high prolactin increases dopamine secretion by hypothalamus;
dopamine secretion in turn inhibits prolactin secretion
Why might bromocriptine be withheld from a woman who is trying to breast feed?
bromocriptine is a dopamine agonist;
will decrease prolactin secretion;
therefore decrease breast milk production
A woman being treated for schizophrenia complains of breast fullness and amenorrhea; what is the likely cause?
antipsychotics are dopamine-antagonists;
resulting in increased prolactin secretion
Why are women less likely to become pregnant when breastfeeding?
prolactin inhibits gonadotrophin-releasing hormone;
thus prevents ovulation
A woman has had her thyroid surgically removed and is not taking thyroid hormone replacement. She now complains of amenorrhea; what is the likely cause?
hypothyroidism may cause increased prolactin;
prolactin causes amenorrhoea
How does prolactin affect fertility in men?
Decreases synthesis and release of gonadotrophin-releasing hormone;
therefore inhibits spermatogenesis
Which enzyme is responsible for converting cholesterol to pregnenolone?
desmolase
Which enzyme catalyzes conversion of testosterone to dihydrotestosterone?
5-reductase
What is the effect of congenital adrenal enzyme deficiencies on adrenal size?
Decreased cortisol production and loss of negative feedback causes increased adrenocorticotropic hormone stimulation leading to bilateral adrenal enlargement
Which enzyme catalyzes the conversion of testosterone to estradiol?
aromatase
What stimulates aldosterone synthase to convert corticosterone into aldosterone?
angiotensin 2
Name five functions of cortisol
Blood pressure maintenance;
Bone formation;
anti-Inflammatory;
inhibits Immune functions;
Gluconeogenesis, lipolysis, proteinolysis
[“cortisol is BBIIG”]
What hormones are involved in regulation of cortisol secretion?
corticotrophin-releasing hormone in hypothalamus;
stimulates adrenocorticotropic hormone release in anterior pituitary;
which stimulates cortisol production by adrenal gland.
In what form is cortisol found in the bloodstream?
bound to corticosteroid-binding globulin
Which cells normally produce parathyroid hormone?
chief cells of parathyroid gland
What is the effect of parathyroid hormone on serum calcium and phosphate?
increases serum calcium;
decreases serum phosphate
What are the functions of gastrin?
- increases acid secretion
- promotes growth of gastric mucosa
- increases gastric motility
What are stimuli for the release of gastrin?
distension
amino acids
vagal stimulation
What serves as negative feedback for gastrin release?
acid secretion;
pH <1.5
What two amino acids are especially potent stimulators of gastrin release?
tryptophan
phenylalanine
Which cells in the gastrointestinal tract make cholecystokinin?
I cells of duodenum and jejenum
What are the actions of cholecystokinin?
stimulates gallbladder contraction;
stimulates pancreatic enzyme secretion;
inhibits gastric emptying
What stimulates cholecystokinin release?
presence of fatty acids and amino acids in the duodenum
Secretin is produced by which cells?
S-cells of duodenum
What are the actions of secretin?
increases bicarbonate secretion;
increases bile acid secretion;
decreases gastric acid secretion
Which two substances stimulate secretin release within the duodenum?
acid;
fatty acids
Somatostatin is made by which cells?
D-cells of pancreatic mucosa;
gastrointestinal mucosa
Does somatostatin lead to increased or decreased pepsinogen secretion? Increased or decreased gastric acid secretion?
decreased, decreased
Does somatostatin increase or decrease pancreatic secretions?
decrease
Does somatostatin increase or decrease fluid secretions in the small intestine?
decrease
What effect does somatostatin have on the gallbladder?
decreases gallbladder contraction
The presence of what substance in the gut lumen causes increased somatostatin release?
acid
What functions to inhibit somatostatin release?
vagal stimulation
Given the functions of somatostatin, why is it classified as an antigrowth hormone?
inhibits digestion and absorption of nutrients;
preventing the body from receiving the nutrient
Glucose-dependent insulinotropic peptide is made by which cells?
K-cells of duodenum and jejenum
What is the exocrine regulatory effect of glucose-dependent insulinotropic peptide?
decreased secretion of gastric acid
What is the endocrine regulatory effect of glucose-dependent insulinotropic peptide?
increased release of insulin
What increases glucose-dependent insulinotropic peptide secretion?
fatty acids;
amino acids;
oral glucose load
Why is an oral glucose load used more rapidly by the body than an equivalent load that is given intravenously?
oral, and not IV, glucose stimulates glucose-dependent insulinotropic peptide secretion; thus faster insulin release
Intrinsic factor is a product of what cells?
parietal cells of stomach
What is the function of intrinsic factor?
vitamin B12 binding;
vitamin B12 absorption in ileum
The destruction of parietal cells is seen in which two conditions?
chronic gastritis;
pernicious anaemia
What cells make gastric acid?
parietal cells of stomach
What is the main action of gastric acid?
decrease stomach pH
What is the action of pepsin?
protein digestion
What stimulates pepsin release?
acid in stomach;
vagal stimulation
Inactive pepsinogen is converted to pepsin by _____ _____.
low pH
Which cells secrete bicarbonate?
mucosal cells throughout GIT;
Brunner glands in duodenum
By secreting bicarbonate within the stomach and the duodenum, mucosal cells prevent what harmful process from occurring?
autodigestion
Bicarbonate secretion within the gastrointestinal tract is increased by what substance?
secretin
HCl and intrinsic factor are released by _____ cells in the _____ of the stomach.
parietal;
body
_____ is produced by chief cells in the _____ of the stomach.
pepsinogen;
body
Define and classify pain
Pain is an unpleasant sensory and emotional experience with actual or potential tissue damage
It can be classified according to its acuity or its nature:
Acute - recent onset and limited duration. Identifiable cause
Chronic - Persists beyond time of healing. No clearly identifiable cause
Nociceptive - results from stimulation of nociceptors
Neuropathic - results from dysfunction of the nervous system
Describe the pain pathway that are activated when injury occurs
- Nociceptors within damaged tissue are activated
- Action potential generated, travels along afferent sensory neurones
- Synapses in dorsal horn within rexed laminae
- Interneurones (secondary neurones) decussate the spinal cord and travel within the ascending pain pathways (spinothalamic tracts)
- Ascending tracts travel up the spinal cord and synapses within the thalamus
- Tertiary neurones project into the somatosensory cortex
- Some ascending spinal pathways project directly into the reticular-activating system and then into higher centres
- Descending fibres from the cortex/thalamus/brain stem exert an inhibitory effect on pain transmission in the dorsal horn
- An immediate polysynaptic withdrawal reflex occurs
Describe: ascending pain pathways
- SPINOTHALAMIC TRACT
- Anterior (light touch) and lateral (pain and temperature)
- Made of both fast and slow fibres - SPINORETICULAR TRACT
- slow fibres that terminate the reticular formation and thalamus - SPINOMESENCEPHALIC TRACT
- terminate in the midbrain and periaqueductal grey mater - DORSAL COLUMNS
- Pressure/vibration/ proprioception carried by Abeta fibres
- Not involved in pain transmission
What are nociceptors;
how are they classified
Nociceptors are unmyelinated nerve endings that respond to thermal, mechanical and chemical stimuli
They are classified according to their sensitivity to different stimuli.
- Unimodal (thermo-mechanoreceptors) - respond to pin-prick and sudden heat
- Polymodal - respond to pressure, heat, cold, chemicals and tissue damage
How do noxious stimuli activate pain transmission
Tissue damage leads to release of endogenous chemicals that stimulate nociceptors such as:
- bradykinin, histamine, serotonin, acetylcholine, H+ & K+
Other chemical mediators lower the threshold for excitation - eg prostaglandins, leukotrienes, substance P, neurogenic A and calcitonin gene-related peptide
Stimulation causes an influx of sodium and calcium which causes depolarisation and the generation of an action potential
This then propagates down pain pathways
What is the substantia gelatinosa
Otherwise known as the laminae II and III in the dorsal horn
A point of significant pain modulation
Main neurotransmitters at the primary sensory afferent neurons
Excitability neurotransmitters - glutamate, aspartate and substance P
What are the main neurotransmitters of the interneurones or secondary afferent neurones?
NMDA,
AMPA;
Neurokinin 1;
adenosine 1 and 2