Part 4 METABOLIC PHYSIOLOGY (PER SUBSTANCE) .pdf



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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

EVOLUTIONARY PHYSIOLOGY SCRIPT

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

PART 4
METABOLIC PHYSIOLOGY
(PER SUBSTANCE)

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

TABLE OF CONTENTS
TABLE OF CONTENTS ................................................................................................................................................. 2
4.
4.1.
4.2.
4.3.

METABOLIC PHYSIOLOGY (PER SUBSTANCE) .......................................................................................... 1
GLUCOSE METABOLISM................................................................................................................................. 1
LIPID METABOLISM: ..................................................................................................................................... 12
PROTEIN - PEPTID METABOLISM ............................................................................................................... 25

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

METABOLIC PHYSIOLOGY (PER SUBSTANCE)
CARBOHYDRATES, LIPIDS AND PROTEINS
4.1. GLUCOSE METABOLISM
Digestion is a subject that we’ll elaborate during organ and digestive physiology later on, for now, just know that the
polysacharids we eat are digested or depolymerized up the level of disacharids
These are:
- maltose
- sacharose
- lactose
Each of these will be broken down into monosacharids by the specific enzymes of the gut epithelium, where they are
produced. The monosacharids will be absorbed and turn up in our chapter now metabolic physiology
- maltose  maltase  glucose absorbed actively together with Na+
- sacharose  sacharase  fructose absorbed passively
- lactose  lactase  glucose and galactose actively absorbed, but the problem is that the enzyme is not longer
produced after the age of 24 months in humans, the genes are sealed off. If lactose continues to be taken it will irritate
the GALT and MALT1 parts of the mucous membrane and can lead to a lot of trouble (see later)
The sugars will be transported by the cytoskeleton through the epithelial cells and released in the connective matrix of
the lamina basalis, as the concentrations will rapidly increase, diffusion towards the capillaries follows automatically.
From hereon they will enter the capillaries and venous vessels which are roughly organized in a series of 4 arcades that
end into the vena mesenteric superior, which itself becomes the vena porta.
Figure 1: The four arcade system of the vena mesenterica superior

Figure 2: The vena porta

1

The digestive tract's immune system is often referred to as gut-associated lymphoid tissue (GALT) and works to protect the body
from invasion. GALT is an example of mucosa-associated lymphoid tissue. About 70% of the body's immune system is found in the
digestive tract. The GALT is made up of several types of lymphoid tissue that produce and store immune cells that carry out attacks
and defend against pathogens. MALT is the same but for all the Mucous membranes.

1

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Important is to note that the two flows do not mix, during digestion when the flow of both is potent they do not mix alike
two rivers that meet. See the satellite photo of the meeting of the waters: Rio Negro and Rio Solimoes in the Amazonas.
(the white dots are clouds)
Do you see how the flows stay separated for a long distance, each keeping its own trajectory?

Through the portal system all sugars arrive in the liver where the uptake is done by the liver parenchymal cells
(epithelial cells), as soon as they enter the cell they are transformed into Glucose-6 phosphate .
This form change of the glucose means just that on the 6 th C atom of the C6 H 12 O6 a phosphate group is added. The
advantage of this form change is that the glucose cannot leave the liver cell anymore, it is chained.
didactic example:
One can compare it to the Daltons in the Lucky Luke comic: as soon as the Daltons enter in the prison (liver
parenchym cell), they get around their ankle (6th Carbon atom) a chain with a big and heavy metal ball (phosphate
group) which withholds them of climbing over the prison wall (the cell membrane). Only when the prisonwarden
(hormone glucagon) gives the order to the prison guard (enzyme glucose-6 phosphatase) to release them, the chain
and ball at their ankle (phosphate group) will be taken off and they can move freely again.(the glucose becomes
membrane permeable again). As soon as this happened of course they take a run and jump the wall. (glucose moves
through the cell membrane) And the Daltons run off because the world is so big, away from prison (glucose does this
because when glucagons comes around it means that the glucose levels in the blood are running low, thus
concentration difference and electrochemical potential difference) The Daltons run away on the roads and then hide
in the countryside. (glucose runs in the bloodvessels and then dives out towards the interstitial and matricial
countryside).
The glucose 6 phosphate when it arrives in the liver cells stands in line
for 5 different metabolic pathways.

2

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

These different pathways will be more or less active according to the needs of the organism: the coordination is neurohormonal control, and how many enzymes are free in that pathway, it often happens that when there is too much cueing
in one path way another is used.

4.1.1. THE 5 INTRAHEPATIC METABOLIC PATHWAYS




Hydrolysis in glucose and phosphate by the enzyme glucose 6 phosphatase (This enzyme is only present in
the liver, so other cells do not release their glucose), the glucose is thus released and goes its way with the
bloodstream (in a peripheral city jail there is no warden so once the Daltons get in there they never get
out again except if the cell is destroyed)
A part of the glucose 6 phospahte will immediately undergo glycolysis, and thus serve as combustion energy to
make ATP



Another part will be transformed through the cycle of the pentose phosphates to make for instance NADPH
(nicotinamide dinucleotide phosphaat ), this is done by taking one C atom away from the molecule. Thus
intermediate metabolites are made for other functions like the pentose for the nucleic acids



The biggest part will be stored in the liver parenchymal cells after it underwent a polycondensation reaction to
form glycogen. Glycogen is a polysacharid, glucoses linked to each other to form a chain. The advantage is that
1 molecule glycogen has the same osmotic pressure as 1 molecule glucose, thus the glucose leaves its coat of
water molecules. Water is not produced as in the protein synthesis, but electrochemically “bond” water is set
free.



If all the other metabolic pathways are saturated by the abundant arrival of glucose, then the rest glucose will be
transformed into lipids with the help of acetyl co-enzyme A .
The advantages of this pathway are:
water and osmotic pressure are not a problem anymore, lipids are apolar.
potentially there is more energy stored for less weight and mass.

4.1.2. THE
4
PATHWAYS

EXTRAHEPATIC

INTRACELLULAR

METABOLIC

When the glucose molecules leave the bloodstream and arrive in the interstitial fluid they will be absorbed by the cells
(actively = with the help of insulin); as soon as they enter the cell they get a phosphate group attached immediately on
the 6 th C atom again. Thus they become glucose 6 phosphate again and as there is no glucose 6 phosphatase in these
cells, glucose is there to stay and be metabolized.
Glucose will be absorbed by all possible cells and then turn into one of these 4 metabolic pathways:
 glycolysis through the krebscyclus : ATP + phosphocreatine + CO2 + H2O (the energy stored in the
phosphor bonds, PHOSPHORYLATION).
 polycondensation into glycogen for storage purposes (This happens in all cells but in the striated muscle cells
the amounts are so big that the glycogen corns are visible with a microscope.).
 transformation for catalytic purposes, sugar groups that are on receptors or enzymes.
 transformation or polymerization for structural purposes: glycosaminoglycans, proteoglycans, glycoproteins .

4.1.3. THE OXIDATION OF THE CARBOHYDRATES
When the oxidation goes together with oxygen shortage (sports , disturbed arterial supply by so called somatic
dysfunctions, respiratory problems etc.) , the Krebscyclus does not function. From the intermediate step of Pyruvate;
there will be lactic acid formation. The lactic acid has then to be transported back to the liver, where it will be
retransformed into glucose.(cycle of Cori). We will come back immediately to this as it is a part of the hepatic
neoglucogenesis.

3

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

When the metabolic demand of glucose is higher than the delivery through depolymerization of glycogen in the liver
permits, than the liver cells will get hormonal commands that activate another set of pathways that are usually assembled
under one name: hepatic neoglucogenesis, meaning building new glucose molecules out of something different:
- lactate or lactic acid (cycle of Cori).
- amino acids (out of alanine for instance)
- tri-acylglyceroles or triglycerides: glycerol + fatty acids (see biochemistry lipids, saponifiable lipids)

4.1.3.1. FORMATION OF AND OXIDATION OF GLUCOSE BY THE NERVOUS SYSTEM
Neurons can in case of emergency transform acetoacetic acid into glucose, and then phosphorylate it in order to make
ATP. Thus it can provide itself with glucose for some time, but the problem is that this pathway leads directly to cellular
and tissular alkalosis, by the disappearance of acid in the environment and increase in acetone or ketone bodies.
addendum:
Acetoacetic acid (also known as 3-oxobutanoic acid or diacetic acid) is a beta-keto acid of the keto acid group, its
empirical formula is C4H6O3 or CH3COCH2COOH. It is unstable at room temperature, decomposing to acetone and
carbon dioxide.
It is a weak organic acid and can be produced in the human liver and neurons under certain conditions of poor
metabolism leading to excessive fatty acid breakdown (diabetes mellitus leading to diabetic ketoacidosis), it is then
partially converted to acetone by spontaneous decarboxylation and excreted either in urine or through respiration. This
sent can be detected in patients. The acid is also present in the metabolism of those undergoing starvation or prolonged
physical exertion as part of gluconeogenesis. It is not the major ketone produced by the body (that being betahydroxybutyrate). Its pKa in pure water is 3.77.
When ketone bodies are measured by way of urine concentration, acetoacetic acid, along with beta-hydroxybutyric acid
(BHB) or acetone, is what is detected. This is done using dipsticks coated in nitroprusside or similar chemicals.
Nitroprusside changes from pink to purple in the presence of acetoacetate, the conjugate base of acetoacetic acid, and
the colour change is graded by eye. The popular dipstick used to detect ketone bodies in urine "Ketostix" by Bayer, only
detects acetoacetate, not BHB or acetone.
For example in hunger strikes this has to be closely supervised because once this pathway is activated the point of no
return is rapidly reached; when this is the case it is practically irreversible even with glucose injections. Metabolic
alkalosis is lethal for the organism.
I f there is not to much hurry (amount of hormones triggering), the liver preferentially uses non esteric fatty acids that
are stored “en masse” in the adipocytes of the connective tissue, they are transported by the plasmatic albumin.
5

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4.1.4. NEUROHORMONAL CONTROL OF THE GLUCID METABOLISM
The homeostatic balance is as follows:
When the glycaemia increases too much, the BBB will be thorn apart and stay open for hours, and the kidneys are not
fast enough during the tubular re-absorption phase, thus a massive amount of urine with valuable minerals and nutrients
will be lost. (pollakisuria)
When the glycaemia decreases too much, chaos comes over all cells, because they get in trouble with their energy
household. This phenomenon will quickly turn into metabolic alkalosis, coma and death.
Addendum
Metabolic alkalosis results from altered metabolism. It is the most common acid-base disorder seen in hospital in the
United States.
Is a result of decreased hydrogen ion concentration leading to increased bicarbonate and carbon dioxide concentrations,
or alternatively a direct result of increased bicarbonate concentrations.
Causes
There are four mechanisms of metabolic alkylosis:
1. Loss of hydrogen ions Most often occurs via two mechanisms, either vomiting or renally. Vomiting results in the
excretion hydrogen ions and the retention of bicarbonate. Renal losses of hydrogen occur when excess aldosterone
induces the retention of sodium and hence the excretion of hydrogen.
2. Shift of hydrogen ions into intracellular space. Seen in hypokalemia. Due to a low extracellular potassium
concentration, potassium shifts out of the cells, and in order to maintain electrical neutrality, hydrogen shifts into the
cells, leaving behind bicarbonate.
3. Alkalotic agents Alkalotic agents, such as bicarbonate, administered in excess of excretion capabilities by the kidney
can lead to an alkylosis.
4. Contraction alkalosis This results from a loss of water in the extracellular space which is poor in bicarbonate,
typically from diuretic use. Since water is lost while bicarbonate is retained, the concentration of bicarbonate increases.
Compensation
The body attempts to compensate for the increase in pH by retaining carbon dioxide (CO2) through hypoventilation
(respiratory compensation). CO2 combines with elements in the bloodstream to form carbonic acid, thus decreasing pH.
Renal compensation for metabolic alkalosis consists of increased excretion of HCO3- (bicarbonate), because the filtered
load of HCO3- exceeds the ability of the renal tubule to reabsorb it.
Metabolic acidosis is a state in which the blood pH is low (less than 7.35) due to increased production of H+ by the
body or the inability of the body to form bicarbonate (HCO3-) in the kidney. Its causes are diverse, and its consequences
can be serious, including diarrhea, coma and death. Together with respiratory acidosis, it is one of the two general types
of acidosis.
Signs and symptoms
Symptoms are aspecific, and diagnosis can be difficult unless the patient presents with clear indications for arterial
blood gas sampling. Symptoms may include chest pain, palpitations, headache, altered mental status, decreased visual
acuity, nausea, vomiting, abdominal pain, altered appetite (either loss of or increased) and weight loss (longer term),
muscle weakness and bone pains. Those in metabolic acidosis may exhibit deep, rapid breathing called Kussmaul
respirations which is classically associated with diabetic ketoacidosis. Rapid deep breaths increase the amount of carbon
dioxide exhaled, thus lowering the serum carbon dioxide levels, resulting in a compensatory respiratory alkalosis.
Extreme acidosis leads to neurological and cardiac complications:
Neurological: lethargy, stupor, coma, seizures.
Cardiac: arrhythmias (ventricular tachycardia), decreased response to epinephrine; both lead to hypotension (low blood
pressure).
Physical examination occasionally reveals signs of disease, but is otherwise normal. Cranial nerve abnormalities are
reported in ethylene glycol poisoning, and retinal edema can be a sign of methanol (methyl alcohol) intoxication.
Longstanding chronic metabolic acidosis leads to osteoporosis and can cause fractures.

6

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Diagnosis
Arterial blood gas sampling is essential for the diagnosis. The pH is low (under 7.35) and the bicarbonate levels are
decreased (<12 mmol/l). In respiratory acidosis (low blood pH due to decreased clearance of carbon dioxide by the
lungs), the bicarbonate is elevated, due to increased conversion from H2CO3. An ECG can be useful to anticipate
cardiac complications.
Other tests that are relevant in this context are electrolytes (including chloride), glucose, renal function and a full blood
count. Urinalysis can reveal acidity (salicylate poisoning) or alkalinity (renal tubular acidosis type I). In addition, it can
show ketones in ketoacidosis.
To distinguish between the main types of metabolic acidosis, a clinical tool called the anion gap is considered very
useful. It is calculated by subtracting the chloride and bicarbonate levels from the sodium plus potassium levels.
Anion gap = ( [Na+]+[K+] ) - ( [Cl-]+[HCO3-] )
As sodium is the main extracellular cation, and chloride and bicarbonate are the main anions, the result should reflect
the remaining anions. Normally, this concentration is about 8-16 mmol/l (12±4). An elevated anion gap (i.e. > 16
mmol/l) can indicate particular types of metabolic acidosis, particularly certain poisons, lactate acidosis and
ketoacidosis.
As the differential diagnosis is narrowed down, certain other tests may be necessary, including toxicological screening
and imaging of the kidneys.
Causes
lactic acidosis
ketoacidosis
chronic renal failure (accumulation of sulfates, phosphates, uric acid)
intoxication:
organic acids (salicylates, ethanol, methanol, formaldehyde, ethylene glycol, paraldehyde, INH, toluene)
sulfates, metformin (Glucophage®)
massive rhabdomyolysis
The mnemomic MUDPILES is commonly used to remember the causes of Increased anion gap metabolic acidosis.
M-Methanol
U-Uremia
D-Diabetic Ketoacidosis
P-Paraldehyde
I-Infection, Iron, Isoniazid
L-Lactic acidosis
E-Ethylene Glycol, Ethanol
S-Salicylates
ERGO: There have to be released as many glucose molecules from the liver as the cells in the organism take
away; in order to have a stable glycaemia.
Mainly two hormones regulate this, with one mastering hormone:
- glucagon that provokes hyperglycaemia (Some other hormones have the same type of effect but not so
massively).
- insulin that provokes hypoglycaemia (Some other hormones have the same type of effect but not so
massively ).

A third important hormone that plays an important role is somatostatin, which rules both (mastering hormone), so that
there is no inefficient built up of insulin against glucagon. (overproduction)
The neoglucogenesis and distribution of glucose from the liver is stimulated by glucagon.
Glucagon induces a brutal activation of Cyclic AMP intrahepatically, what induces through the second messenger
cascade a massive glycogenolyse. (See cascade effect in Histology /NEURAL TISSUE).

7

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Glucagon induces also lipolysis , and secondarily an hypofysial reaction via Somatotrope Hormone. (STH) and AdenoCorticotrope Hormone. (ACTH).
Both of these are going to increase the neoglucogenesis by extra metabolization of :AA and triglycerides(lipids);
glycerol and fatty acids.
These massive reactions like they are described normally only happen in stress conditions under the influence of stress
hormones or catecholamines.
Addendum:
Catecholamines are chemical compounds derived from the amino acid tyrosine containing catechol and amine groups.
Some of them are biogenic amines. Catecholamines are water soluble and are 50% bound to plasma proteins, so they
circulate in the bloodstream. The most abundant catecholamines are epinephrine (adrenaline), norepinephrine
(noradrenaline) and dopamine, all of which are produced by phenylalanine and tyrosine. Tyrosine is created from
phenylalanine by hydroxylation thanks to the enzyme, phenylalanine hydroxylase (tyrosine is also ingested directly from
dietary protein). Tyrosine is then sent to catecholaminesecreting neurons. Here many kinds of reactions convert it to
dopamine, to norepinephrine and epinephrine eventually. Catecholamines as hormones are released by the adrenal
glands in situations of stress such as psychological stress or low blood sugar levels.
Production
Catecholamines are produced mainly by the chromaffin cells of the adrenal medulla and the postganglionic fibers of the
sympathetic nervous system. Dopamine, which acts as a neurotransmitter in the central nervous system, is largely
produced in neuronal cell bodies in two areas of the brainstem: the substantia nigra and the ventral tegmental area.
Function&Modality
Two catecholamines, norepinephrine and dopamine, act as neurotransmitters in the central nervous system and as
hormones in the blood circulation. The catecholamine norepinephrine is a neurotransmitter of the peripheral sympathetic
nervous system but is also present in the blood (mostly through "spillover" from the synapses of the sympathetic
system).
High catecholamine levels in blood are associated with stress, which can be induced from psychological reactions or
environmental stressors such as elevated sound levels, intense light, or low blood sugar levels.
Extremely high levels of catecholamine (also known as catecholamine toxicity) can occur in CNS trauma due to
stimulation and/or damage of nuclei in the brainstem, particularly those nuclei affecting the sympathetic nervous system.
In emergency medicine, this occurrence is widely known as catecholamine dump.
Effects
Catecholamines cause general physiological changes that prepare the body for physical activity (fight-or-flight
response). Some typical effects are increases in heart rate, blood pressure, blood glucose levels, and a general reaction
of the sympathetic nervous system. Some drugs, like tolcapone (a central COMT-inhibitor), raise the levels of all the
catecholamines.
Degradation
They have a half-life of approximately a few minutes when circulating in the blood.
Monoamine oxidase (MAO) is the main enzyme responsible for degradation of catecholamines.
Methamphetamine and MAOIs bind to MAOs to inhibit their action of breaking down catecholamines. This is primarily
the reason why the effects of amphetamines last longer than cocaine and other substances. Amphetamines not only
causes a release of dopamine, epinephrine, and norepinephrine into the blood stream, but also keeps it working there for
a long time.
In basal metabolism these reactions are eventually moderately involved. In other words, you should try to imagine that
these reactions happen in a slow modulated pattern like waves of hormones rising and lowering again like the tide of the
ocean. Until a sudden massive effort or stress situation appears that demands lots of glucose. Or for instance after
digestion when there is a massive increase of glucose ion the bloodstream (a soft drink = 15 - 20 lumps of pure sugar),
from that moment on the hormone levels won’t be like a tide coming in and out but like a big rock you throw in a pond:
rapid high waves that will in time slowly die out and return to a slow tide. Keep this living image in mind! And think
of it next time you order a coke or fanta…

8

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Insulin shall play an essential role in the in the uptake of the masse of glucose that runs in the bloodstream. Thus all this
hormones work together and not separately!
Insulin is produced in the  cells of the isles of Langerhans (cell groups of micro endocrine glands distributed in the
exocrine pancreas gland. See epithelial tissue / glands).
Insulin is produced as a polypeptid chain called proinsulin. In the middle there is a peptid bridge called peptid C, when
Insulin must be released (neuro-hormonal control) two endopeptidase enzymes will cut the peptid C out of the chain
while a ligase enzyme raccomodates the the two proinsulin chains into an active insulin hormone. From that moment on
it is active and released by the cells into the matrix and interstitium, from which it will penetrate the blood vessels
(concentration difference = diffusion). The peptid C is also secreted and is not metabolized by any other cell anymore,
not even in the liver or kidney. This is thus a perfect laboratory bloodtest to know how much insulin is still produced by
the body, even when the patient injects himself with synthetic insulin. Although the injections of synthetic insulin lead
directly to an inhibition of own production, usually within a year the Langerhans cells are then destroyed and the patient
is dependant for life on insulin injections. If diabetic patients start to inject themselves usually you have about a year to
reduce, stop or inverse the process (depending on the correctness of the diagnosis 2) once this period is through, it is
useless. Although Osteopathic treatment can often achieve, by a better homeostatic regulation, that the patient can
reduce the doses of his injections.

Figure 3: schema of insulin production

Insulin accelerates the transmembranous transport of glucose, inhibits the glucose production and release through an
inhibition of glucose 6 phosphatase enzyme and last but not least stimulates the polymerization or polycondensation
reactions to form glycogen.
The secretion of insulin is triggered by hyperglycemia and by hormones such as gastro-intestinal peptide (GIP )
glucagon and some AA like leucine .
Insulin and glucagon keep each other in dynamic balance helped by somatostatin.
Somatostatin is produced in the pancreas and by some hypothalamo-hypofysial cells
2

In many cases, as soon as the sugar levels get to high, doctors tend to turn too rapidly without exact diagnosis to insulin therapy, although not every
sugar level disturbance is due to endocrine pancreas destruction; liver-gall and suprarenal problems are often involved and often treating the cause
sees to a “miracle healing”. It is not a miracle just a bad diagnosis.

9

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

It has an inhibiting action on the release of STH , TSH , gastrin, glucagon en insulin.
The general effect of somatostatin is a decrease of the blood sugar levels. Somatostatin is probably the actor in the
sudden hypoglycaemia after massive vegetative reactions. (visceral, gynaecological, or fascial manipulations and
sometimes after thrust techniques in the upper cervical region!)
Some catecholamines like adrenalin and noradrenaline provoke a massive hyperglycemia, while they inhibit the insulin
secretion. They accelerate the cellular glycolysis, this makes the catecholamines probably phylogentically younger than
insulin.
Cortisol and the glucocorticoïds provoke similar reactions as the catecholamines: hyperglycemia, while the storage and
peripheral consumption is slowed down, thus water storage and typical “moonface” during the regular intake of
corticoïds

10

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Figure 4: hormonally induced glucides metabolism

11

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4.2. LIPID METABOLISM:
The most striking characteristic of the lipids is that they are only solutionable in apolar or organic solvents like ether ,
ammoniac, chloroform, alcohol etc.; thus water repels them (See biochemistry) Because of these hydrofobic
characteristic they are problematic in a biologic organism, they always have to be coupled to a hydrophilic substance;
and when used in a structure they will turn to the inside of the hydrophilic molecules because the polar water
environment pushes them away.
Figure 5:cell membrane

12

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4.2.1. THE ELEMENTARY LIPOID MOLECULES
The elementary lipoid molecules are:
- fatty acids
- glycerol
- mono, di, tri -glycerides: glycerol, esterized & with 1, 2, 3 fatty acid chains.
- steroids: lipids with a gonan core (precursor steroid hormones)
- phospholipids: glycerides with a phosphatic acid restgroup, lecitine, choline
The fatty acids: are made of chains of mainly C and H atoms up to 24 C’s long. for example arachidic acid
CH3(CH2)18COOH
The saturated ones have only single bonds between the groups, while the unsaturated ones have at least one double
bond. If this double bond is after the 9th C atom it is an essential fatty acid for humans, because we lack the enzymes to
make that kind of fatty acids. for instance: linolic acid, linolenic acid and arachidic acid
The glyceroles: are alcohols that are esterized, the basic building block of all saponifiable lipids, and of.
monoacylglycerol (1 ester bond), diacylglycerol(2 ester bonds) and triacylglycerol. (3 ester bonds)
The acylglyceroles or mono-, di- or triglycerides, espescially these last ones are very volimunous molecules, because
their apolarity and their size combined they are the most strongly reppelled by water. Therefore they are the ideal energy
storage molecules.
monoglycerides are: monoacylglycerol + one fatty acid chain
diglycerides are: diacylglycerol + two fatty acid chains
triglycerides are: triacylglycerol + three fatty acid chains
The steroids are molecules that have a gonan core see biochemistry. The steroids will essentially play a role in the
constitution of the cell membranes and as precursors of the steroid hormones and bilary salts and acids. Who took most

anabolic steroids you think?
The phospholipids are glycerides that have an extra phosphor acid rest group, with at least one negative charge, and
thus become polar on that side of the molecule.
Because of this characteristic semi polar and thus semi hydrophilic-semi hydrophobic they will be the ideal structural
molecule for cell membranes and liposomes, lipoproteins etc.
Everywhere, in plasma and lymphatics the lipids will be enveloped in and or bound to proteins and are thus always
called lipoproteins.

13

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4.2.2. THE PROTEINS THAT TRANSPORT LIPIDS
The pure protein is called APO-lipo-protein. It is produced by the epithelial cylindrical cells of the mucous membrane
that delimit the lumen of the gut and by the liver parenchymal cells.
 The small fat drops (micelles) that are the result of the digestion are absorbed in the small intestine. In general the
transport is not per molecule, but per drop or micelle, which is surrounded where necessary by apolipoproteins: We are
thus talking about very voluminous complexes. This complex with its protein coat is called a chylomicron. The
chylomicrons will be taken up by the lymphatics and so be transported towards the bloodstream and liver, while
glycerol and short fatty acid chains will be transported directly by the vena mesenterica superior to the liver. As the
chylomicrons go with the lymphatics they first take a grand tour around the body with the bloodstream before coming to
the liver. Look where it enters the bloodstream.

14

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Figure 6: chylomicron or lipoproteins

4.2.3. THE
FOUR
ESSENTIAL
ULTRACENTRIFUGATION

TYPES

LIPIDS

IN

We must shortly return in history (biology and physiology and medicine) to get this right:
This is really important because you can see for yourselves how much sense the actual medical politics have
towards lipaemia, diet and medication. (statins or cholesterol reducers)
1830 - 1840: Liebig develops techniques for quantitative analysis and applies them to biologic systems and organisms.
1905: Knoop finds through deduction the  oxidation of fatty acids
1935: Schoenheimer & Rittenberg use as first ones, isotopes as tracers to study the intermediary metabolism of the lipids
in living beings.
1935: Davson & Danielli postulate as first ones the model of the cell membrane (bilipid layer and the fluidic mosaic
model).
1942: Bloch & Rittenberg discover that acetate is the precursor of gonan and as such the pre-precursor of cholesterol.
As you can see, chemistry and biochemistry specific to the lipids has a very young history. In order to understand lipids
within living organisms and more specifically in humans, there was no much choice left for the physiologists than use
ultra-centrifugation of plasma.
During ultracentrifugation of the blood plasma, the heaviest parts are projected far away; the lighter ones will be
projected less far of the rotation axis. By using this technique they had the possibility to classify the unknown lipids
according to density and as they were found in living human. The thus proposed terminology is still in use today,
although it is in my opinion not very specific, nor physiologic (in the sense of living physiology) and even narrow
minded: it is one form of perception, but no more than that. The terminology is chemically incorrect because it uses
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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

synthetic names based on density for different lipids and proteins. Actually each group contains all the ingredients but in
other proportions. (See figure 7)

“The tragedy of science is the slaying of a beautiful hypothesis by an ugly fact.”

TH

Huxley
The cholesterol controversy of atherogenesis
During the 70s and 80s, some researchers and practitioners considered the lipid hypothesis as unverified due to the lack
of proof at that time that lowering blood cholesterol levels results in decreased risk for atherosclerosis. Some skeptics
were openly questioning its validity by pointing out flaws in the studies supporting it. This discussion is also referred to
as the "cholesterol controversy." Predictions were made that further research during the 80s and 90s would help settle
this controversy. However, even after the Coronary Primary Prevention Trial and the NIH Consensus Conference in the
mid 80s, criticism persisted in a vocal minority of the scientific community questioning the statistical significance of the
trials and the conclusions of the panel.
In the following years, studies with lipid- and cholesterol-lowering drugs such as statins provided further evidence in
support of the lipid hypothesis although the mortality statistics did not change. Further studies were designed
specifically to affirm the validity of the lipid hypothesis and settle the controversy. Despite the general consensus
regarding the lipid hypothesis as a proven fact within the scientific community by the turn of the century, resistance to its
acceptance still persists in a small minority of the scientific and medical communities, who argue that it is based on false
premises and misrepresented data, and has thus not been scientifically validated at all.
Ravnskov, Uffe (2000). The Cholesterol Myths: Exposing the Fallacy that Saturated Fat and Cholesterol cause Heart
Disease. United States: New Trends Publishing. ISBN 0967089700
Stehbens WE (2001). "Coronary heart disease, hypercholesterolemia, and atherosclerosis I. False premises". Exp Mol
Pathol 70: 103-119. PMID 11263954
Stehbens WE (2001). "Coronary heart disease, hypercholesterolemia, and atherosclerosis II. Misrepresented data". Exp
Mol Pathol 70: 120-139. PMID 11263955
Ultracentrifugation:
- chylomicrons: lipoproteins in the form of big drops, coming directly from the gut after digestion.
- very low density lipoproteins: mainly triglycerides (big volume but poor mass).
- low density lipoproteins: mainly cholesterol (± 50 % of the total mass)
- high density lipoproteins: mainly phospholipids & apo-proteins
- The fifth type: Remnants, are transformed in the liver into VLDL, they are in fact the leftovers of the
chylomicrons after their grand tour through the body in the bloodstream.
-The sixth type: Intermediate density lipoproteins: are a kind of intermediate stage between VLDL, HDL &
LDL in other words they have an almost equal mix with an intermediated density.

The chylomicrons circulate through the whole body and during every contact with cells; they will be aggressed by the
lipoprotein lipases on the cells. What these enzymes do is so to say take handfuls grab in the lipid pool passing by.
These fats will be metabolized in the cell or stored as energetic stash. (heart, intermuscular & intramuscular adipocytes
etc.)
The remnants (the leftovers of our chylomicrons after every one had his grab, during its journey in the circulation) are
absorbed by the liver cells parenchymal cells and fat storing cells (see liver) and will be used in different ways there:
- metabolized for the neoglucogenesis
- metabolized for the formation of precursors
- tanked up again with triglycerides to make VLDL’s that will be sent out
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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

The HDL’s (phospholipids) will be degraded in glycerol, phosphates and fatty acids during the digestion process, the
enterocytes reconstruct the phospholipids. Because of their polar group they will be solved in the portal and even caval
circulation, the rest is used in the VLDL or in the chylomicrons (lymphatics) where they help to coat the strongly apolar
molecules and glue the chylomicrons together (See Figure 6)

Figure 7: schematic constitution of the ultra centrifuged groups.

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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Free cholesterol
Triglycerides or triacylglyceroles
Apo-protein parts
Cholesterolesters
Phospholipids

4.2.4. ENDOGENOUS ORIGIN OF LIPIDS:
The liver is capable as well to metabolize lipids as it is to synthesize lipids from non lipid molecules.
This is not chemically correct but an excellent mnemonic to keep it in mind according to chemical complexity:
(from complex to simple)
Proteins = mainly CHONSP
Sugars = mainly CHO
Lipids =
mainly CH
The liver can without any problems descend the line (deconstruct): turn AA into sugars and sugars into lipids.
Or it can transform within the same line: transform one AA into another AA as long as it is not an essential AA for
humans: Nutritional essentiality is characteristic of the species, not the nutrient. Nine amino acids are generally regarded
as essential for humans. They are: isoleucine, leucine, lysine, threonine, tryptophan, methionine, histidine, valine
and phenylalanine. A mnemonic used to remember these acids runs: I Like Light That Tries Making Home Varied&
Pretty. In addition, the amino acids arginine, cysteine, glycine, glutamine and tyrosine are considered conditionally
essential, meaning they are not normally required in the diet, but must be supplied exogenously to specific populations
that do not synthesize it in adequate amounts
Transform one sugar into another: glycogen- glucose- pentose
Transform lipids into another, or from sugars:
The units with 2 C units (Acetyl co A) are recruited from the glucids, proteins or even from ethylic alcohol. (When this
excessive it develops stheatosis or fatty liver3.)
The two C units are connected to form fatty acids (non essential fatty acids), these fatty acids will than be attached to
glycerol to form triglycerides. After that the liver can assemble them to form a VLDL that is send to the bloodstream via
the liver sinusoids, and the vena hepatica to the heart.
The hepatocytes will also associate Acetyl co A in order to built cholesterol with it. (Remember acetate is the precursor
of gonan and as such the pre-precursor of cholesterol.) In a genetically normal functioning organism the livers’
endogenous cholesterol production will be reciprocally inversed to the arriving exogenous cholesterol from the
digestion (chylomicrons-remnants), the excess cholesterol will be metabolized if the bilary system functions normally.
So maybe the first reasonable diet prescription is not: “leave all animal fats away!”
Think about it.? Cholesterol is used peripherically for the synthesis of organelles, steroïdhormones,
vitamine D, cell membrane etc

3

In cellular pathology, steatosis (also called fatty change) is the process describing the abnormal retention of lipids within a cell. It reflects an
impairment of the normal processes of synthesis and breakdown of triglyceride fat. Excess lipid accumulates in vesicles that displace the cytoplasm.
When the vesicles are large enough to distort the nucleus, the condition is known as macrovesicular steatosis, otherwise the condition is known as
microvesicular steatosis. Whilst not particularly detrimental to the cell in mild cases, large accumulations can disrupt cell constituents, and in severe
cases the cell may even burst.

18

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Figure 8 SCHEMA OF THE V.L.D.L. METABOLISM:

Figure 9 SCHEMA H.D.L. METABOLISM(return of the excessive cholesterol to the liver)

19

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Figure 11: INSULIN AND THE ADIPOCYTE METABOLISM :

20

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4.2.5. HORMONES AND ENZYMES OF THE LIPID METABOLISM:


Lipoprotein lipase (LPL)
Lipoprotein lipase is found in vascular endothelium. It is activated by insulin, ACTH, TSH, glucagon and
thyroid hormone. Its activity is enhanced by heparin. As discussed above, lipoprotein lipase hydrolyzes CM
and VLDL to free fatty acids and glycerol and VLDL-remnants, respectively. Apolipoprotein C is essential for
activation of LPL.



Hepatic lipase
This enzyme hydrolyzes surface phospholipids on lipoproteins and is responsible for converting VLDL to
LDL.



Hormone sensitive lipase
This enzyme is responsible for lipolysis (mobilization of triglycerides from adipose tissue to yield free fatty
acids and glycerol). The enzyme is stimulated by catecholamines, growth hormone, thyroxine, corticosteroids
and prostaglandins. It is inhibited by insulin. Fatty acids are transported to the liver (free or albumin-bound),
where they are taken up and used for energy (beta oxidation), combined with triglycerides to form VLDL or
incorporated into ketones. Therefore, lipolysis will increase VLDL production.

In the adipocytes, it will be the amount of triglycerides in storage that will direct the balance in favor for hydrolysis or
re-synthesis of the triglycerides.
The hydrolysis is dependant of the hormone sensitive lipase via adenylcyclase.
The hydrolysis is thus activated by:
- adrenalin
- cortisol
- Less by STH (Somatotrope hormone)
- glucagon
She will be inhibited by insulin. (As the arrival of glucose in the adipocyte stimulates the synthesis of triglycerides).

21

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

The effects of increasing male hormones with age, baldness and…

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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

This is a review before starting with the proteins

23

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

4.3. PROTEIN - PEPTID METABOLISM
4.3.1. PROTIDS IN SMALL MOLECULAR FORMS IN BLOOD AND URINE:
4.3.1.1. DEFINITIONS.

Protids are all substances that are a part of the Nitrogen pool, substances that contain a (N) nitrogen, they are classified
after size and complexity. (See biochemistry)
- amino acids and derivates
- peptides: linked AA by a peptide bond molecular mass < 10.000
- proteins: peptide chain with a complex three-dimensional organization and a molecular mass > 10.000

4.3.1.2. DIGESTION
The digestive mechanism of the proteins will be looked at in detail during the organ physiology, but as you can probably
imagine: they will be deconstructed until the level of dipeptides. (Like the sugars almost, except that the organs that do
this are different)
From duodenum 2 on to the ileo-caecal junction they will be absorbed actively. (a small amount will be absorbed –in the
stomach.)
All transport systems are active, sometimes coupled with the Na+ uptake. The epithelial cells produce peptidases. These
will liberate single AA which will be send to the interstitium and taken up by the vena mesenterica superior.)
Via the vena porta to the liver and then they start a journey through the body with the bloodstream.
As this mechanism should be known now because we touched it already, let us look at the derivates (from a
laboratory view) so you get the physiology and the practical laboratory knowledge in one. But that is not all, if
you want to extract the full Osteopathic potency of this part of the course you need to understand and think in
several directions with the same text:
-paste it in your physiology knowledge at all the dimensions from molecular to systemic
-see the laboratory practice, Semiology: what is the indicator, what does it mean not only in itself but also in
the chain reaction?
-Osteopathic practice: When you find an impaired function or form in your patients functioning, reverse
the thought: what does these organs and their metabolic functions provoke as chain events and what other
tissue, organs or systems are directly victim of this impairment. Does this corroborate with what you find in
all dimensions?

4.3.2. DERIVATES OF THE AMINO ACIDS
When humans get too many AA in their nutrition the liver will react:
The liverparenchymal cells mainly will split of the Nitrogen radical and oxide what is left over of the AA (transform it in
sugar and you know now what happens with the sugar.
The N radical (if not in the liver it will be send to the liver) itself will be transformed in urea. Urea is less toxic as waste
product and chemically neutral; reaction = CO2 + diamide (2 NH2) → structure = (NH2)2CO
The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions occurring in many animals that
produces urea from ammonia (NH3). This cycle was the first metabolic cycle discovered (Krebs and Kurt Henseleit,
1932). In mammals, the urea cycle takes place only in the liver. Organisms that cannot easily and quickly remove
ammonia usually have to convert it to some other substance, like urea or uric acid, which are much less toxic.
Insufficiency of the urea cycle occurs in some genetic disorders (inborn errors of metabolism), and in liver failure. The
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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

result of liver failure is accumulation of nitrogenous waste, mainly ammonia, which leads to hepatic encephalopathy. In
other words neural symptoms can be a lack of disposal from the biliary and liver system, check these always!
Urea will be eliminated for +- 1/3 over the biliary system directly, the other 2/3 will diffuse in the blood and will tour
around the bodies connective system through the blood circulation, the part that arrives in the kidney will be excreted by
the urine.
The part that is excreted by the biliary system will partly be changed in ammoniac by the urease bacteria of the gut.
Possible cause for sigmoid tumors. The ammoniac will be disposed of by the liver again.
Laboratory value, Semiology: Urea in blood :
- Too many proteins in nutrition
- Kidney hypofunction
- Biliary hypofunction
- Too little water uptake or water loss dysentery (water loss if severe can be seen by the sunken eyes and
tested by the skin fold pinch slow return, especially in important in pediatrics, where the evolution of the
situation can go form moderate to lethal within hours!)

In
humans, dehydration can be caused by a wide range of diseases
and states that impair water homeostasis in the body. These include:
External or stress-related causes
Prolonged physical activity without consuming adequate water, especially in a hot and/or humid environment
Prolonged exposure to dry air, e.g., in high-flying airplanes (5-15% r.h.)
Survival situations, especially desert survival conditions(Travel in desertic area)
Blood loss or hypotension due to physical trauma Diarrhea
Hyperthermia
Shock (hypovolemic)
Vomiting
Burns
Lacrimation
Infectious diseases
Cholera
Gastroenteritis
Shigellosis
Yellow fever
Malnutrition
Electrolyte disturbance
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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Hypernatremia (also caused by dehydration)
Hyponatremia, especially from restricted salt diets
Consumption of alcohol, caffeine or other diuretic substances.
Fasting
Recent rapid weight loss may reflect progressive depletion of fluid volume. (The loss of 1 L of fluid results in a weight
loss of 1 kg.)
Patient refusal of nutrition and hydration
Other causes of obligate water loss
Severe hyperglycemia, especially in Diabetes mellitus
Glycosuria

4.3.2.1. CREATINE AND CREATININE (plasmatic):
Is synthesized in the lever from AA (arginine, glycine, and methionine) and goes through the blood circulation mainly to
the striated musculature.( 95% of it is later stored in the skeletal muscles, with the rest in the brain, heart, and testes., in
females the creatine concentration is normally higher than in males due to the muscle mass).
The excretion through the kidney is higher in females than in males but the tubular reabsorption is almost 100% .
In the tissues, creatine will get a phosphate group from an ATP, so phosphocreatine is generated. This is the energy
provider for a few seconds.
A fraction will, as the P is taken away during the energy delivery, form a cyclic ring structure; this substance is called
creatinine and is no longer usable. It will leave the muscle, brain or testis, and through the circulation, until it is
eliminated by the nefrons.
Laboratory value, Semiology: The concentration is stable according to the muscular mass, if it increases in the blood or
lowers in the urine, it indicates a kidney hypofunction. (More relevant value than the urea values!)

4.3.2.2. AMMONAEMIA :
As seen under UREA, is NH3 a derivate of the oxidative catabolism of AA., normally it is transformed in urea and
excreted by the biliary system and kidneys, a part will be retransformed in the gut by the urease bacteria that metabolize
it. While ammonia is as well water as fat soluble it can go all ways. Ammonaemia or increased NH3 values in the blood
are laboratory values for liver hypofunction, or strong fermentation in the colon paired with liver-biliary hypofunction.
Ammoniac is an extremely toxic waste, for all tissues but the most sensituive to it is the neural tissue (CNS and PNS)

4.3.3. DERIVATES OF THE NUCLEIC ACIDS:
4.3.3.1. URIC ACID
Uric acid is formed in the liver from the purines surplus (bases of the nucleic acids).
Uric acid is only slightly water solvent and will thus tend to concentrate in the connective tissue, when the concentration
reaches the crystallization point it will turn into crystals in the tissues. This is known as gout.
The crystallization coefficient is dependant on:
-temperature
-concentration
- pressure
The crystallization process consists of two major events, nucleation and crystal growth.
Nucleation is the step where the solute molecules dispersed in the solvent start to gather into clusters, on the nanometer
scale (elevating solute concentration in a small region), that becomes stable under the current operating conditions.
These stable clusters constitute the nuclei. However when the clusters are not stable, they redissolve. Therefore, the
clusters need to reach a critical size in order to become stable nuclei. Such critical size is dictated by the operating
27

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

conditions (temperature, super saturation or concentration, pressure.). It is at the stage of nucleation that the atoms
arrange in a defined and periodic manner that defines the crystal structure — note that "crystal structure" is a special
term that refers to the internal arrangement of the atoms, not the macroscopic properties of the crystal: size and shape.
The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical cluster size. Nucleation
and growth continue to occur simultaneously while the supersaturation exists. Supersaturation is the driving force of the
crystallization, hence the rate of nucleation and growth is driven by the existing supersaturation in the solution.
Depending upon the conditions, either nucleation or growth may be predominant over the other, and as a result, crystals
with different sizes and shapes are obtained (control of crystal size and shape constitutes one of the main challenges in
industrial manufacturing, such as for pharmaceuticals). Once the supersaturation is exhausted, the solid-liquid system
reaches equilibrium and the crystallization is complete, unless the operating conditions are modified from equilibrium so
as to supersaturate the solution again.
Many compounds have the ability to crystallize with different crystal structures, a phenomenon called polymorphism.
Each polymorph is in fact a different thermodynamic solid state and crystal polymorphs of the same compound exhibit
different physical properties, such as dissolution rate, shape (angles between facets and facet growth rates), melting
point, etc.
The higher the concentration (super saturation) and the lower the temperature, the faster the nucleation point is reached.
That is why uric acid crystals are usually found in the connective tissue of the periphery of the human organism: hands,
feet, nose and ears. When the pressure on the tissue is regularly high the crystallization will tend to speed up. On the
other hand, crystals will be enveloped by adipocytes to protect the surrounding tissue, but if the pressure is high enough
it will provoke inflammation, therefore gout is known to give mainly symptoms on the toes and feet.
Origin: liver dysfunction, kidney dysfunction, exaggerated purine rich nutrition. (meat)
medication = colchicine.
Colchicine is a highly poisonous alkaloid, originally extracted from plants of the genus Colchicum (Autumn crocus, also
known as the "Meadow saffron"). Originally used to treat rheumatic complaints and especially gout, it was also
prescribed for its cathartic and emetic effects. Its present medicinal use is mainly in the treatment of gout; as well, it is
being investigated for its potential use as an anti-cancer drug. Colchicine inhibits microtubule polymerization by binding
to tubulin, one of the main constituents of microtubules. Availability of tubulin is essential to mitosis, and therefore
colchicine effectively functions as a "mitotic poison" or spindle poison. Since one of the defining characteristics of
cancer cells is a significantly increased rate of mitosis, this means that cancer cells are significantly more vulnerable to
colchicine poisoning than are normal cells. It should be noted, however, that the therapeutic value of colchicine against
cancer is (as is typical with chemotherapy agents) limited by its toxicity against normal cells.
Apart from inhibiting mitosis, a process heavily dependent on cytoskeletal changes, colchicine also inhibits neutrophil
motility and activity, leading to a net anti-inflammatory effect.
Short metabolic review of the most significant plasma proteins:

4.3.3.2. ALBUMIN:
Metabolism: synthesis in the liver cells in the form of pro-albumin. Albumin has a
molecular mass of 80.000 and is the most important initiator of the osmotic
pressure (60 % of the plasma proteins are albumin) and functions as a transport
molecule for substances like: bilirubin, free fatty acids, calcium and some drugs.
Albumin has in its form many active openings or bind sites; by the bond with the
transported substances the toxicity of them is reduced or neutralized. Catabolism of
albumin is in all tissues but mainly in the liver. (Half-life time 20 days)
Laboratory value, Semiology:
 shortage of synthesis
- Protein shortage: mal absorption, malnutrition, mal digestion.(See the Onge
girl: steatopygia4 and abdominal edema enough fat but protein shortage.)

decrease:

4

Steatopygia is a high degree of fat accumulation in and around the buttocks. The deposit of fat is not confined to the gluteal
regions, but extends to the outside and front of the thighs, forming a thick layer reaching sometimes to the knee. This development

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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

- Hepato-cellular dysfunction: cirrhosis , extreme icterus
- Competition synthesis: increased synthesis of other proteins  dysglobulinaemia, cancer.
 increased loss
- Renal loss: a healthy nefrons’ filter has an opening of 70.000 molecular mass, in nefrotic problems albumin will be
the first protein to be lost to the urine.
- Tissular loss: in large wounds like burnwounds or abrassion wounds.
4.3.3.3. -GLOBULINS:

Schematic representation of a protein electrophoresis gel
Alpha Globulins are a group of globular proteins in plasma, which are highly mobile in alkaline or electrically charged
solutions. They inhibit certain blood protease and inhibitor activity.

4.3.3.3.1.

-FOETOPROTEINS (FP OR AFP) :

Metabolic synthesis by the hepatocytes during the fetal development, in children and adults the production is stopped
unless: tumors of the gut or liver develop than the production starts again.
Laboratory value, Semiology: Increase
- Liver cirrhosis
- Liver cancers
- Teratoma5: embryological tumor can pass unnoticed and start to grow by hormonal stimuli during puberty for instance.

4.3.3.3.2.

1 ACID GLYCOPROTEIN (OROSOMUCOIDE )

Metabolic synthesis by the liver, it is a very stable protein that is very water soluble because of its glucid-chains.

constitutes a genetic characteristic of the Khoisan. It is specially a feature of the women, but it occurs in a lesser degree in the males
(in most genetic variations of Homo sapiens, females tend to exhibit a greater propensity to adipose tissue accumulation in the
buttock region as compared with males). It has also been noted among the Pygmies of Central Africa and the Onge-tribe of the
Andaman Islands.
5

A teratoma is a type of neoplasm (specifically, a tumor). The word teratoma comes from Greek and means roughly "monstrous
tumor". Definitive diagnosis of a teratoma is based on its histology: a teratoma is a tumor with tissue or organ components
resembling normal derivatives of all three germ layers. Rarely, not all three germ layers are identifiable. The tissues of a teratoma,
although normal in themselves, may be quite different from surrounding tissues, and may be highly inappropriate, even grotesque
(hence the monstrous): teratomas have been reported to contain hair, teeth, bone and very rarely more complex organs such as
eyeball, torso, and hand. Usually, however, a teratoma will contain no organs but rather one or more tissues normally found in organs
such as the brain, thyroid, liver, and lung.

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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Laboratory value, Semiology: Increase
Typically first phase of protein increase during inflammation, is a better reference than sedimentation rate.
ERGO: Most  globulins say something on the hepato-cellular condition, tumor formation and or inflammation
see Immunoglobulins; in the course labo & fluida.
4.3.3.4. ENZYMES AND ISOZYMES6:
Enzymes are mainly within the cells, or outside of the cells in the lumen of the gut (digestion). When the plasmatic
concentration raises this means usually increased cell death or cell destruction. (trauma, infection, inflammation or
tumors)
In the laboratory tests organ specific enzymes will be investigated:

4.3.3.4.1.

 AMYLASE

Organ specific for the pancreas and salivation glands.
- acute pancreatitis
- tumor process in the pancreas.
- tumor process in the salivation glands
- acute salivation gland inflammation like parotiditis

4.3.3.4.2.

TRANSAMINASE

Organ specific for the liver (AA metabolism)7
- liver cell destruction (statins)
- acute hepatitis
-tumor process in the liver

4.3.3.4.3.

ALKALIC PHOSPHATASES

Are present in almost all tissues, but they are present in very high concentrations in the osteoblasts. They are eliminated
through the liver by the biliary system.
semiological value:
- Bone disease with increased osteoblastic activity:
(Paget , hyper parathyroidia, bone metastases)
- Hepato-biliary dysfunctions :
6

Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction.
These enzymes usually display different kinetic parameters, or different regulatory properties. The existence of isozymes permits the
fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage (for example lactate dehydrogenase
(LDH)). In biochemistry, isozymes (or isoenzymes) are isoforms (closely related variants) of enzymes. In many cases, they are coded
for by homologous genes that have diverged over time. Although, strictly speaking, allozymes represent enzymes from different
alleles of the same gene, and isozymes represent enzymes from different genes whose products catalyse the same reaction, the two
words are usually used interchangeably.
7

Transaminase or an aminotransferase is an enzyme that catalyzes a type of reaction between an amino acid and an α-keto acid.
Specifically, this reaction (transamination) involves removing the amino group from the amino acid, leaving behind an α-keto acid,
and transferring it to the reactant α-keto acid and converting it into an amino acid. The enzymes are important in the transformation
of various amino acids, and measuring the concentrations of various transaminases in the blood is important in the diagnosing and
tracking liver damage.

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Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

(hyper haemolysis, cirrhosis , Hodgkin, biliary obstruction or dimuinished excretion)

4.3.3.4.4.

CREATINE KINASE (CREATINE PHOSPHOKINASE OR CPK OR CK)

Mainly specific enzyme for skeletal and heart musculature, it is present everywhere but in massive amounts in the
musculature. Is normally excreted by the kidneys
semiological value:
-myocard infarction
-acute renal failure
-destructive myopathy
-can increase massively after muscle fatigue or non habitual physical strain.
semiology
<study> The science or art of signs. The art of using signs in signaling.
<medicine> Specifically: The science of the signs or symptoms of disease; symptomatology.
Origin: Gr. Shmeion, a mark, a sign. Source: Websters Dictionary
Blood tests are laboratory tests done on blood to gain an appreciation of disease states and the function of organs.
Since blood flows throughout the body, acting as a medium for providing oxygen and other nutrients, and drawing waste
products back to the excretory systems for disposal, the state of the bloodstream affects, or is affected by, many medical
conditions. For these reasons, blood tests are the most commonly performed medical tests. Blood is obtained from one
of the patient's veins by venipuncture or fingerprick, except for tests such as Arterial blood gas.
Blood is useful as it is a relatively non-invasive way to obtain cells, and extracellular fluid (plasma), from the body to
check on its health. Although the term blood test is used, most routine tests (except for most haematology) are done on
plasma or serum.
Urine test checks different components of urine, a waste product made by the kidneys. A regular urine test may be done
to help find the cause of symptoms. Urine has hundreds of different body wastes. What you eat, drink, how much you
exercise, and how well your kidneys work can affect what is in your urine.
More than 100 different tests can be done on urine. A regular urinalysis often includes the following tests.
Color. Many things affect urine color, including fluid balance, diet, medicines, and diseases. How dark or light the color
is tells you how much water is in it. Vitamin B supplements can turn urine bright yellow. Some medicines, blackberries,
beets, rhubarb, or blood in the urine can turn urine red-brown.
Clarity. Urine is normally clear. Bacteria, blood, sperm, crystals, or mucus can make urine look cloudy.
Odor. Urine does not smell very strong, but has a slightly "nutty" odor. Some diseases cause a change in the odor of
urine. For example, an infection with E. coli bacteria can cause a bad odor, while diabetes or starvation can cause a
sweet, fruity odor.
Specific gravity. This checks the amount of substances in the urine. It also shows how well the kidneys balance the
amount of water in urine. The higher the specific gravity, the more solid material is in the urine. When you drink a lot of
fluid, your kidneys make urine with a high amount of water in it which has a low specific gravity. When you do not
drink fluids, your kidneys make urine with a small amount of water in it which has a high specific gravity.
pH. The pH is a measure of how acidic or alkaline (basic) the urine is. A urine pH of 4 is strongly acidic, 7 is neutral
(neither acidic nor alkaline), and 9 is strongly alkaline. Sometimes the pH of urine is affected by certain treatments. For
example, your doctor may instruct you how to keep your urine either acidic or alkaline to prevent some types of kidney
stones from forming.
Protein. Protein is normally not found in the urine. Fever, hard exercise, pregnancy, and some diseases, especially
kidney disease, may cause protein to be in the urine.
Glucose. Glucose is the type of sugar found in blood. Normally there is very little or no glucose in urine. When the
blood sugar level is very high, as in uncontrolled diabetes, the sugar spills over into the urine. Glucose can also be found
in urine when the kidneys are damaged or diseased.

31

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Nitrites. Bacteria that cause a urinary tract infection (UTI) make an enzyme that changes urinary nitrates to nitrites.
Nitrites in urine show a UTI is present.
Leukocyte esterase (WBC esterase). Leukocyte esterase shows leukocytes (white blood cells [WBCs]) in the urine.
WBCs in the urine may mean a UTI is present.
Ketones. When fat is broken down for energy, the body makes substances called ketones (or ketone bodies). These are
passed in the urine. Large amounts of ketones in the urine may mean a very serious condition, diabetic ketoacidosis, is
present. A diet low in sugars and starches (carbohydrates), starvation, or severe vomiting may also cause ketones to be
in the urine.
Microscopic analysis. In this test, urine is spun in a special machine (centrifuge) so the solid materials (sediment) settle
at the bottom. The sediment is spread on a slide and looked at under a microscope. Things that may be seen on the slide
include:
Red or white blood cells. Blood cells are not found in urine normally. Inflammation, disease, or injury to the kidneys,
ureters, bladder, or urethra can cause blood in urine. Strenuous exercise, such as running a marathon, can also cause
blood in the urine. White blood cells may be a sign of infection or kidney disease.
Casts. Some types of kidney disease can cause plugs of material (called casts) to form in tiny tubes in the kidneys. The
casts then get flushed out in the urine. Casts can be made of red or white blood cells, waxy or fatty substances, or
protein. The type of cast in the urine can help show what type of kidney disease may be present.
Crystals. Healthy people often have only a few crystals in their urine. A large number of crystals, or certain types of
crystals, may mean kidney stones are present or there is a problem with how the body is using food (metabolism).
Bacteria, yeast cells, or parasites. There are no bacteria, yeast cells, or parasites in urine normally. If these are present,
it can mean you have an infection.

RESEARCH ARTICLE
Advance Online Article September 27, 2005

INTERSTITIAL
FLUID
FLOW
INDUCES
MYOFIBROBLAST
DIFFERENTIATION AND COLLAGEN ALIGNMENT IN VITRO
Summary
The differentiation of fibroblasts to contractile myofibroblasts, which is characterized by de novo expression of αsmooth muscle actin (α-SMA), is crucial for wound healing and a hallmark of tissue scarring and fibrosis. These
processes often follow inflammatory events, particularly in soft tissues such as skin, lung and liver. Although
inflammatory cells and damaged epithelium can release transforming growth factor β1(TGF-β1), which largely mediates
myofibroblast differentiation, the biophysical environment of inflammation and tissue regeneration, namely increased
interstitial flow owing to vessel hyperpermeability and/or angiogenesis, may also play a role. We demonstrate that low
levels of interstitial (3D) flow induce fibroblast-to-myofibroblast differentiation as well as collagen alignment and
fibroblast proliferation, all in the absence of exogenous mediators. These effects were associated with TGFβ1 induction, and could be eliminated with TGF-β1 blocking antibodies. Furthermore, α1β1 integrin was seen to play an
important role in the specific response to flow, as its inhibition prevented fibroblast differentiation and subsequent
collagen alignment but did not block their ability to contract the gel in a separate floating gel assay. This study suggests
that the biophysical environment that often precedes fibrosis, such as swelling, increased microvascular permeability and
increased lymphatic drainage – all which involve interstitial fluid flow – may itself play an important role in
fibrogenesis.
Key words: Fibrosis, α-Smooth muscle actin, Transforming growth factor β, β1 Integrin, Shear stress

Introduction
Myofibroblasts play a key role in both physiological wound healing and pathological fibrocontractive conditions such as
various forms of fibrosis and desmoplasia (Gabbiani, 2003; Serini and Gabbiani, 1999). During early wound healing,
growth factors released by inflammatory cells stimulate fibroblasts to migrate into the provisional clot matrix, where
they proliferate and reconstitute a collagen-rich extracellular matrix (ECM) (Martin, 1997). The gradual increase in
ECM stiffness by fibroblast tractional forces is mandatory for their further evolution into myofibroblasts (Hinz and
Gabbiani, 2003b), which actively close the wound by contraction. Once epithelium has covered the wound,
myofibroblasts normally disappear by apoptosis and the granulation tissue eventually evolves into a scar containing few
cells (Desmouliere et al., 1995). Under pathological conditions of fibrosis, however, the myofibroblasts do not undergo
apoptosis but instead proliferate and overproduce ECM. Fibrosis is the pathologic hallmark of many common
32

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

fibrocontractive diseases, including pulmonary fibrosis (Thannickal et al., 2004), hepatic cirrhosis and chronic
glomerulonephritis (Desmouliere et al., 2003), systemic sclerosis (scleroderma) (Varga and Jimenez, 1995) and
desmoplastic stromal response (Desmouliere et al., 2004; Mueller and Fusenig, 2004; Walker, 2001).
Myofibroblasts are generally characterized by expression of α-smooth muscle actin (α-SMA) protein, the actin isoform
typical of smooth muscle cells, conferring a high contractile activity to these cells (Hinz et al., 2001a), although α-SMA
is not required for collagen gel contraction in vitro (Grinnell, 1994; Hinz and Gabbiani, 2003a; Vanni et al., 2003). The
primary inducer of fibroblast-to-myofibroblast differentiation is transforming growth factor β1 (TGF-β1) (Desmouliere
et al., 1993; Ronnov-Jessen and Petersen, 1993), acting either via paracrine release by inflammatory, epithelial or tumor
cells (Werner and Grose, 2003) or via autocrine regulation (Kim et al., 1990). Mechanical factors that either provide
resistance to matrix contraction or exert tensional forces on the fibroblast cytoskeleton can also modulate fibroblast
differentiation. For example, when wound granulation tissue fibroblasts were subjected to mechanical tension in vivo by
immobilizing the edges of full-thickness wounds, α-SMA expression was upregulated; tension release by frame removal
led to stress fiber disassembly and downregulation of α-SMA expression (Hinz et al., 2001b). In vitro, fibroblasts
cultured in three-dimensional (3D) collagen gels exhibit increasing levels of α-SMA expression with increasing matrix
stiffness and/or externally applied stretch (Grinnell et al., 2003) but do not differentiate in free-floating gels (Arora et
al., 1999). Thus, mechanical forces are strongly implicated in myofibroblast differentiation.
Here, we demonstrate that low levels of interstitial flow (i.e. fluid flow through a 3D matrix) can itself induce collagen
alignment and fibroblast-to-myofibroblast transition via autocrine upregulation of TGF-β1. We previously reported that
human dermal fibroblasts align under interstitial flow in 3D collagen gel cultures, perpendicular to the direction of flow
(Ng and Swartz, 2003). As aligned fibroblasts and matrix fibers are often seen in wound and fibrotic tissues (Darby et
al., 1990; Hinz et al., 2001b), we proposed that interstitial flow could itself contribute to fibrosis even in the absence of
inflammatory cells as observed in idiopathic pulmonary fibrosis (Pardo and Selman, 2002; Thannickal et al., 2004).
Interstitial flow is present in soft tissues as an important component of the microcirculation between blood and
lymphatic vessels, and interstitial flow is increased during events such as inflammation and wound healing where an
influx of inflammatory cells and active angiogenesis both contribute to increased fluid flux into the surrounding tissues.
The levels of flow we imposed reflect probable pathological values, as they were three to ten times higher than those
reported for normal tissue (Chary and Jain, 1989). In the context of desmoplastic stroma, the high interstitial pressure of
tumors may lead to an increased outflow of tumor interstitial fluid into the stromal tissues surrounding the tumors
(Heldin et al., 2004; Jain, 2001; Swabb et al., 1974). Thus, our findings suggest that the biomechanical environment
associated with inflammation (which is accompanied by cytokines), vascularized tumors, remodeling blood vessels or
increased lymphatic flow (which is not necessarily associated with cytokines), can itself stimulate myofibroblast
differentiation.

Fig. 1.
Experimental set-up and alignment determination. (A) Design features of radial interstitial flow tissue culture chamber.
The chamber is made of porous polyethylene and surface-modified glass materials to anchor the ECM and allow direct
visualization. (B) Algorithm for image quantification of alignment and orientation. The confocal image (i) is modified
(ii) to remove edge effects. (iii) A FFT transformation is performed to obtain a power spectrum from which (iv) an
intensity frequency histogram is plotted and an alignment index (=(δ/(Δ + δ))/(δ/(Δ + δ))ideal) and the peak angle
(θpeak) extracted.
Materials and Methods

Culture of human dermal fibroblasts
CCD1079sk neonatal human dermal fibroblasts (HDF, American Type Culture Collection, Manassa, VA) were
expanded in α-MEM supplemented with 10% fetal bovine serum (GIBCO BRL, Grand Island, NY) and 1%
penicillin/streptomycin (Sigma, St Louis, MO) and used in passages 7-9.
33

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Preparation of fibroblast-populated matrices and application of interstitial flow
Collagen gels (2 mg/ml), seeded with 5×105 fibroblasts/ml, were cast in an interstitial flow chamber (Fig. 1A) as
previously described (Ng and Swartz, 2003). The set-up was immersed in media overnight for cell attachment at 37°C,
5% CO2 in an incubator. For the induction of flow, the chamber was connected to a reservoir of growth medium via a
peristaltic pump and a pressure manometer. The flow was delivered at 0.012 ml/minute, leading to a weighted average
velocity of 6.3 μm/second. Medium surrounded the chamber and could diffuse through the outer and inner PE rings.
Two static controls were used, one mechanically constrained and the other floating. In the first, the gel-filled chamber
was set up as before but with no connection to the flow delivery apparatus. The mechanically relaxed static control
consisted of the cell-populated gel seeded into an eight-well Lab-Tek coverslip chamber system (Nalge Nunc,
Naperville, IL) and allowed to contract freely throughout the experiment. All cultures were maintained in a humidified
37°C, 5% CO2 incubator.

Immunofluorescence staining
The entire gel was fixed by immersion in 2% paraformaldehyde in PBS for 30 minutes and permeabilized in 0.5%
Triton X-100. To detect f-actin and α-SMA, it was immersed overnight at 4°C in 150 nM Alexa 488-conjugated
Phalloidin (Molecular Probes, Eugene, OR) and 5 μg/ml monoclonal Cy3-conjugated mouse anti-human α-SMA
antibody (clone 1A4, Sigma). In some cases, the gels were also incubated in 500 nM TOTO-3 (Molecular Probes) for
nuclear counterstaining. To detect proliferation, gels were incubated with 0.8 μg/ml monoclonal mouse anti-human Ki67 (clone MIB-1, DakoCytomation, Carpinteria, CA) and then 10 μg/ml Alexa 546-conjugated rabbit anti-mouse IgG
(Molecular Probes), followed by counterstaining with Phalloidin and TOTO-3. To visualize TGF-β1 protein expression,
gels were incubated with 20 μg/ml rabbit anti-human TGF-β1 (Promega), followed by incubation with 2.5 μg/ml Alexa
647-conjugated goat anti-rabbit IgG (Molecular Probes).

Confocal fluorescence and reflectance microscopy
Images were taken using laser-scanning confocal microscopy (Leica LCS SP2 laser microscope system, Mannheim,
Germany). Confocal reflectance contrast microscopy was performed to visualize collagen fibers using a 40× (1.25 NA)
oil objective lens with modifications based on a previous protocol (Brightman et al., 2000; Friedl et al., 1997). To detect
Phalloidin and reflectance simultaneously, samples were excited with a 488 nm Ar laser and both the respective
emission signal and reflected light passed through an RT 30/70 beam splitter and collected in two separate channels.
Two other channels were used to detect emission signals from the α-SMA and TOTO-3 stains, which were excited by
He-Ne lasers (543 nm and 633 nm respectively). Samples were vertically scanned from the bottom coverslip with a total
depth of 20-100 μm and a pinhole diameter of 40-70 μm. The sequential images were collected at a step depth of 0.3-2.0
μm and reconstructed using Leica LCS (Leica) or Volocity (Improvision, Lexington, MA) software.

Bioneutralization studies
For bioneutralization studies, antibodies with known function-blocking activity were added to the cell suspension and
incubated for 30 minutes at 37°C prior to seeding in the collagen matrices. Concentrations were chosen in accordance
with previously demonstrated blocking concentrations. They were also maintained in the culture medium throughout the
experiment at a lower concentration as indicated by pre-incubation and experiment: mouse anti-human α1β1 integrin
(clone SR84, BD Biosciences Pharmingen, 10 and 2 μg/ml) (Rettig et al., 1984; Setty et al., 1998), mouse anti-human
α2β1 (clone BHA2.1; Chemicon; 20 and 10 μg/ml) (Li et al., 2003) and rabbit anti-human TGF-β1 (Promega; 0.8
μg/ml) (Zatelli et al., 2000).

Image analysis quantification
Fibroblast proliferation, density, spreading and expression of α-SMA and TGF-β1 were quantified using ImageJ (NIH,
Bethesda, MD). All cells in each image (typically 50-200) were evaluated, using three images per experiment, with three
to five experiments per condition. Particle counting was used to determine the number of proliferating (Ki67+) cells
normalized to the total cell number (TOTO-3+). α-SMA and TGF-β1 were quantified by calculating the projected areas
of their signals and normalizing those to the f-actin signals. To quantify the projected areas, each image was first
converted into a binary image using the threshold function with fixed limits determined from sample images; these were
then despeckled, and the total area of signal (α-SMA or TGF-β1) was divided by the total cell area (i.e. the total area of
f-actin signal). Cell density (number of cells/mm3) was quantified directly from cell counts (TOTO-3+) whereas cell
spreading was expressed as the fraction of projected cell area (f-actin signal) per total projected image area.

34

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Fast Fourier image analysis and quantification of cell and collagen fiber orientation and
alignment
Fast Fourier transform (FFT) analysis, an indirect method previously applied to quantify collagen fiber alignment in
SEM and histological images of ligaments, sclerodermal lesions and scar tissues (Chaudhuri et al., 1987; Nishimura and
Ansell, 2002; Pourdeyhimi et al., 1997; van Zuijlen et al., 2002), was used here to evaluate the orientation distribution
of structures in confocal images. We developed a MATLAB program to perform the analysis. First, an image was
imported as a matrix array and Welch windowing was applied to reduce edge effects caused by discontinuities in the
imperfect periodic images. The FFT algorithm then transformed the windowed image into a power spectrum, which was
highly contrasted before the intensity frequencies were summed to determine the orientation intensity distribution
histogram.
From each orientation histogram, the peak angle, or angle of highest frequency, was determined. However, although this
indicates the angle at which the most cells or fibers are aligned, it does not reflect how many objects (cells or fibers) are
aligned at this angle; if the objects were perfectly randomly oriented, the peak angle would be arbitrary and irrelevant.
Thus, we also defined an alignment index to reveal the fraction of cells or fibers that were aligned within 20° of the peak
angle and this was normalized to the fraction of randomly oriented fibers that would lie within this range (i.e.
40°/180°=0.22). A randomly aligned matrix would have an alignment index of 1; the higher the value, the higher the
fraction of cells or fibers aligned near the peak angle.

Statistical analysis of parametric and non-parametric data
Normally distributed data were represented by bar graphs showing the mean and s.d., and unpaired Student's or
Welch's t-tests were used to compare mean differences between data with unequal or equal variances, respectively
(equality of the variances were assessed using an F-test). Comparisons of three groups or more were performed using
ANOVA with Dunnett's post-test. For non-normally distributed data, which were represented by medians and 95%
confidence intervals using box plots, the nonparametric Mann-Whitney test was used to compare median differences
whereas the Krushal-Wallis test with Dunn's post test was used to compare three groups or more.

Results
Interstitial flow induces cell and matrix alignment
To investigate the effects of interstitial flow on fibroblast and matrix organization, fibroblast-populated collagen gels
were subjected to an average interstitial flow velocity of 6.3 μm/second (leading to an approximate shear stress of 0.10.3 dyn/cm2; see Discussion for details). Fibroblasts aligned perpendicular to the direction of flow within 2 days ,
correlating with the alignment of the surrounding collagen fibers in the same direction. Analysis of FFT intensity
frequency histograms confirmed these qualitative observations of cell and matrix alignment: the alignment index, a
measure of the fraction of cells or ECM fibers that are aligned to within 20° of the peak angle, showed significant
differences between both cells and collagen fibers under flow compared to those in static conditions. Furthermore, under
interstitial flow, the peak angle was oriented at 83±18° for cells under flow but were randomly aligned (peak angle
58±46°) in static conditions; similar trends were seen with fiber orientation, with peak angles of 90±10°, and 84±52° for
flow and static conditions, respectively.

Interstitial flow promotes myofibroblast differentiation and proliferation
To investigate whether the aligned cells were proliferating and differentiating into myofibroblasts, we immunostained
fibroblast-populated collagen gels with Ki67 (a proliferation marker) and α-SMA and quantified confocal images as
described in Materials and Methods. Interstitial flow induced α-SMA expression in about 5% of fibroblasts after 2 days
and 97% after 5 days. In contrast, virtually all fibroblasts in static conditions remained undifferentiated after 2 days and
only 14% were α-SMA-positive after 5 days. In addition, interstitial flow increased the proliferating cell fraction
compared to both static controls. Thus, it can be concluded that interstitial flow (order of microns per second) enhances
myofibroblast differentiation and proliferation in collagen matrices.

TGF-β1 mediates flow-induced myofibroblast differentiation
As TGF-β1 is the major known inducer of myofibroblast differentiation, we examined whether TGF-β1 was involved in
the interstitial flow response. First, we observed by immunostaining that interstitial flow strongly induced TGFβ1 protein expression by the fibroblasts, whereas none could be detected under static conditions. This was consistent
with the expression of α-SMA shown in Fig. 3B. When an anti-TGF-β1 blocking antibody was introduced into the flow

35

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

media, α-SMA expression was eliminated, suggesting that the mechanism by which interstitial flow induces α-SMA
expression is mediated through the upregulation of TGF-β1.

Fig. 2.
Alignment of human dermal fibroblasts in a collagen matrix subjected to radial interstitial flow. Confocal images of cells
(A) and matrix fibers (B) with their corresponding FFT analyzed intensity frequency histograms. f-actin is labelled green
with the confocal reflection in red; arrow indicates flow direction. These observations were quantified by alignment
index (C) and peak angle (D) for cell and matrix alignment, respectively. Unpaired t-tests were used for statistical
analysis of the means; significant differences (**P<0.01) were observed in alignment index in both cells and matrix
under flow conditions compared to that measured under static conditions using Mann-Whitney test. Bar, 200 μm (A); 20
μm (B).

Fig. 3.
Interstitial flow induces α-SMA expression in fibroblasts. (A) Confocal images of cells at 2 and 5 days showing α-SMA
expression under mechanically constrained static and interstitial flow conditions (green, f-actin; red, α-SMA; arrow
indicates flow direction). (B) Significantly higher levels of α-SMA expression are seen in fibroblasts undergoing
interstitial flow at both time points (**P<0.01 using Dunn's test). Box plot shows 95% confidence intervals with midline
showing the median. (C) The percentage of proliferating (Ki67+) cells after 2 days was higher under interstitial flow
conditions than either constrained or relaxed static controls (bar represents the mean value and error bars, s.d.; **P<0.01
using Dunnett's test). Bar, 200 μm.
Furthermore, TGF-β1 neutralization abolished flow-enhanced cell density and spreading without affecting cell
alignment. Without the blocking antibody, cell density and spreading (i.e. projected cell area per cell) were both
increased by interstitial flow after 2 days, but with TGF-β1neutralization, no increase in density or spreading was seen.
Interestingly, blocking TGF-β1 only slightly affected the flow-induced cell alignment: the alignment index was not
changed but there was a greater distribution in peak angle. In contrast, collagen fiber alignment was reduced, indicating
that α-SMA is required for the cells to align the matrix but not to align themselves. Taken together, these results indicate
that interstitial flow causes an upregulation of TGF-β1 expression, which induces α-SMA expression, which in turn
causes matrix alignment.

Interstitial flow effects are mediated through α1β1 integrin
Matrix remodeling depends on the transmission of intracellular contractile forces to the ECM at sites of integrin-type
cell-matrix adhesions. Fibroblasts are known to mechanically interact with collagen fibers primarily through
β1 integrins, particularly α1β1 and α2β1 (Heino, 2000). To investigate whether ligation and signaling through these
β1 integrins were important in mediating the fibroblast differentiation response to interstitial flow, blocking antibodies
were used to specifically target α1β1 and α2β1 integrins. We found that although both reversed the effects of flow on αSMA expression, cell density and cell spreading, α1β1 integrin blocking completely neutralized α-SMA expression. The
differences in α-SMA expression between blocking of α1β1 and α2β1 were significant (P=0.0002 using Mann-Whitney
test), indicating that α1β1was a more potent regulator of α-SMA expression than α2β1. Furthermore, blocking of
36

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

α1β1 integrin had a smaller effect than α2β1 blocking on cell density and spreading. Importantly, specific blocking of
α1β1 had little effect on the ability of the fibroblasts to attach to the gel and contract it, in contrast to blocking of
α2β1 integrins, which caused significant cell detachment and prevented matrix contraction. Finally, blocking
α1β1 prevented both cell and matrix alignment; the lack of change in cell alignment during α2β1 blocking was probably
due to the fact that very few cells remained attached to the matrix when α2β1 is blocked. These results suggest that
ligation of α1β1 integrins is necessary for interstitial flow-induced myofibroblast differentiation and subsequent cell
proliferation, cell alignment and matrix alignment.

Fig. 4.
TGF-β1 mediates flow-induced myofibroblast differentiation. (A) Confocal images showing TGF-β1 and α-SMA
expression in mechanically constrained static (top) and flow conditions, without (middle) and with (bottom) TGFβ1 neutralizing antibody (red, f-actin; green, TGF-β1; blue, α-SMA; arrow indicates flow direction). (B) TGFβ1 expression quantification (**P<0.01 using Dunn's test) in fibroblasts under constrained static and interstitial flow
conditions for 2 days. Box plot shows 95% confidence intervals with midline showing the median. (C) α-SMA
expression quantification in normal compared to bioneutralized flow conditions (**P<0.01 using Mann-Whitney test).
(D,E) Quantification of cell density in terms of number of cells per mm3 and cell spreading in terms of projected cell
area (μm2/cell) (**P<0.01 using Dunnett's test). (F) Alignment and (G) orientation of the cells and matrix fibers under
each condition (*P<0.05; **P<0.01 using Dunnett's test). Bar, 200 μm.

Discussion
Much information has recently emerged about the cellular and biochemical events that underlie tissue remodeling
towards a fibrotic phenotype. For example, the development of fibrosis follows a similar pathway to normal wound
healing except in most cases there is a chronic progression of the disease which is characterized by: (1) a continuous
insult or stimulus (chemical or mechanical); (2) excessive synthesis of collagen and other ECM components; and (3) a
decrease in resolution owing to a downregulation of matrix-degrading enzymes (Mutsaers et al., 1997). In addition, in
many tissues fibrosis is typically preceded by chronic inflammation, which provides a source of inflammatory cellderived cytokines such as TGF-β1 that are crucial mediators of fibrogenesis. However, fibrosis can also follow noninflammatory events such as swelling in lymphedema (Campisi and Boccardo, 2002) and in idiopathic pulmonary
fibrosis (Pardo and Selman, 2002); the mechanisms of myofibroblast differentiation and subsequent progression to
fibrosis are still not clear in such pathologies, and the role of interstitial flow that typically accompanies such processes
has not previously been addressed.

Fig. 5.
37

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

Interstitial flow effects are mediated through α1β1 integrins. (A) Confocal images of cells under normal, α1β1- and
α2β1-neutralized flow conditions for 2 days (green, f-actin; red, α-SMA; arrow indicates flow direction). (B) Bar graphs
indicating α-SMA expression levels for the respective normal and bioneutralized conditions (**P<0.01 using Dunn's
test); box plot shows 95% confidence intervals with midline showing the median. (C,D) Effects of integrin blocking on
cell spreading and density (**P<0.01 using Dunnett's test). (E,F) Effects of integrin blocking on the alignment and
orientation of cell and collagen fibers under the various experimental conditions (*P<0.05; **P<0.01 using Dunnett's
test). Error bars indicate s.d. (G) Contraction of free-floating gels by fibroblasts after no treatment (control) or treatment
with either of the integrin-blocking antibodies as indicated; only neutralizing α2β1 integrin prevents contraction. Bar,
200 μm.
Our results here suggest that interstitial flow alone may be sufficient to induce and sustain fibrosis, even in the absence
of TGF-β1 secretion by other cells such as inflammatory, epithelial or tumor cells, and correlates with key features of
the progression of an inflammatory state to a fibrotic pathology. We show that fibroblasts in 3D collagen cultures
undergoing somewhat superphysiological levels of interstitial flow (6 μm/second, or roughly 1-3 dyn/cm2 shear stress)
exhibit features commonly observed in tissues of fibrotic phenotype such as scar tissue and the desmoplastic stroma
around tumors: the cells proliferate, differentiate into myofibroblasts, and align parallel to each other. Our observation
of cell and fiber alignment here is consistent with in vivo observations during wound healing where collagen fibers and
fibroblasts become aligned within the wound bed (Ehrlich and Krummel, 1996; Tomasek et al., 2002); furthermore,
perpendicular cell alignment to fluid flow has previously been seen in 2D shear studies on smooth muscle cells (Lee et
al., 2002). The flow-induced increase in cell proliferation also mimics the pathological condition: whereas in normal
wound repair, myofibroblasts eventually undergo apoptosis while the granulation tissue evolves into a scar with a sparse
cell population (Tomasek et al., 2002); in fibrotic conditions, they continue to proliferate and overproduce ECM,
leading to elevated fibroblast density (Ehrlich and Krummel, 1996).
To investigate the mechanism underlying this interstitial flow-induced myofibroblast differentiation, we examined the
role of TGF-β1, a potent and well-known inducer of α-SMA expression (Arora et al., 1999; Dugina et al., 2001; KunzSchughart et al., 2003; Vaughan et al., 2000) as well as a stimulus of collagen production (Roberts et al., 1986) and
inhibitor of collagen proteolysis (Mutsaers et al., 1997). First, we saw that interstitial flow triggered the autocrine
production of TGF-β1 in fibroblasts, correlating with the increased α-SMA expression, and that blocking TGF-β1 with
antibodies completely prevented the flow-induced α-SMA expression. Furthermore, this TGF-β1 induction is probably
responsible for the flow-enhanced cell proliferation, as TGF-β1 has been shown to prevent apoptosis (Phan,
2002; Zhang and Phan, 1999) and induce cell proliferation, at least in vascular smooth muscle cells (although it also has
been found to inhibit proliferation under certain conditions) (Gibbons, 1994).

Fig. 6.
Proposed mechanism of interstitial flow-driven myofibroblast differentiation and matrix remodeling. First, flow itself
can impose shear stress on the cells directly and strain on the cells via stresses on the ECM fibers to which the cells
attach. Either of these may trigger TGF-β1 expression, the latter through α1β1 integrin signaling. TGF-β1 drives α-SMA
expression as the fibroblasts differentiate into myofibroblasts and align the matrix fibers.
To further explore how flow triggers TGF-β1 signaling, we examined the roles of β1 integrins. These vital cell matrix
adhesion molecules play crucial roles in transducing ECM strain to the cell (Chiquet et al., 2003; Eckes et al., 1999) and
are known to be important in tissue repair and fibrosis by regulating cell proliferation, survival, differentiation,
migration, matrix deposition and wound contraction (Danen and Sonnenberg, 2003; Eckes et al., 1999; Mutsaers et al.,
1997). Although several different integrins can mediate collagen matrix remodeling, including α1β1, α2β1, α11β1, and
αvβ3 (Tamariz and Grinnell, 2002), we focused on α1β1 and α2β1 integrins because β1 integrins have been associated
with tensional force generation in fibroblasts within a collagen type I matrix (Jenkins et al., 1999; Schiro et al., 1991)
and because α1β1 and α2β1integrins are largely responsible for regulating ECM remodeling in fibroblast-populated
collagen gels (Langholz et al., 1995). The results from our bioneutralization experiments suggest that β1 integrins do
indeed play a specific role in mechanical force-regulated formation of α-SMA, particularly α1β1, as blocking antibodies
against α1β1 completely inhibit the flow-induced expression of α-SMA without interfering with the ability of the
fibroblasts to contract the gel. Thus, although our gel contraction data is consistent with earlier reports that α2β1 integrin
38

Physiology: Introduction
M.Girardin D.O., Evost Fellow, Pro-sector
1995, reeditions 2007, 2014, 2015

is specifically required for gel contraction (Jenkins et al., 1999; Schiro et al., 1991), it also demonstrates that the
α1β1 integrin is specifically involved in flow-induced differentiation but not required for ECM adhesion or contraction
by the fibroblasts. This indicates that the α1β1integrin plays a specialized function specific to fibroblast differentiation
and is not surprising as α1β1 is abundant in smooth muscle cells (Heino, 2000) and is thought to be the sole integrin
utilized by contracting myofibroblasts in wound healing in vivo (Racine-Samson et al., 1997).
Thus, these studies suggest a mechanism of flow-induced myofibroblast differentiation and matrix remodeling as
illustrated in Fig. 6. As for the fundamental mechanotransduction mechanism triggering these responses, it is still
unclear exactly how the cell might sense such low levels of interstitial flow. It has been established that mechanical
forces like stretch can drive fibroblast differentiation toward a myofibroblast phenotype (Arora and McCulloch,
1994; Hinz et al., 2001a; Hinz and Gabbiani, 2003b), although the mechanism remains unclear; furthermore, 2D shear
stress can induce autocrine TGF-β1 expression in vascular smooth muscle cells both in vitro on confluent cell
monolayers (Ueba et al., 1997) or in vivo after experimental artery injury (Song et al., 2000). Here, the levels of
interstitial flow imposed are extremely small: based on a measured average hydraulic conductivity of 1×10–9 cm2 at the
beginning of the experiment that decreased to 2×10–10 cm2 after 5 days of interstitial flow owing to matrix remodeling
(Ng and Swartz, 2003), we estimated the average fluid shear stress on the cells to vary between 0.15 and 0.33
dyn/cm2 (Wang and Tarbell, 1995). It is not known whether such small shear stresses can be sensed by the cells,
although 2D stresses as low as 0.1 dyn/cm2 imposed on endothelial cell monolayers can elicit gene upregulation
(Barakat and Lieu, 2003). Little to no stretch would be expected to be imposed on the cell as the ECM is anchored in all
directions, although non-affine deformation behavior in a collagen matrix can lead to nonuniformly distributed strain;
however, this would tend to decrease, rather than increase, imposed strain on the cell (Pedersen and Swartz, 2005).
Other possible mechanisms may include small changes in the local extracellular biochemical environments: for example,
changes in extracellular distribution and transport of cell-secreted cytokines (Swartz, 2003).
In conclusion, our data demonstrate the influence of interstitial flow on myofibroblast differentiation, fibroblast
proliferation and matrix alignment; all of which are distinct and important characteristics of fibroblasts in fibrotic
tissues. Its strong ability to induce myofibroblast differentiation occurs without exogenous inflammatory mediators. This
suggests that interstitial flow may help to modulate fibroblast phenotypes and drive the progression of fibrotic diseases,
including organ fibrosis (lung, liver, renal or heart), defective wound healing like Duputryen's contacture (Tomasek et
al., 2002) and connective tissue diseases like artherosclerosis, scleroderma and asthma. Interestingly, we have previously
found that the same range of interstitial flow also enhances endothelial morphogenesis (Ng et al., 2004), which is
another essential component of the wound healing process (Ehrlich and Krummel, 1996). Taken together, these results
suggest that the biophysical environment of tissues undergoing chronic inflammation and/or swelling may significantly
affect long-term tissue remodeling towards a fibrotic state. Our results also have a potential use in regenerative medicine
and may be useful in the design of therapeutic approaches to prevent fibrotic disease or to promote wound healing.

4.3.4. ACKNOWLEDGEMENTS
This work was funded by grants from the Whitaker Foundation (RG-01-0348) and the National Science Foundation
(BES-0134551). The authors are grateful to John Pedersen for assistance with the FFT analysis.

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