Thyroid Hormone Therapy: Cutting the Gordian Knot
Thyroid Hormone Therapy: Cutting the Gordian Knot
Supplement to The Art of Getting Well
“Medical data is for informational purposes only. You should always consult your family physician or one of our referral physicians prior to treatment - The Arthritis Trust of America
Article provided by:
The Arthritis Trust of America
Sources are given in references.
Authors of contributions\quotations are alphabetically arranged; major author, if any, is underlined.
Broda O. Barnes, M.D., John Baron, D.O., James A. Carlson, D.O., Anthony HJ. Cichoke, D.C., O.P.
Dimball, M.D., Shirley Holmstead, Lita Lee, Ph.D., Howard Loomis, D.C., Raymond F. Peat, Ph.D.,
Gus J. Prosch, Jr., M.D., E. Denis Wilson, M.D. Jonathan V. Wright, M.D./Responsible editor/writer
Anthony di Fabio.
Copyright 1994
All rights reserved by The Roger Wyburn-Mason and Jack M. Blount Foundation for the Eradication of
Rheumatoid Disease AKA The Arthritis Trust of America,
7111 Sweetgum Road, Suite A, Fairview, TN 37062-9384
Thyroid: Master Gland & Regulator
The human body, from one perspective, is fundamentally a carbon/oxygen engine. It burns carbon with
the use of oxygen, producing heat and other forms of energy.
The rate at which this engine burns its fuel (carbon) has a certain efficiency, and is measured by a
concept called “basal metabolism”.
Basal metabolism represents the energy expended to maintain respiration, circulation, peristalsis, muscle
tone, body temperature, glandular activity, and the other vegetative functions.
If the rate of burning is higher than normal, one is called hyperthyroid, and if the rate is lower than normal,
one is said to be hypothyroid.
Thanks to Broda O. Barnes, M.D. (deceased) and his research and book, Hypothyrodisim: The
Unsuspected Illness1 we now know that improper thyroid functioning is the basis to many disease states,
as well as the key to wellness.
The reason is simple to explain: Proper enzyme functioning depends upon proper temperature. Too high
or too low, and the enzymes do not function, or do not function properly. As tens of thousands of
enzymes are the very essence of our proper functioning cells, organs, and systems, improper functioning
of enzymes also causes improper functioning of cells, organs and systems.
According to E. Denis Wilson, M.D., there are perhaps 60 different symptoms that stem from improper
enzyme functioning, many of them also named as diseases in their own right.
Wilson’s Syndrome: Multiple Enzyme Deficiencies
There’s a simple treatment that just might cure, or at least improve, more than 60 different disease
symptoms by permitting enzymes to function properly. The various disease symptoms include: Fatigue,
Headaches, Premenstrual Syndrome, Irritability, Dry Hair and Hair Loss, Decreased Memory and
Concentration, Insomnia and Narcolepsy, Anxiety and Panic Attacks, Heat and/or Cold Intolerance,
Depression, Fluid Retention, Inappropriate Weight Gain, Constipation and Irritable Bowel Syndrome,
Dry Skin, Allergies, Asthma, Itchiness, Hives, Unhealthy Nails, Acid Indigestion, Decreased Sex Drive
and Joy of Living (Anhedonia), Irregular Periods and Menstrual Cramps, Infertility, Decreased
Self-esteem, Decreased Wound Healing, Increased Skin Infections and Acne, Hemorrhoids,
Hypoglycemia, Low Blood Pressure, Food Cravings, Fatigue and Sleepiness After a Meal (Increased
Post-Prandial Response), Elevated Cholesterol Levels, Recurrent Infections, Carpal Tunnel Syndrome,
Lightheadedness, Dry Eyes and Blurred Vision, Psoriasis, Changes of Skin and Hair Pigmentation,
Flushing, Arthritis and Muscular Joint Aches, Increased Bruising, Musculoskeletal Sprains, Ringing in the
Ears (Tinnitus), Abnormal Throat and Swallowing Sensations, Canker Sores, Bad Breath, Inhibited
Sexual Development, Cold Hands and Feet and Raynaud’s Phenomena, Lack of Coordination, Food
Intolerances, Sweating Abnormalities, Increased Susceptibility to Substance Abuse.
Hypothyroidism means, simply, low functioning of the thyroid gland. This gland regulates the rate at
which we burn fuel - carbon - to provide us with the energy to move, to repair ourselves, and, in general,
to live, all mediated by enzymes.
There is an interaction between the thyroid, pituitary and hypothalmus that is complex, and while the
pituitary, in a real sense, is the “master” gland that makes all else function at the proper level, the thyroid
gland is the great “fuel-burning” regulator. The pituitary - via a substance titled TSH, or Thyroid
Stimulating Hormone - tells the thyroid gland when to produce thyroid which then regulates the body’s
metabolism.
According to Dr. Barnes, 40% of all Americans suffer from hypothyroidism.
The quantity of thyroid hormone produced from the thyroid gland, like many other measures in nature,
will follow a “normal distribution,” which means that some folks will produce more and some less, but,
like the wide variation in the measure of people’s heights, a certain percentage of those measured will be
tall, some short, and some in between. When the whole population of humans are considered, one can
develop a neat “frequency of occurrence” curve called “normal.”
Most medical statistics will quote an “average” of all the measurements as being where “norm” is, and
then arbitrary limits are set on both sides of this average which, if you fall within those limits, will also be
considered “acceptable.”
Most laboratory analysis of blood specimens and other biological tests are interpreted in the same
manner - if outside the arbitrary limits it’s called abnormal. If inside the arbitrary limits, it’s called normal.
People’s production of thyroid follows a gradient scale, from very low in thyroid production to very high
in thyroid production, depending on genetics and other factors.
It is that group of people just below the arbitrary limits of “normal” to “very low,” that Dr. Broda Barnes
classified as among the 40% who are deficient in thyroid.
He said that of all problems that can affect physical or mental health, none is more common than thyroid
gland deficiency, that none is more readily and inexpensively corrected, and that none is more often
untreated, even unsuspected, by the medical profession.
Assuming there is no damage to hormone-producing glands, numerous other factors can cause
hypothyroidism, among which, according to Lita Lee, Ph.D.,2 chemist, enzyme therapist, nutritional
consultant, lecturer, and author, are the use of fluoride, synthetic and genetically engineered hormones
(estrogen and others) found in meat, dairy products, poultry and eggs, and in birth control pills that block
the release of thyroid hormone from the gland, dietary excess of poly-unsaturated fats, such as soybean,
safflower and corn oils, excess iodine found in bread-dough conditioners and iodized salt, and endurance
exercises. E. Denis Wilson, M.D.3 has added stress, cortisone (and some other drugs), illness, and
fasting.
E. Denis Wilson, M.D. has expanded upon Dr. Barnes’ work, defining “Wilson’s Syndrome” also the
name of his book, Wilson’s Syndrome.3 As the functioning of enzymes—vital to life—depend on a
proper carbon/oxygen temperature of 98.40 to 98.60, and as the temperature is a function of proper
thyroid utilization, a deficiency in thyroid utilization produces enzyme dysfunction which, in turn, produces
about eighty different disease symptoms. Multiple Enzyme Deficiency is Wilson’s Syndrome.
With hypothyroidism, people will suffer from low energy and fatigue, chronic headaches, repeated
infections, menstrual disturbances of many kinds, memory disturbances, concentration difficulties,
depression, paranoid symptoms, unyielding skin problems or circulatory difficulties. Hypothyroidism can
be a major factor in arthritis, heart disease, lung cancer, emphysema, and it is responsible for emotional
and mental disturbances.
Many overactive children, for example, whose lives are being ruined by the use of psychiatric
mood-altering drugs, are simply suffering from lack of thyroid, or, as may also be the case, overuse of
sugar and/or food allergies gone unrecognized.
According to Gus J. Prosch, Jr., M.D., with thyroid deficiency “the tendency to gain weight (sometimes
called obesity), constipation, and higher cholesterol counts are very common, as well as cold hands and
feet, dry skin and swelling around the eyes on awakening each morning.”
Dr. E. Denis Wilson believes that all of the above symptoms and their corresponding diseases can have a single underlying causation, namely an underproduction of thyroid of the right kind, and is the most common and easily treated cause.3
Fortunately severe cases of hypothyroidism are rare, but it is the low or marginal cases that go
unaddressed, for the most part, by the medical profession.
Hypothyroidism, affecting as it does the basal metabolism - the rate at which people produce energy - is
it any wonder that it can also affect the so-called incurable disease called Rheumatoid Arthritis, and other
forms of arthritis?
“Forty percent of the American people - four of every ten children and adults - today are suffering
needlessly and many are dying for lack of an ingredient vital for health,” reported Dr. Barnes.
Dry skin 97 79
Coarse skin 97 70
Lethargy 91 85
Slow speech 91 56
Edema (swelling) eyelids 90 86
Sensation of cold 89 ‘ 95
Decreased sweating 89 68
Cold Skin 83 80
Thick tongue 82 60
Edema of face 79 95
Coarseness of hair 76 75
Heart enlargement 68 --
Pallor of skin 67 50
Impaired memory 66 65
Constipation 61 54
Gain weight 59 76
Loss of hair 57 41
Pallor of lips 57 50
Labored/difficult breathing 55 72
Swelling of feet 55 57
Hoarseness 52 74
Loss of appetite 45 40
Nervousness 35 51
Excessive menstruation 32 33
Deafness 30 40
Palpitations 31 23
Poor heart sounds 30 --
Pain over the heart 25 16
Poor vision 24 --
Changes in back of eye 20 --
Painful menstruation 18 --
Loss of weight 13 9
Emotional instability 11 --
Choking sensation 9 --
Fineness of hair 9 --
Cyanosis (skin bluish) 9 --
Difficulty in swallowing 3 --
Brittle nails -- 41
Depression -- 60
Muscle weakness -- 61
Muscle pain -- 36
Joint pain -- 29
Burning/tingling sensation -- 56
Heat intolerance -- 2
Slowing of mental activity -- 49
Slow movements -- 73
Some symptoms that would logically be ascribed to hyperthyroidism (overactive production of thyroid)
are illogically and surprisingly found in hypothyroidism. Barnes described his reasons for believing that
hypothyroidism is on the rise, and also the reasons why the medical problem has been so long
overlooked.
Briefly - but not the whole answer - part of the cause of hypothyroidism lies with the fact that people
tend to select mates of the same emotional activity - which means spouses that have similarly low
hypothyroid states. This selection, then, tends to create children that inherit the underactivity of thyroid
gland [or its proper utilization] from both parents.
Various clinical tests have been devised to determine thyroid functioning, which have included the thyroid
releasing hormone, T3 uptake, T4 index, T7, RT3, PBI (protein-bound iodine) and others. Dr. Wilson
explains that “these tests have their uses and are directed at assessing various levels of the thyroid system
function but are not extremely useful in predicting the onset and/or resolution of the symptoms of
decreased thyroid hormone system function.”
The first chemical test was PBI, a measure of Protein-Bound Iodine circulating in the blood.
Unfortunately iodine from iodized salt can combine with other proteins so that the PBI test mistakenly
measures these. Many physicians swore by the PBI test as an accurate index of thyroid function which it
was not.
Later, the thyroid hormone protein was broken up, yielding a simple iodine-containing material called
Thyroxin, or T4. Many physicians began using this test as the answer, which it was not. Triiodothyronine,
or T3 was discovered, an event that embarrassed those that insisted T4 was the test.
The test T3 could detect T3 but not T4; The test T4 could detect only T4, and the PBI test could detect
neither.
All the tests developed over the years seem to fail to do what counts, which is to determine the amount
of hormone stored in the cells of the body. That is, the hormone’s actual expression at the cell receptor
sites is most important. According to Dr. Wilson, “Tests can measure how much hormone is in the blood,
but can’t measure how much is in the cells, and certainly can’t measure what’s most important: the actual
amount of thyroid stimulation of the cell (except by body temperature.)”
As an example, among the 60 or so enzyme deficiency diseases caused by thyroid deficiency may be that
of overweight problems. Dr. Gus J. Prosch4 says “I’ve treated over 20,000 overweight patients . . . and
I’ve found over 90% produce normal thyroid (T3, T4), yet they usually display most or a great majority
of hypothyroid symptoms.
“They go to their regular doctors who tell them ‘obviously, your thyroid is low.’ Their physicians then
perform thyroid blood studies which come back ‘normal,’ and the patient is then told ‘well, it is not your
thyroid, so keep following your 1,000 calorie diet.’ “The patients follow the diet, and often gain weight anyway.
“What those physicians are overlooking is that people that tend to gain excess weight have normal
production of amounts of T3 and T4, therefore . . . they have the low thyroid symptoms even though they
have normal blood studies.”
Dr. Broda Barnes, who performed much research on thyroid functions, advocated a simple test that
anyone can use. We can make a determination for ourselves whether or not we are utilizing the correct
amount of thyroid, and, if not, alert our family physician to the possible need for thyroid hormone
supplements, to be given in a manner developed by E. Denis Wilson, M.D., to be described. Dr. Barnes’ simple test is described in two parts, one for males and one for females between menarchy
and menopause.
Males and Females Outside of Menarchy and Menopause
Temperature is taken each morning before arising out of bed. This is important, because, according to
Dr. Barnes, one needs to measure the lowest state of energy as a baseline, the “basal metabolism
temperature”.
What kind of thermometer to use is up to you but insure that you’ve calibrated it by comparing it against
several other thermometers, selecting the one that is consistent with other readings. You may wish to
measure any one of three places, oral, anal, or beneath the armpit (axillary fold).
The anal temperature will tend to be about 10 F higher than either the armpit or oral measure.
According to Broda Barnes, M.D., the armpit and oral measure should be the same, except when you
have a sickness or fever, in which case the oral temperature will no longer be accurate. However, E.
Denis Wilson, M.D. feels that the armpit temperature is generally lower by 0.8 degrees, and you might
want to check this for yourself before deciding which one to use.
The temperature is to be taken each morning before moving from bed (or other physical exertions), and
recorded by date. Over a period of a week or so, you will begin to notice some ups and downs, but not
much. The criteria (under armpit) recommended by Barnes is this: (1) If the temperature is consistently
between 97.8 and 98.2 then you are “normal” i.e., don’t worry over your state of thyroid; (2) If above
98.2, then you are hyperactive (too much thyroid being produced); (3) If below 97.8 you are
hypothyroid (too little thyroid being produced.)
Lita Lee, Ph.D., says that “Another way to tell [if you are hypothyroid] is to measure your resting pulse.
The healthy resting pulse should be about 85 beats per minute. The national average is around 72. If your pulse is less than 80, you may have an underactive thyroid. Babies have a pulse greater than 100 until around the age of eight years when the pulse slows down to around 85.”2
For Women Between Menarchy and Menopause
Women in this category often keep track of the onset of ovulation, and so taking the temperature, as
described above, is perfectly OK until the onset of ovulation. On the onset of ovulation, the temperature
drops about 0.20 F, then the temperature increases about 0.60 F for several days, then decreases to the
base line about the 2nd or 3rd day after the flow starts, after which the rules as previously described can
be followed.
Pregnant Woman
Pregnant woman will have a higher temperature because of progesterone production. Best to work
directly with your obstetrician in determining thyroid deficiency by this method.
According to James A. Carlson, D.O.4, Knoxville, TN, “Prior to menarchy after menopause there is not
enough hormone activity to stimulate a period and it is not going to influence the temperature significantly.
Woman following menarche up to menopause could have some significant reasons using the “10-4
model” -- 10-4 model means that the period is 4 days, then 10 days until ovulation, 4 days of ovulation
and then 10 days before menses again.
“There are changes that occur and are the basis for [some] Catholics through the years using as one birth
control measure the pre-ovulatory daily temperatures to determine the onset of ovulation. There is about
a 0.2 degree decrease in temperature with the onset of ovulation. Following the ovulation there would be
a slight increase in temperatures and if the patient was a significant hypothyroid candidate she is also a
potential chronic candidiasis candidate.
“The candida milieu is best or optimal just before the menses begins. The toxin given off by the candidia
makes the heart work more (an ionotrophic effect) as well as a pseudo-estrogen effect.
“The pseudo estrogen effect does effect the free estrogen levels by occupying receptor sites. The
ionotrophic effect increases the heart rate thereby increasing the workload and increasing the
temperatures sometimes significantly and is the basis for the premenstrual symptoms (PMS) frequently
seen.
“After the menses begins, the progesterone effect rapidly decreases and the estrogen effect rapidly
increases and there is a period then of greatest temperature stability. . . . if she is extremely healthy and
not a hypothyroid candidate and not a chronic candidia candidate and does not have a low grade
infection, . . . her temperatures would be fairly stable. These are not the type of people that we
commonly see in our [medical] practice and therefore the baseline for the patient to help us monitor her
progress should best be taken throughout the month so that [we] would afford a better look at her from a
standpoint of the potential candidia patient.
“It would give us the data to help the patient if they were using this as a birth control method and would
help establish the basal temperature record on the most optimal days. Unfortunately, it is difficult to wait
a month before instituting therapy. [The] patient should see if the side effects from hypothyroid decreases
the immune system, [and/or] a decreased digestive system with malabsorption particularly of the B
vitamins and calcium, etc. . . . The variation in the temperature gradient should improve throughout the
month as the patient establishes a normal condition (euthyroid) state.
“If a true normal (euthyroid) state, the days of ovulation should be the only true days of temperature gradient baring infections be they bacterial or fungal or of states of anemia which would have a decrease in oxygen carrying capacity and would decrease the temperature in its own right.”5
According to Dr. Prosch,4 for accuracy,”It is important to use a regular oral thermometer, not a digital,
and to be sure to shake the thermometer down the night before to below 940.” He also believes that,
with the Barnes method, one should use the axillary fold (armpit), leaving the thermometer there 10
minutes, read it, and then arise in the morning.
Although Dr. Prosch may use the Broda Barnes treatment method, treatment procedure of first choice is
that of Dr. E. Denis Wilson’s method. It is much simpler and may result in more permanency for the
patient. Dr. Prosch describes his clinical procedure for those who wish to use the Broda Barnes’ method.
Dr. Prosch has the patient read the axillary fold (armpit) temperature for three or four days, having them
measure their temperature before coming to his office on their first visit when possible. When he sees the
patient the first time and has not been able to get their temperature measured prior to this visit, he asks
the patient to take their temperature in the above manner between their first and second visits.
Based on succeeding measurements for a period of three weeks, a small amount of natural thyroid is
administered, and measurements continued daily. This process is repeated until the armpit measurement
has reached normality, which amount now being taken defines the amount of hormone replacement
required to fuel the hypothyroid body, bringing it back to an adequate temperature to ensure proper
enzyme functioning.
According to John Baron, D.O.,1 the natural porcine (pig) thyroid as furnished by Armour
Pharmaceuticals is preferable to the synthetic levothyroid. Thyroid tablets as furnished by Armour
provide 38 mcg T4 (thyroxine sodium: levothyroxine) and 9 mcg T3 (thyronine sodium) per grain of
thyroid.
Dr. Baron also relates that alligator thyroid is equivalent to 50 porcine thyroids or 100 cows.
Dr. Prosch1 reports that he has found that synthetic levothyroxine cannot be tolerated by some patients.
A Catalog of physician approved sources may be obtained from The Arthritis Fund/The
Rheumatoid Disease Foundation 5106 Old Harding Road, Franklin, TN 37064. Please send a
tax-exempt donation and self-addressed, stamped, legal-sized envelope.
One word of caution: though Barnes emphasizes temperature as an accurate index to the functioning of
the thyroid gland, the end objective is not “normal” temperature, but rather a decrease or total absence of
disease state and disease symptoms. By the same reasoning, it is not that we treat the thyroid, but that
we seek improvement of the body’s metabolic efficiency.
This process takes time and patience. Bodily or system changes may take as long as three months of the
euthyroid (normal) condition or perhaps as long as twelve before showing major change.
Further caution: if it is determined that you are hypothyroid, then thyroid supplements may be required
for the remainder of your life by this method.
However, there is great hope for your body reversing a hypothyroid state so that you will not be
dependent upon daily intake of thyroid. E. Denis Wilson, M.D. has developed a unique method. His
“Wilson’s Syndrome,” or “Multiple Enzyme Dysfunction,” treatment program will be described.
It is considered dangerous to supplement with hormones of any kind, without close medical supervision,
and without knowing what is going on.
As in the case of any end product hormone, such as cortisone or testosterone, or thyroid, one can shut
down one’s own hormonal manufacture with drastic results, including atrophy of the producing gland and
life-threatening dependency on artificial or substitute hormones thereafter.
The Barnes’ technique relies on an accurate test - temperature - to determine whether or not you are
taking too much or too little thyroid. When a determination is finally made as to exactly how much thyroid
to take to reach euthyroid (normality), then the amount your body produces plus the supplement is the
total required.
In other words, it is important that the amount of thyroid supplemented does not replace what your body
already produces, but merely adds to, and supplements, to achieve an amount exactly correct for you.
The principal effect of all this is to increase the metabolic rate of body tissues: Enhances oxygen
consumption of most tissues of the body, increases the basal metabolic rate, and the metabolism of
carbohydrates, lipids, and proteins; exerts a most profound influence on every organ system in the body,
including the development of the central nervous system.
The basal temperature test is not a perfect test for thyroid function as there are conditions other than
hypothyroidism that may produce low readings such as pituitary deficiency, or adrenal gland deficiency
or starvation, and some thyroid deficiency can occur for other reasons.
O.P. Dimball, M.D., over a ten-year study (five years of which were carried out at the famed Cleveland
Clinic and five years in private practice) concluded that hypothyroidism cannot exist without some
myxedema, a condition marked by dropsy-like swelling, especially of the face and hands, smallness of
the thyroid gland, slowing of the pulse rate, dryness and wrinkling of the skin, falling of the hair, dulling of
mental activity, sluggishness of movement and retardation of the rate of basal metabolism (called also
Gull’s disease). A trained physician can probably spot these characteristics and correlate symptoms with
your temperature records.
Compensation for Hypothyroidism
While fatigue is an obvious symptom of suffering from hypothyroidism it is generally not recognized that
people compensate for this problem. They will pour their energy reserves into some activity that is
necessary for survival or which is pleasurable, and then slump thereafter. This explains academic and
athletic successes in the absence of normal thyroid functioning. A student or adult will unconsciously
meter effort where important, and then default at activities that are unimportant, or go home to rest
through the fatigue slump.
The opposite from fatigue can also occur especially in school children who exhibit tremendous activity, to
the point where they disturb others. It is the author’s personal opinion that rather than harm the child for
life by use of Ritalin or related mind-destroying drugs, the school system would be better advised to (1)
check for hypothyroidism, (2) check for allergies, (3) review dietary factors, (4) check for candidiasis
and/or (5) seek counseling via Church of Scientology techniques that apply L. Ron Hubbard’s processes
for self-confrontation of buried emotion and pain until such is discharged and no longer the cause of the
hyperactivity. These approaches could require involvement of the whole family, not just the child’s
participation.
Contrary to reported results of some poorly designed studies on the subject, Gus J. Prosch, Jr., M.D.1
reports that, “I’ve learned it is the excess consumption of sugar, sweets and processed foods, plus the
imbalance of essential fatty acids that cause the problem of hyperactivity, by resulting in an imbalance of
good and bad prostaglandins which are the major factors in hyperactivity.”
A subnormal temperature which is characteristic of low thyroid function can contribute to anemia by its
effects on blood cell production in the bone marrow, according to Barnes. Many of his patients who had
been diagnosed as anemic lost the condition on correcting the thyroid deficiency.
When fatigue is associated with hypothyroid states, migraine headaches may also disappear on
correction.
Are you always complaining of the cold? Especially when others are comfortable? Possibly
hypothyroidism.
Physicians have long been aware that total removal of the thyroid gland affects behavior and emotional
states. They haven’t always understood what a small deficiency might cause. A marked deficiency
occurring at an early age may lead to growth failure and dwarfism; yet a minor deficiency also may allow
growth to proceed at a normal rate and then to accelerate, producing a seven-footer.
There are similar paradoxical effects on energy and behavior.
Mild degrees of hypothyroidism can set the stage so that brief loss of adequate sleep or a brief period of
undue stress may do what it would not otherwise be capable of doing in anyone with normal thyroid
function, according to Dr. Barnes. It adds just enough to the lowering of resistance to permit infection,
and also such diseases as Rheumatoid Arthritis and candidiasis. Barnes lists ear, rheumatic heart
infections, pneumonias, influenzas, bladder, bone infections (osteomyelitis), colds, and many other
problems of infection.
Skin problems and/or infections loom large, with thyroid deficiencies, including boils, carbuncles, acne,
impetigo (a skin condition), cellulitis, erysipelas (a contagious disease caused by staphylococcus),
“Winter Itch,” eczema, ichthyosis, Systemic Lupus Erythematosus and psoriasis.
Lupus is one of the connective tissue diseases which include Rheumatoid Arthritis, Progressive Systemic
Sclerosis, polymyositis, amyloiditis, necrotizing arteritis, and rheumatic fever. All of these diseases are
associated with deposition of mucopolysaccharides in the connective tissues. As Barnes says,
“Considering the fact that thyroid deficiency leads to deposition of mucopolysaccharides in connective
tissue and other tissue, it is not surprising, or shouldn’t be, that thyroid therapy can be beneficial.”
Increased blood circulation through the skin is one benefit of thyroid correction.
Barnes has also associated thyroid deficiencies with menstrual irregularities, miscarriages, infertility,
including sexual depression, and correction of thyroid deficiencies with avoidance of dilatation and
curettage (D & C) and hysterectomies.
Hypertension and heart problems have been improved via use of thyroid supplements.
There is much damage to various tissues and systems when one has Rheumatoid Disease, and anything
that can help repair that damage is important for arthritics to look over and perhaps try, if applicable.
Since proper thyroid functioning determines the rate at which one can repair damage, it is most important
for arthritics to consider the possibility of thyroid supplementation if hypothyroid.
Barnes says, “Actually, many arthritic patients I have seen not only have a history of infections, easy
fatigability, and other indications of possible low thyroid function but for many years they have
experienced minor joint aches and pains for which they did not seek medical help until their discomfort
became acute.”
Barnes also points out that use of cortisone depresses thyroid function.
In Gouty Arthritis, the uric acid level of the blood is often high in hypothyroid patients, and this level
usually falls when thyroid therapy is instituted.
Barnes also cites cases of diabetes and hypoglycemia, obesity lung cancer and emphysema that are
improved through thyroid therapy.
To summarize Broda Barnes’ view:
1. Everyone should test for thyroid deficiencies.
2. Clinical laboratory tests are unnecessary, and may give faulty readings, whereas use of an ordinary
thermometer before rising in the morning will develop the trend data required.
3. If the trend temperature readings (armpit temperatures) are below 97.80 F, suspect hypothyroidism,
and visit your family doctor for corrective action. If above 98.20 F, suspect hyperthyroidism - and visit
your doctor.
4. Do not expect changes with thyroid therapy, once begun, short of months of treatment.
5. Almost every human condition or disease can be improved by thyroid supplements, if the condition is
hypothyroid.
6. It is never too late to supplement and bring about relief.
7. Thyroid supplement, if indicated, is a low-cost, effective treatment, and, following the Barnes’ method,
often must be done for the remainder of one’s life.
There is hope, however, in changing a dIsfunctioning thyroid imbalance, which will be described in
“Wilson’s Syndrome: Multiple Enzyme Dysfunction,” which follows.
Insert Sidebar:
E. Denis Wilson, M.D. says, “The T3 therapy I recommend can often rest a person’s metabolism so that
the patient can maintain his own temperature on his own even after the therapy is discontinued. But
should the patient be subjected to another severe stress, as in one of Dr. Prosch’s patients [to be
described], the patient can relapse. So people may get confused by words like ‘permanently,’ ‘solved’,
‘cured,’ etc.
“Treatment might fix this episode of Wilson’s syndrome, but T3 therapy cannot remove a person’s tendencies toward having this sort of problem, and obviously can’t make them immune from further insult, or stress. Hopefully, the person can remain improved forever, but there is always a chance that the circumstances that caused a patient to have this problem in the first place, can cause the person to have the problem again and relapse.”6
As an arthritic, consider the Broada Barnes’ test. It can’t harm you, and it may help tremendously.
Further, if you are not hypothyroid, you will have spent no money learning the fact of no deficiency, but
rather a small amount of time each morning for several mornings before arising, taking your temperature.
What could be fairer? Or cheaper?
Wilson’s Syndrome: Multiple Enzyme Dysfunction
Cutting the Gordian Knot of Hypothyroidism3
Gus J. Prosch, Jr., M.D. provides about 100,000 people with his experiences through WDJC radio
station (93.7 FM) out of Birmingham, Alabama. His fifteen minute radio program has call-ins two days a
week, and the other five days he discusses, diseases, herbs, treatments—indeed, a wide variety of
topics.
Many doctors, including Gus J. Prosch, Jr., M.D. have found that the E. Denis Wilson, M.D. method is
more reliable than the Broda Barnes, M.D. method and leads directly to a technique that can result in
improvement that can persist even after the treatment has been discontinued—a sort of “resetting
phenomenon.”
In advising his patients and his radio audience, Dr. Prosch says that “At least twenty percent of American people have Wilson’s syndrome, the effects of thyroid dysfunction.”173
The good news is really good, because until Dr. Wilson’s work was known, one most likely would have
had to take thyroid supplements for the remainder of their lives, as recognized in the Dr. Broda Barnes
method. Since Dr. Wilson’s method has become known, it’s possible to “reset” proper thyroid functioning
unless stress or other factors again cause a dysfunction.
Dr. Wilson has done a brilliant piece of detective work, standing on the pioneer accomplishments of Dr. Broda Barnes, and a summary of his findings and methodology follow:
According to Dr. Wilson, the reason why thyroid dysfunction underlies almost all disease states is
because of the effect of that apparent deficiency on enzymes.
Enzymes are the catalysts of our carbon/oxygen engine, without which life could not exist.
But, enzymes are temperature dependent. They behave slow or fast depending upon the temperatures
found in each individual cell. In fact, the only single direct measure of the quantity of utilizable thyroid that
is known is the body’s temperature, so closely are thyroid/temperature/enzymes related.
How well an enzyme functions depends upon its shape, and the shape of an enzyme depends upon its temperature. In other words, an enzyme’s shape can change according to temperature which then determines its effectiveness in biochemical behavior. Dr. Wilson writes that enzymes are “like a twisted telephone receiver cord that will untwist when you answer the phone and pull the cord tight, and then twist back into its previous shape when you put it back on the hook.”3
When enzymes are too hot, they get too loose; when they’re too cold, they get too tight. There is an
optimum geometry for each enzyme, which, of course, is dependent upon an optimum temperature. In
general, as with most chemical reactions, the colder the temperature, the slower the action, and vice
versa.
Ninety-eight point six degrees fahrenheit (oral temperature) is the magic number, because it’s the number
or temperature at which enzymes operate at their peak efficiency, which, in turn, permits each cell, each
tissue, each organ and each system to operate at peak efficiency.
When body (oral) temperature is persistently low—below 98.40 -98.60 fahrenheit—many enzymes
lose their ability to function properly, and a “dysfunction” of cells, tissues, organs and bodily systems
takes place. Dr. Wilson has named this condition “Multiple Enzyme Dysfunction” (MED) syndrome.
E. Denis Wilson, M.D. writes: “Of all chronic medical problems, I believe that Wilson’s Syndrome is the most common and has the greatest impact and is the easiest to address and is the most likely to be remedied and is the most rapidly responding and has the most inherent or non-foreign of treatments. For these reasons Wilson’s Syndrome should be the first of impairments to be considered in the treatment of patients rather than the last.”3
Multiple Enzyme Dysfunction also describes the basic source of so many apparently unrelated disease states as already described. And, of course, Multiple Enzyme Dysfunction is caused, in its turn, by low temperature, which, in turn, is caused by a deficiency in thyroid, the carbon/oxygen engine stoker
Thyroid Linkage to Life
The Metabolism—How It Works
At the brain certain hormones (thyrotropin releasing hormone, TRH) are released and travel to the
pituitary gland causing the production of another hormone called thyroid stimulating hormone (TSH).
Thyroid stimulating hormone (TSH) enters into our blood stream, travels to the thyroid gland at the base
of our neck and stimulates a substance called thyroxine, also known as T4.
Thyroxine (T4) is converted to liothyronine (T3) by an enzyme, 5’-deiodinase, which is found in most of
our bodily tissues.
Thyroxin (T4) is converted to liothyronine (T3) outside of the thyroid gland inside the body’s tissues. This
fact is one of the major keys to understanding why measures of glandular thyroid are virtually
meaningless, except where there is a problem with the gland itself.
Liothyronine (T3) has its action at the nuclear membrane receptors of the cells of the body. A cascade of
chemical reactions within each cell occurs which affects the metabolic rate of each cell. Metabolism (or
metabolic rate) is the energy expended to maintain respiration, circulation, peristalsis, muscle tone, body
temperature, glandular activity, and other functions.
The metabolic rate of the cells determines the metabolic rate of the body.
The metabolic rate of the body, together with the amount of surface area of each person’s body,
environmental conditions, and other factors determines the body’s temperature.
The body’s temperature determines the function and activity of the enzymes which are responsible for
most of the important chemical reactions in the body.
These chemical actions are the basis to the body’s functions.
All of the above is a description of why thyroid hormone is so vitally important for proper bodily
functioning, and for achieving wellness with any disease state, not just arthritis.
Without thyroid hormone, the body would not live.
Where Thyroid Goes Awry!
The Structures of T4, RT3, and T3
The second key to understanding Multiple Enzyme Dysfunction lies with a fact long overlooked in
medical circles. Dr. Wilson reports that thyroxin (T4) is factually converted to liothyronine (T3) in two
ways. As one of these two ways is a reverse image of the other, one is called liothyronine (T3) and the
other is called reverse liothyronine (RT3). Chemically, they look similar, but their biochemical activity is
totally different. Thyroxin (T4) and liothyronine (T3) have the capacity to stimulate the cells’ thyroid
hormone receptors, with thyroxin (T4) having a small effect on the cells—liothyronine (T3) having four
times more effect than thyroxin (T4), and reverse liothyronine (RT3) having no effect whatsoever.
Thyroxin (T4), then, can be converted into two different hormones, one being liothyronine (T3) and the
other being reverse liothyronine (RT3). These two hormones are distinct and different metabolites of
thyroxin (T4). They can both occupy the same sites on the cells’ thyroid hormone receptors, thereby
competing for position. Since reverse liothyronine (RT3) has no biochemical activity it cannot and does
not fuel the metabolism of the cell. If a large number of thyroid hormone receptors are filled with reverse
liothyronine (RT3), then cellular functions will decrease also causing the whole body to decrease in its
ability to function. This condition, then, causes a lowered body temperature, decrease in enzyme activity,
decrease in bodily functions, and Wilson’s Multiple Enzyme Dysfunction syndrome, displaying itself with
many clinical and subclinical disease states.
The Major Multiple Enzyme Deficiency Problem
Apparently nature has invented reverse liothyronine (RT3) as a means of slowing down our metabolisms,
a damping effect, so to speak, on the furnace stoker of our body.
Whenever we are placed under stress, fasting, illness, cortisol usage and some other medicines, our
bodies began to manufacture more of the reverse liothyronine (RT3). Dr. Prosch says that “The body slows down to give the body more energy to handle the stress or illness. “The body fools itself by changing the form of liothyronine (T3) to reverse liothyronine (RT3). Reverse
liothyronine (RT3) is a mirror image of liothyronine (T3), and, while it fits the cell’s thyroid membrane receptors, it has no metabolic activity.”7
When conditions are reversed, and there is less stress, no longer fasting, no illness, and we’ve quit using
the damaging medicines, our bodies should revert back to producing liothyronine (T3) instead of reverse
liothyronine (RT3). However, this does not happen with many of us. Consequently, we have established
a new homeostasis, a new balance between thyroxin (T4), liothyronine (T3), and reverse liothyronine
(RT3) -- and our metabolism is permanently lowered. The result—Wilson’s Multiple Enzyme
Dysfunction, and subsequent clinical and subclinical illnesses.
According to Dr. Prosch, the energy level drops, skin gets dry, hair gets brittle, the immune system weakens (it’s suppressed so that you start getting more allergies), fatigue increases—there’s sixty some different symptoms that Dr. Wilson has been able to identify, that people with Wilson’s Multiple Enzyme Dysfunction have one or, usually, a number of them.”7
The Solution to Over-saturation of Reverse Liothyronine (RT3)
Gus J. Prosch, Jr., M.D. now uses Wilson’s method as a first trial for solving Multiple Enzyme
Dysfunction, having found it superior to the Broda Barnes method.
The faulty homeostasis—faulty balance—can be reversed in most people who are otherwise organically
sound. It is done by supplying the individual with an appropriate amount of liothyronine (T3) in a
controlled program, until the supplemental liothyronine (T3) is no longer required.
By increasing the individual’s intake of liothyronine (T3), a greater proportion of liothyronine (T3)
compared to reverse liothyronine (RT3) is experienced by each cell, which causes more of the
liothyronine (T3) to take up position in the thyroid receptor membranes of the cells.
Thyroxin (T4 ) also decreases, which decreases production of reverse liothyronine (RT3).
Over a period of weeks, this procedure has a very good chance of reversing the process, improving
metabolism, increasing average daily temperature, optimizing enzyme functioning, decreasing Multiple
Enzyme Deficiencies, and decreasing clinical and subclinical conditions.
How Gus J. Prosch, Jr. M.D. Uses Liothyronine (T3) In Solving Wilson’s Syndrome
1. The patient establishes a baseline by accurately measuring oral temperature at three chosen intervals
during the day, say, 9:00 a.m., 1:00 p.m. and 5:00 p.m. These temperatures are recorded and averaged,
producing the average for the day.
2. When a sufficient number of averages have been recorded (usually two weeks), Dr. Prosch looks
them over, along with other factors, and determines whether or not his patient is a candidate for this
therapy.
3. If the patient is a candidate for this process, then s/he will be given a small amount of liothyronine (T3)
to take orally each day; and s/he will continue recording daily average temperature. Caution: the
liothyronine (T3) must be made up special by a compounding pharmacist, called “T3SR,” (T3
compounded with a sustain release agent to be taken every 12 hours) according to Dr. Prosch.
The liothyronine (T3) on the commercial market, packaged for non-compounding pharmacists, is not
used because a sustained release liothyronine (T3) must be used that is compounded in the correct
dosage increments. Dr. Prosch reports that “We have 13 different doses of the T3SR,” (7.5 mcg, 15.0
mcg, 22.5 mcg, 30.0 mcg, 37.5 mcg, 45.0 mcg, 52.5 mcg, 52.5 mcg, 60.0 mcg, 67.5 mcg, 75.0 mcg,
82.5 mcg, 90.0 mcg).
For a listing of physician approved compounding pharmacists, send a tax-exempt donation and a
self-addressed, stamped envelope to The Arthritis Fund/The Rheumatoid Disease Foundation, 7111A
Sweetgum Road, Fairview, TN 37062-9499, or contact the Wilson’s Syndrome Foundation,
(800)-621-7006.
4. The amount of liothyronine (T3) will be increased incrementally each day; and s/he will continue
recording average daily temperature.
5. If at any time from the first dosage forward, the patient’s symptoms resolve—temperature reaches
between 98.4 0 and 98.60 Fahrenheit—then the medication is maintained at that dosage for three
weeks, after which it is slowly discontinued by decreasing it incrementally, twice as slow as it was
increased incrementally, and s/he continues recording average daily temperature.
6. Although somewhat simplified, if all has worked well, the body’s thyroid homeostasis should be
reversed. One should be able to discontinue the medicine—the patient will be down to zero dosage by
then—and one’s Multiple Enzyme DIsfunctioning should be solved, along with a disappearance of any
one, or all, of some 60 different symptoms.
7. However, there are some who have to repeat through this total cycle several times before their body gets the message, inhibiting an over-production of reverse liothyronine (RT3), and increasing one’s own liothyronine (T3) production.173,175
Dr. Prosch has many people who have Wilson’s Multiple Enzyme Deficiency syndrome. He reports, “I’ve
good results in about 70 to 80 percent of the people that I can get to follow this program. It’s very
involved, and one must take care to do everything just right, and it’s a lot of work keeping track of
temperatures and dosages on time.
“One patient, the worst case I’ve seen, had to go through 14 cycles before her body reset.”
E. Denis Wilson, M.D., a dedicated pioneer, has given up his practice to spread the good news. He’s
picked out 200 doctors to teach, and Dr. Prosch was one of the first to learn.
How to Quickly and Easily Obtain Proper Treatment for Wilson’s Syndrome is available from the
Wilson’s Syndrome Foundation, PO Box 916206, Longwood, Florida 32791-6206; (800) 621-7006.
This patients’ guide helps you to find treatment in your area, discussing each of the three easy ways you
can obtain treatment for Wilson’s Syndrome.
Dr. E. Denis Wilson’s book, Wilson’s Syndrome: The Miracle of Feeling Well, is available through this
foundation for a tax-exempt donation of $25 or more.
Thyroid-Stimulating Foods
According to Lita Lee, Ph.D., fruits or fruit juices, which provide magnesium that works with thyroid to
moderate stress, also helps modulate blood sugar and calm down adrenal glands. Fruit juices can also
help to stimulate increased production of liothyronine (T3).
Raymond F. Peat, Ph.D., recommends a salad recipe consisting of grated carrots, vinegar, coconut oil
and salt. Fatty acids in the coconut oil are thyroid-stimulating. Carrot fiber tones the bowel and binds
toxins.
Salt mobilizes glucose and calms adrenal glands, according to Peat.
Coconut oil has several thyroid-promoting effects. It contains butyric acid which helps thyroid hormone move into the brain [ liothyronine (T3 ) uptake into glial cells]. It opposes anti-thyroid unsaturated oils. It contains short and medium chain fatty acids which help modulate blood sugar, is anti-allergic, and protects mitochondria against stress injuries.195
Of course, while useful, unless reverse liothyronine (RT3) has been minimized at the cellular level, these
nutritional assists will be of lessened value.
The Case of Shirley Holmstead
Shirley Holmstead had suffered from Chronic Fatigue Syndrome—a condition very similar to
Fibromyalgia—and other complaints for many years. After consulting with Dr. Prosch, and being placed
on Wilson’s Multiple Enzyme Deficiency syndrome treatment, Shirley found that she had to cycle through
the treatment several times before her body became normalized.
After normalization, Shirley did very well for two years, when she again encountered a very stressful situation. Her body flopped a second time, producing an over-abundance of reverse liothyronine (RT3) and she immediately went to Dr. Prosch to resume the treatment that would normalize her metabolism.8
Once thyroid utilization has been normalized, determination of enzyme deficiencies can be important. Lita
Lee, Ph.D.9 uses a 24-hour urinanalysis developed by Howard Loomis, D.C. which will determine the
following:
Acidity/alkalinity (pH), specific gravity, too little or too much volume, chloride, calcium, food intolerances
and nutritional deficiencies, indican (determines need for colon cleansing and other conditions), sediment
(determines need for multiple enzymes), and abnormal solutes.
Anthony J. Cichoke, D.C.,10 writing in Townsend Letter for Doctors & Patients, says that “It is
generally agreed that the disease [Rheumatoid Arthritis] could be lessened by the early detection,
degradation, and disposal of immune complexes (which have penetrated the joint cartilage from the
synovial fluid). . . . Immune complexes can act as antigens and provoke the plasma cells to synthesize
antibodies resulting in the formation of gamma globulins (contained in these immune complexes). . . .
some physicians and researchers feel that enzyme mixtures are superior to all anti-rheumatic drugs
because of their comparable efficacy but minimal side-effects. . . . Fibrin is at least indirectly associated
with the cause of a rheumatic disease. But, proteolytic enzymes degrade fibrin (built up by the
tissue-immobilized immune complexes). The result is that the protective cloak is removed from the
immune complexes. Above all, this fibrin degradation ensures that the immune complexes are detected,
degraded, dissolved, and subsequently eliminated. In this way, the mechanisms leading to the
inflammatory process are more rapidly stopped and potential for further deterioration is reduced.”
References
1. Anthony di Fabio, The Master Regulator, The Arthritis Fund/The Rheumatoid Disease Foundation,
5106 Old Harding Road, Franklin, TN, 37064, 1989; also Broda O. Barnes, M.D., Lawrence Galton,
Hypothyroidism: The Unsuspected Illness, Harper & Row, New York, 1976.
2. Lita Lee, Ph.D., “Hypthyroidism, A. Modern Epidemic,” reprint from Earthletter, Spring 1994, 2852
Willamette St., #397, Eugene, Oregon.
3. E. Denis Wilson, M.D., Wilson’s Syndrome, Cornerstone Publishing Company, 4524 Curry Road,
Suite 211, Orlando, Florida 32812, 1991.
4. Anthony di Fabio, Rheumatoid Diseases Cured at Last, The Arthritis Fund/The Rheumatoid Disease
Foundation, 5106 Old Harding Road, Franklin, TN, 37064, 1985.
5. Personal correspondence from James Carlson, D.O.
6. Personal communication with E. Denis Wilson, M.D.
7. Personal interview Gus J. Prosch, Jr., M.D. November 21, 1995.
8. Personal interview with Shirley Holmstead (fictious name), November 21, 1995.
9. Lita Lee, Ph.D., “The 24-Hour Urinalysis According to Loomis,” reprinted from Earthletter, Volume
2, Summer 1994, 2852 Willamette St., #397, Eugene, Oregon
10. Anthony J. Cichoke, D.C., “Chiropractic & Nutrition,” Townsend Letter for Doctors & Patients,
911 Tyler St., Port Townsend, WA 98368-6541, January 1996, p. 32.
More on Thyroid
Our last two newsletters featured Drs. Broda Barnes’ and E. Denis Wilson’s views on the importance of
thyroid functioning for sixty diseases, among which can be found some forms of arthritis.
Jonathan V. Wright, M.D., who was a featured practicing physician (Kent, Washington) in our June
1993 newsletter, is a consultant for Meridian Valley Clinical Laboratory and also president of the
National Health Federation. “Thanks for the ‘thyroid series’ you’ve been publishing,” he recently wrote to
us. “For years, I’ve been bugging Meridian Labs to put out an accurate, inexpensive thyroid panel
including Thyroid Stimulating Hormone (TSH), Thyroxin (T4), Liothyronine (T3), and Reverse
Liothyronine (RT3). They’ve finally done it, and for [only] $85!”
Insufficient thyroid utilization may not be reflected by normal thyroid glandular tests. One of the problems
that was faced by those who use the E. Denis Wilson, M.D. program to help patients in an attempt to
reverse their thyroid utilization (hypothyroidism) was the lack of any definitive Reverse Liothyronine
(RT3) blood test. And so an extensive series of accurate temperature measurements was required by the
patient for implementation of Wilson’s recommended treatment.
This new laboratory test when used properly may prove to be a major clinical indicator, as the amount of
Reverse Liothyronine (RT3) produced by our cells determines our temperature (metabolism), and our
bodily temperature determines how well tens of thousands of essential enzymes function, and those
enzymes determine the health of our cells, organs, systems, and overall bodily processes.
Before asking your doctor to obtain this new test, they should read Wilson’s Syndrome. The book is
available through this foundation for a tax-exempt donation of $25.
Meridian Valley Clinical Laboratory requests that your doctor “draw blood in a Serum Separator Tube
(SST) and allow it to clot for 20 minutes, then centrifuge it for at least 10 minutes. The serum should then
be poured into a transfer tube. A minimum of 3.0 ml of serum is required. It should be frozen and
shipped via Overnight Mail in a prepaid kit. Monday through Thursday delivery only,” they advise.
Meridian Valley Clinical Laboratory can be contacted at 515 W. Harrison Street, Ste. #9, Kent, Wa
98032; (800) 234-6825; fax (206) 859-1135.
01 MACROMOLECULES
1. All the items listed on this slide are required by the FDA (Food & Drug Administration) to be included on any packaged food item. Excluding water, fats, carbohydrates, and protein make up virtually all of any food product (notable exceptions include alcoholic beverages where alcohol is a major caloric contributor). In addition, many manufacturers will voluntarily add the amount of mono-unsaturated and poly-unsaturated fat. Dietary fibers, usually from the carbohydrate, cellulose, are not included in the total carbohydrate. This is because we cannot digest cellulose and therefore do not absorb the glucose from which it is made into our blood. Cellulose contributes no calories to your food. The difference between the total carbohydrate value and the sugar value is typically due to the presence of starch, a digestible glucose polymer.
2. (Not shown in handout) An example two nutrition labels from soup cans. The top label is for Cream of Chicken Soup; the lower is for Split Pea Soup. Note that there are about 9 calories per gram of fat and only about 4 calories per gram of carbohydrate or protein. You should be able to estimate the caloric content of food from food labels.
3. Carbohydrate, fat, and protein are the major components of food because all food is derived from living organisms, and these macromolecules are the major components of all living tissue. Lipids include more than simply fat (triglycerides), but most of the lipid content in our food is from fat (or oils). Not indicated on food labels because it is a minor contributor to the total food content are nucleic acids. These molecules are nonetheless critical to the storage and expression of genetic information in a cell. The dry weight of a cell (which is ~ 90% water) contains 50% protein, 10% lipid, 15% carbohydrate, and 15% nucleic acid. Most plant & animal tissue suitable for food will have much higher protein, lipid and carbohydrate content. For example the cells in the tuber part of a potato are designed to store starch for later plant growth and will have much more carbohydrate than an average cell. Also the muscle tissue that makes up a hamburger will be higher in protein etc. You should know the types of macromolecules. You should be able to classify specific molecules we discuss later into one of these four categories.
4. Carbohydrates, fat, and proteins contained in living organisms have a variety of functions, but three functional categories show up time and again. 1) They are the materials used to make the physical structures of cells. 2) They can contribute to the cells energy needs. 3) They also contribute to the chemical communication required to maintain multi-cellular organisms. For example lipids perform a structural role as the primary component of cell membranes. Triglycerides are the principal energy reserve molecule used in plants (vegetable oil) and animals (fat). Many hormones, including the steroid hormones, are lipids that fulfill a communication role in the body. You should know the function for each type of molecule we introduce in this class as well as its macromolecular classification.
5. The carbohydrate cellulose, a polymer of glucose, is the major component of plant cell walls, and thus is probably the most abundant polymer on earth. Animal cells do not have cell walls, and thus the role of carbohydrates in animal cell structure is much less prominent. Starch (in plants) and glycogen (in animals) are both glucose polymers like cellulose, but serve as glucose reserves to meet the organism’s energy demands. Carbohydrates, usually as components of glycoproteins or glycolipids in cell membranes, also perform important signal recognition functions.
6. Protein is the structural material used heavily in the architecture of both the intracellular cytoskeleton and extracellular matrices of an organism’s cells. Examples include the collagen that is a major component of bone, skin, and connective tissue. Dietary protein not required for making new protein, and during fasting, any body protein can be oxidized to provide about the same amount of energy as carbohydrate. In principle you could have a carbohydrate free diet (e.g. Atkins diet), but not a protein free diet. This is because protein is the only useful source of the element N in our diet. Many proteins perform communication roles in organisms. Examples include signal molecules like the hormone, insulin as well as the class of proteins responsible for receiving signals from hormones, and neurotransmitters, called receptors.
7. Living tissue is largely composed of organic molecules, which includes all the polymers listed above. More than 95% of the atoms in these compounds will be H, C, N, or O, listed in red on the periodic table in this slide. The elements shown in blue are major components of living tissue, but not nearly abundant. Many elements not listed are important in trace amounts. You should know the typical # of bonds formed by H (1), C (4), N (3), and O (2) in most organic compounds (including macromolecules). This will be useful in understanding molecular formulas.
8. Organic chemicals are categorized by functional groups. These are molecules that share a certain combination of elements bonded in the same way. For example, proteins are polymers of amino acids. Amino acids are so named because they contain both an amine functional group (-NH2), and a carboxylic acid functional group, or simply a carboxyl group (-COOH). You should learn to recognize the functional groups found in each of the macromolecules I will introduce.
9. In a generic amino acid, both the amino and carboxyl groups are attached to the same carbon. This carbon is referred to as the α-carbon (because it is the first carbon after the carboxyl group). There are 20 (genetically determined) amino acids. They differ only in the ‘R’ group attached to the α-carbon. This is referred to as the side chain. For example in alanine (Ala), the side chain is a methyl group (-CH3).
10. Amino acid side chains are characterized according to their polarity. Nonpolar molecules have almost entirely C & H atoms (hydrocarbons). Polar side chains that cannot ionize typically have a hydroxyl group or an amide functional group. Polar side chains that can form ions are separately classified as acidic (carboxyl groups) or basic (amine) side chains. Given the name and structure of an amino acid side chain, you should be able to supply the correct classification based on these rules.
11. (See amino acid handout) Here are examples of some nonpolar goups. Note that an aromatic ring alone is nonpolar, as it contains only C & H. Sulfur, S, is nonpolar in a thio ether (C-S-C) connection, as in methionine.
12. (See amino acid handout) Here are examples of polar (but not ionizable) side chains. Note that S in a sulfhydryl group (-SH) is polar, as in cysteine, Cys.
13. An acid is a proton donor. Any molecule containing a carboxyl group is an acid, and can react to give up its extra proton (H+). Thus there is an equilibrium mixture of the unionized (acid form) of the carboxyl group, and the (–) charged (basic form) of the carboxyl group. This is often referred to as the carboxylate ion. The amount of each of these forms depends on the pH of the solution. The pH at which the amounts of each form are equal is called the pK of that particular group. Given the pH and pK for a group, and knowing the principles shown on this slide, you should be able to indicate which form of the carboxyl group predominates. For example the side chain of aspartic acid has a pK of 3.9. In blood (pH = 7.4) the pH > pK and thus almost all aspartic acid side chains are in the basic or (–) charged form. The aspartic acid ion is referred to as aspartate.
14. The same principles apply to amine groups that can ionize to form a (+) charged (acid form) ammonium group.
15. This slide illustrates both the acid and basic forms of aspartic acid and lysine. Apartic acid (or aspartate in its – charged form), as its name implies, is an acidic amino acid. Lysine is a basic amino acid. You should be able to recognize carboxylic acids and amines and understand their acidic and basic properties. Given a pK and a pH for an acidic or basic group, you should know its predominant form.
02 TRANSGENIC SOY BEANS – PROTEIN SEQUENCE
1. Review of the general structure of an amino acids
2. One property of amines and carboxylic acids is that they will react with each other in a dehydration reaction (removing water). The –OH coming from the carboxylic acid, while the –H comes from the amine. This leaves the Carboxyl carbon with only 3 bonds (it wants 4), and the amine nitrogen with only 2 bonds (it wants 3), so they join together ....
3. …. to form an amide functional group, which is characterized by a carbonyl group (C=O), sometimes referred to as a keto group, bonded directly to an N-H.
4. If two amino acids are connected by an amide bond in a dehydration reaction …..
5. …. the resulting molecule is called a dipeptide, and the amide bond connecting the two amino acids is referred to as a peptide bond. Note that the word peptide can be either an adjective (describing a type of bond), or a noun (defining a certain type of molecule). And you thought scientists didn’t remember any basic English!
6. Note that the possibility exists to continue to add another amino acid to a dipeptide to make a tripeptide, and so on. If there are many amino acids in a single chain the chain is referred to as a polypeptide chain. A protein is a functional molecule composed of one or more folded polypeptide chains. If a polypeptide has no function it will not be referred to as a protein, but simply a polypeptide. You should be able to draw the structure of polypeptides, understanding the nature of the peptide bond.
7. The sequence of a polypeptide chain is simply a list of the amino acids, in order, starting at the amino end of the chain. You should be able translate a sequence given by amino acid names only into a structure and vice-versa.
8. The genes of an organism determine the sequence of the proteins that organism can make. Later we will discuss the ability of a polypeptide chain to fold into a specific 3D structure, which then allows this molecule to perform its function. Understanding the concepts represented in this slide is essential to an understanding of biochemistry.
9. The digestion of proteins is an example of a hydrolysis reaction. Hydro means water and lysis means to cut, so a hydrolysis reaction cuts a bond while adding water. Of course in your stomach and intestines this process requires enzymes as catalysts and adding proteins to water will not result in spontaneous hydrolysis. The products of the digestion/hydrolysis process are the mixture of amino acids from which the protein or polypeptide chain were made. You should be able to recognize peptide bonds in a polypeptide and understand what products will be produced by hydrolysis.
10. Essential amino acids must be part of your daily diet. Nonessential amino acids can be made from other amino acids and thus are not absolutely required in your diet. However, you must ingest about 60g of protein per day, and this must contain a balanced distribution of essential amino acids.
11. This slide lists the 8 essential amino acids, as well as the other 11 nonessential amino acids. You do not need to memorize these, but you should understand why proteins containing some amino acids are more important in your diet.
12. One reason gelatin, a protein, is a poor source of dietary protein is because it contains few essential amino acids. Its sequence contains repeat units of three amino acids, G-X-P, where G = glycine, X = any other amino acid, and P = proline (often modified to hydroxy-proline). Note this pattern and the few essential amino acids in the sequence segment listed. Egg albumin (the protein in egg whites) and meat, which is largely muscle protein from animals (don’t ask about hot dogs), have a good balance of essential amino acids. However, most vegetarian sources of protein have deficiencies in one or more essential amino acids, which is why it is important for vegetarians to mix their protein sources. For example soy beans are low in methionine, while brazil nuts have a protein (also called an albumin) which is methionine rich.
13. Efforts to transgenically incorporate the gene coding for this protein into soy plants were successful, and the resultant beans expresses the brazil nut protein and thus were a more complete protein source.
14. However, anyone with an allergy to brazil nuts now seemed to be allergic to the transgenic soy beans. For this reason you will not find transgenic soybeans in your grocery store. This is one example that illustrates the concern many individuals have over transgenic crops. These concerns often surface as a blanket fear with no allowance for understanding the biochemistry involved. In this case the research stopped the implementation of transgenic, Met rich, soybeans before they got to filed production levels. Some proteins cause allergies because they resist digestion and are absorbed into the blood whole or in large polypeptide segments that some individuals my recognize as ‘foreign’ protein (like an invading virus) and produce antibodies. You should understand the structural feature (non-digestibility) of proteins that may cause allergic reactions in some individuals. The immune response to such proteins will be a later topic.
15. Thus the saying “You are what you eat” is correct in the sense that our cells are composed of the same macromolecules that make up our food. Horses, cows, plants, people, and even bacteria are all composed of these same categories of macromolecules. However, the maxim breaks down when we consider that we rarely assimilate soy bean protein as soybean protein. Rather we rearrange the amino acids from soy proteins and make our own distinct set of human proteins that in turn determines what we are in a functional sense.
16. All proteins are coded for by genes (DNA segments). The gene sequence determines the amino acid sequence or primary structure of the protein. When proteins are made they fold up into a specific conformation (3D orientation of atoms within the molecule). Just like a pretzel has a characteristic shape, so do proteins. I will use the pretzel shape to symbolize a folded protein called pretzelase. Only in this folded shape will the protein perform its function.
17. See slide 2 in 03 PRIONS AND PROTEIN STRUCTURE.
03 PRIONS AND PROTEIN STRUCTURE
1. See slide 15 in 02 Transgenic Soy Beans – Protein Sequence.
2. Protein structure is defined on four levels and called primary, secondary, tertiary, and quaternary structure. The primary structure of a protein is its sequence, but this leaves us with a view of a protein like a string of yarn. Yarn is purchased in a ball. It would be very awkward if 50 yards of yarn had to be kept in its elongated form, or even if it is randomly clumped together. Likewise, proteins do not exist as elongated threads of polypeptide. The secondary structure describes the pattern of progression of the backbone in the protein. The chains take on a shape. Secondary structure is the result of H-bonding in which a carbonyl (C=O) group in the backbone accepts a hydrogen bond from an amide nitrogen (N-H) somewhere else along the protein chain. Tertiary structure describes how the side chains interact with each other. Finally, quaternary structure occurs when a functional protein is composed of 2 or more folded polypeptide chains or subunits.
3. Hydrogen bonds are very important in living systems. They assist in forming the 3-D structure of proteins required for function. They are also the primary interaction involved in the retention and expression of genetic information in DNA and RNA. Their weakness (about 1/10 the bond strength of a covalent bond) is actually a benefit in that they can be readily broken and reformed over and over. Think of them as the post-it-notes of the molecular world. To have a hydrogen bond, an H must be covalently attached to an atom that is more electronegative, such as N or O. Then this H atom must get close to (about 2-3 å) another electronegative N, or O from another molecule or a different part of the same molecule. C – H groups can never form H-bonds. You should understand the molecular requirements for H-bonding and be able to recognize the potential of a funtional group to form this kind of bond.
4. There are three recurring types of secondary structure found in proteins. 1) α-helix: segments of protein with a coiled appearance; 2) β-sheet: 2 or more adjacent, elongated backbone segments that together make up sheets of protein, and 3) β-turns: in which the direction of the polypeptide chain reverses. These three account for about 80% of the backbone structure of most proteins. The other 20% is simply referred to as coiled. In coiled segments (often called random coils, but there is little randomness about it) there is no pattern to the shape. Many coiled areas also turn, so every turn in a protein is not a β-turn.
5. This slide illustrates the coiled appearance of an α-helix. The coil is always right handed, or counter-clockwise in direction. The nature of the H-bond is shown at the right.
6. α-helix segments are very compact with a distance along the chain of about 1.5å per amino acid (full extension is 3.5å). Therefore they can be stretched.
7. (Not shown in handout) The side chains in an α-helix have no room on the inside of the coil. They all face outward.
8. In β-sheet secondary structure H-bonds connect two or more adjacent sheets of extended, polypeptide chain.
9. This picture emphasizes the sheet-like appearance.
10. (Not shown in handout) Often times flat arrows are used in protein structure cartoons to represent β-sheet segments of the backbone, while cylinders or coils represent α-helix.
11. In a β-turn, H-bonds between the 1st and 4th amino acids in the turn stabilize the chain reversal. You should know the three types of secondary structure in polypeptides, and be able to distinguish their structural characteristics.
12. (Not shown in handout) The plant seed protein cranbin contains an example of each form of secondary structure. The color codes for the secondary structure are; red = helix, yellow = sheet, blue = turn, while white = coiled or undefined 2ndary structure.
13. Most infectious illnesses are caused by a bacteria or virus. These are living organisms that have genetic material, either DNA or RNA, and can reproduce. One type of infectious disease that occurs in animals and humans is thought to be caused by proteins. The term prion signifies a protein that is an infectious agent. Keep in mind that a minority of scientists still are not convinced that proteins are truly infectious agents, and think there is an undiscovered virus that is at fault. This interpretation of the data is becoming less accepted with new information
14. Prion diseases have the general term of Transmissible Spongiform Encephalopathies, or TSE’s, since they are characterized on autopsy, by sponge-like brain tissue. Some examples are Mad Cow Disease, Scrapie in sheep, and a number of human diseases which include CJD (Creutzfeldt-Jakob Disease), Kuru, FFI (Fatal Familial Insomnia), and GSS (Gerstmann-Straussler-Scheinker disease). Although it is difficult to spread this disease between species, it has now been shown that humans can get a form of CJD from ingesting ‘tainted’ beef. Recently you may have read about the threat of CWD (chronic wasting disease) to the deer population in Wisconsin and possibly Minnesota. This is also though to be a prion disease. It is not known if humans can get CJD from diseased venison.
15. The abnormal brain tissue found in TSE’s is due to the death of brain neurons (nerve cells in the brain). These cells have characteristic plaques, made of insoluble protein fiber material. Symptoms of the disease are typical of neurological disorders, but vary from one type of TSE to another depending on which area of the brain is affected.
16. Such plaques are polymers of the protein, PrPsc, which is an abnormal conformational form of the protein PrPc. The function of PrPc in brain neurons is unknown. In order to understand what I mean by conformational form, we need to investigate protein structure in more detail.
17. PrPc and PrPsc are simply two different ways to fold the same protein. The equilibrium typically favors the functional PrPc form. In this form a segment of the polypeptide has two α-helix coils (upper right). In switching to the PrPsc form the this pair of helices become 4 interconnected strands of β-sheet (lower right).
18. In TSE’s the helical form converts to the sheet form. It then can induce a normal PrPc structure to change to the PrPsc form also.
19. Prion proteins do not reproduce in the nature of virus and bacteria. However, there is always normal PrPC present in your brain (and other areas). Introduction of a small amount of PrPSC to the brain can cause the multiplication of this abnormal structural form of the protein which then gives the appearance of having ‘reproduced’.
20. Soon most of the protein will be forming protein aggregates called fibrils.
21. The fibrils then aggregate to form the plaque structures observed in the brain tissue of all TSE diseases. You should know the cause of prion disease and understand its connection to protein structure.
22. Tertiary structure is due to side chain interactions in proteins.
23. These depend on the classification categories of the side chains. Disulfide bonds are covalent bonds between two cysteine side chains. H-bonds can be formed between any two polar side chains (includes acidic or basic). Salt bridges require one acidic and one basic side chain. The pH of the solution must be between the pK’s of each group so they are both charged. The salt bridge is the electrostatic attraction between the opposite charges. Hydrophobic interactions are the week bonds of nonpolar side chains which cluster on the inside of a protein’s structure to avoid water contact.
24. An illustration of a disulfide, a H-bond, and a salt bridge between appropriate side chains. The black represents the backbone. Note that this could additionally be in a helix, sheet, or turn conformation.
25. This slide illustrates the buried nature of nonpolar side chains. Not all side chains take part in tertiary interactions. Polar, acidic, or basic side chains not involved in tertiary interactions usually point outwards and interact with the solvent, water. This does not apply to proteins residing in lipid bilayers where the ‘solvent’ is nonpolar. You should know the four types of tertiary structure, the classification of side chains required for each type, and the structural reason for this connection.
26. Proteins exist in an equilibrium between the functional folded state, and an unfolded, or randomly coiled state. This equilibrium typically favors the folded state.
27. Denaturation occurs when chemical denaturants or heat shift the equilibrium toward the unfolded state.
04 HEMOGLOBIN & COLLAGEN: Quaternary Structure and fibrous Proteins
1. See slide 14 in 02 Transgenic Soy Beans – Protein Sequence. One of the key themes of this course is the connection between genes, protein sequence, protein structure, and cellular functions.
2. Often proteins are categorized as either globular or fibrous. Fibrous proteins differ from globular proteins in three ways. 1) Fibrous proteins are typically insoluble in water, whereas globular proteins are water soluble. 2) Fibrous proteins aggregate into fibers by cross-linking multiple, unfolded polypeptide chains. It is the aggregation that makes them insoluble. Globular proteins fold, and may form small aggregates (quaternary structure), but even these are still water soluble. 3) Ultimately, the insoluble fibrous proteins form structural material in the organism. The fibers can be ‘woven’ into many functional forms in some cases. Globular proteins are mobile due to their solubility and their structure should be thought of as flexible rather than rigid. You should be able to distinguish the properties of fibrous and globular proteins.
3. This slide lists some of the major categories and functions of fibrous proteins encountered in the animal kingdom. Collagen is the most common protein found in animals. Collagen fibers take on multiple forms with a variety of functions, depending on the type of cell that makes the structure.
4. I referred to gelatin before as a protein source deficient in essential amino acids. Gelatin is formed by boiling, and then dehydrating collagen from animal tissue. Vegetarians beware, gelatin is not a vegetarian food! Its sequence contains many Pro residues. You should know the side chain structure and classification of Pro since it is pertinent to Collagen function.
5. One topic illustrated by collagen structure is the existence of amino acid side chains not coded directly by the gene sequences. One such example of this is the amino acid hydroxyproline, which is made by the action of the enzyme proline hydroxylase on pro-collagen. Here a hydroxyl group replaces a H atom at the central C on the side chain ring. You should know how this alters the classification of the side chain.
6. Proline hydroxylase acts in the ER lumen, the site for all sorts of protein processing. The enzyme requires the action of two cofactors, iron, and vitamin C. Vitamin C is a reducing agent which means the same as the currently popular term, anti-oxidant. Vitamin C is important for many biological process, some of which are not known, but its role in making strong collagen fibers is well understood, and functions at the level of proline hydroxylase activity.
7. This sampling of the collagen sequence shows how many of the Pro residues are converted to Hyp residues.
8. Pro is a nonpolar side chain, whereas Hyp is polar, due to the hydroxyl group. This allows two Hyp residues to hydrogen bond with each other. It is this H-bonding between two different polypeptide chains rather than within one folded polypeptide, that is responsible for most of the cross-linking in collagen.
9. (Not shown in handout) This allows three strands (polypeptide chains) of collagen to assemble into a structure called the triple helix.
10. Each triple helix unit can be further cross-linked to form individual collagen fibers.
11. Vitamin C deficiencies in ones diet produces symptoms including joint pain, skin problems, and in extreme loss of teeth. If severe these can lead to the disorder called scurvey. All of these symptoms can be attributed to a weakening in the cross-linking required to form strong connective tissue, which is made of collagen. In the 1500s sailors often would lose their teeth from a lack of vitamin c in their diets until a British Physician made the connection between vitamin c and scurvey, and had the British Navy take limes on board their ships.
12. (Not shown in handout) Vital Signs is a monthly feature of Discover Magazine, that addresses case studies encountered in the Emergency Room. A September, 1999 article about osteogenesis imperfecta, (OI) illustrates the human side of a molecular problem. Also highlighted in the movie ‘Unbreakable’.
13. The disease osteogenesis imperfecta takes on many different forms with varying degrees of severity. Some can be fatal. Only individuals with the milder forms of the disease can survive birth. All are due to a mutation (change in gene sequence) of the gene coding for procollagen. These can have a number of effects including subtle flaws in the structure of collagen fibers that weakens the bones made from these fibers. Such is the case in OI.
14. This slide shows only a few of the many mutations found in procollagen that leads to various forms of OI. Please do not memorize these mutations, they are designed only to illustrate the connection between protein sequence, structure, and function. You should be able to distinguish between the causes of OI and scurvey, while understanding their common connection to collage function. These are good examples of genetic vs. environmental influences on function or as it is often referred to; nature vs. nurture. In many situations the nature/nurture distinction is not so black and white.
15. Hemoglobin (Hb) is an example of a protein containing quaternary structure, the level of protein structure that exists only if proteins are composed of 2 or more polypeptide chains.
16. (Not shown in handout) In addition to a folded polypeptide chain, some proteins require a non-peptide component called a cofactor or prosthetic group to perform their function. An example of a common prosthetic group is the heme group (actually a combination of a metal ion and a porphyrin ring). Examples of heme proteins include hemoglobin, myoglobin, cytochrome c, cytochrome oxidase, and many other cytochromes.
17. (Not shown in handout) Hb & Mb are involved in oxygen binding & transport, while the cytochromes are involved in oxidation/reduction reactions in cells. Myoglobin is a protein that has an iron-heme group (shown in green) as a prosthetic group.
18. (Not shown in handout) The heme group is essential for the activity of both Hb & Mb because it is the location at which the O2 molecule binds. No heme, no O2 binding, no function. This is why a dietary shortage of iron can cause anemia. The amount of Fe available limits the amount of functional Hb the RBC’s can synthesize.
19. Hemoglobin and Myoglobin are examples of two distinct proteins that have a common genetic ancestry, meaning they most likely evolved from the same precursor protein a long time ago. Both have one iron-heme prosthetic group per polypeptide chain. Both bind oxygen. The single polypeptide chain in Mb has an almost identical shape down to the same α-helices in the same positions, as does each subunit in Hb. However, functional Hb requires four subunits (i.e. it has quaternary structure). This structural difference influences its function by making it a poorer oxygen binder. Although you might think this is not an improvement, the weaker binding is better suited to the oxygen transport function of Hb in blood, while tighter binding better suits the requirement for internal oxygen diffusion and storage within individual cells. (slide shown in class but doesn’t print well)
20. This slide lists similarities and differences between myoglobin and hemoglobin. Hb has quaternary structure with four subunits, while Mb is a single polypeptide chain. Hb is expressed in red blood cells while Mb is most abundant in muscle cells. Both proteins bind O2 but Mb binds O2 tighter. P50 = the O2 Pressure needed to have 50% of all Mb (or Hb) molecules bound. The lower P50 the tighter the binding.
21. Hb transports O2 from the lungs to other tissues via the blood, while Mb is involved in the storage and local transport of O2 from the cell surface to the mitochondria. Lastly, Hb is an example of a protein that is regulated by the chemical concentrations of H+, CO2, BPG, and O2 in RBC’s. In Mb, binding to O2 depends only on the amount of O2 and is unregulated. Hb binding is much more complicated and requires a more complicated structure.
22. One way to look at the dependency of binding on the amount of oxygen is called a saturation plot. Note that the curve for Mb is hyperbolic, and the P50 is about 1 torr. Note that the Hb saturation curve is sigmoidal (common for regulatory protein binding), and has a P50 of about 26 torr. Thus it takes much more oxygen to achieve 50% binding. The sigmoidal shape is caused by the cooperative effects of O2 binding to each subunit. That is after it binds to one subunit (and removes BPG) it binds much more readily to the next three subunits. Thus the O2 binding to Hb is “all or none”. The blue line is a fictitious saturation curve showing what might happen if you had the weakened binding without allosteric regulation. The amount of oxygen in lungs is about 100 torr. At this pressure both Hb & Mb are completely saturated. The typical oxygen pressure in a functioning cell is about 20 torr. Note that at this pressure Mb would still not release its O2 load, and thus would make a terrible oxygen transporter in blood. However, because of BPG binding and its sigmoidal shape, Hb will deliver about 75% of its O2 load to the typical cell. You should understand the functional differences between Hb and Mb and how this relates to their ability to bind O2. This will require that you understand what P50 means and be able to ‘read’ the saturation curve diagram.
05 & 06 INTRODUCTION TO LIPID STRUCTURE
1. Lipids are molecules that come from living tissue and are soluble in nonpolar solvents. Water the typical solvent in cells is a polar solvent. Examples of nonpolar solvents would include liquids like gasoline, hexane, benzene, etc. In living systems the inside part of the membrane is essentially a nonpolar solvent. What makes a molecule nonpolar, is a high number of carbons compared to the more electronegative O’s or N’s. Remember that H atoms have a similar electronegativity to C.
2. This slide shows the ratio of C:O in a number of substances. Note the higher numbers for the lipids, stearic acid and cholesterol. For amino acids, the N atom counts like O since it is also more electronegative than C or H. Between the two amino acids the nonpolar amino acid has a higher C:O(N) ratio, but still not close to the lipids shown in green in the table. You should understand the concept of polarity and its effect on both protein and lipid sructure.
3. The structural foundation of biological membranes is the lipid bilayer which is largely composed of phosphoglycerides. IN animal cells this structure is supplemented by the steroid, cholesterol. Our bodies fat, and the oils that are stored in plants are triglycerides which function as fatty acid storage molecules for energy reserve. Steroid hormones and prostaglandins are lipids that function as signal molecules. They turn on or off many cellular processes.
4. Both of these types of lipids are made by combining an alcohol and acid to make an ester.
5. The carboxylic acids found in lipids are called fatty acids, by virtue of their long chains. They typically contain 10-18 carbons. In the representation shown here, each bend represents a CH2 group. The end would then represent CH3.
6. A further distinction is to label fatty acids as saturated or unsaturated. Saturated fatty acids have no carbon-carbon double bonds ( >C=C< ).
7. (Not shown in handout) Stearic acid is an 18 carbon, saturated fatty acid. Notice that it has a straight chain structure. Because of this, stearic acid molecules are easy to stack, and are solids at room temperature.
8. Unsaturated fatty acids have at least 1 >C=C< double bond. In nature, the configuration at this double bond is cis, which means that both of the hydrogens are on the same side of the bond, while both of the larger groups are on the other side. This produces a bent chain structure.
9. (Not shown in handout) Oleic acid is an 18 carbon faty acid with 1 C=C double bond. Because of the bend they do not stack well together and are liquids at room temperature.
10. 18 carbon fatty acids are among the most common. Shown here are four of these. Linoleic and linolenic acid are known as polyunsaturated since they have two or more C=C double bonds. These are only made in plant and bacteria. The polyunsaturates found in lard must come from the pig’s diet. Oleic acid is monounsaturated. It is made in animal tissues. On the whole plant lipids contain a higher degree of unsaturation. The table below indicates some of the more common fatty acids and their natural abundance in two lipids, lard, and soybean oil.
11. These fatty acids are rarely on their own in tissue. Rather they combine with alcohols to form esters.
12. The most commonly used alcohol is the triol (3 –OH groups), glycerol. If each hydroxyl is made into an ester with a fatty acid, then the lipid produced is called a triglyceride. Triglycerides are the chemicals we know as fat (often called oil if it is liquid, e.g. when it comes from a plant). This is what food labels are referring to when they list fat.
13. On the last slide, R, was used to represent the fatty acid chain. This slide gives a better idea of the relative size of these chains. Since they constitute most of the molecule, and are nonpolar, triglycerides are very nonpolar, and therefore water insoluble. This is true of most lipids. In addition a triglyceride can contain any mixture of fatty acids, thus the term triglyceride refers to many different molecules, rather than a single molecule. Usually they include the fatty acids listed in the table below. Animal fats contain many more saturated fatty acids….
14. ….. whereas vegetable oils contain many more unsaturated ones.
15. This slide summarizes some of the key differences (and similarities) between animal fats and vegetable oils. You should understand the chemical basis for saturation vs. unsaturation and the implications of this for the structures of fatty acids and the physical properties of the triglycerides made from these fatty acids.
16. This slide shows a table of some of the more common fatty acids found in natural triglycerides. Note that the lard has higher amounts of saturated fatty acids while soybean oil has much more polyunsaturated ones.
17. Many foods list as one of the ingredients, partially hydrogenated vegetable oil. Hydrogenation is a chemical reaction that adds hydrogen across a C=C double bond. Thus you can make unsaturated fatty acids, saturated (or partially so). This is why crisco is a solid even though it contains no animal fat.
18. During the process of hydrogenation, some triglycerides have their double bonds changed from the cis configuration to the more stable trans form. Trans fatty acids, are not bent, like cis ones, and thus resemble saturated fats more than unsaturated ones. Recent evidence, although still controversial, suggest that margarine is no better for your blood cholesterol levels than butter is (and may in fact be worse), since these trans fatty acids seem to stimulate your body to raise the proportion of ‘bad cholesterol’ (LDL) to ‘good cholesterol’ (HDL). For years the popular media has taken the simplistic approach that less cholesterol in the diet means less in your blood. However, doing this by making unsaturated vegetable triglycerides into more saturated ones by hydrogenation really doesn’t work. This is evidently because the higher saturated fats stimulate your body to simply make more of the ‘bad’ cholesterol. Thus it is healthy to reduce the use of spreads like margarine, butter, cream cheese, etc., but it is probably no help to simply replace butter with margarine (however, it is cheaper). You could however, simply pour vegetable oil on your toast. (see the abstract of the article on trans fatty acids included with your notes)
19. This slide shows the linear structure of trans oleic acid (lower left) compared to the bent (and therefore liquid) structures of cis-oleic, linoleic, and linolenic acids.
20. Soap is made by hydrolyzing triglycerides (animal fat) with lye (NaOH). Most hydrolysis reactions are done in acid, but NaOH is a base. Thus this process is a base hydrolysis, also called saponification. The products are the salts of fatty acids (and glycerol). Because they are salts they are (partially) soluble in water.
21. The resultant ionized fatty acids are the major component of soap. The (-) carboxyl group likes water, but the nonpolar tail does not. This is emphasized in the schematic drawing where the (–) carboxyl is simply a red circle.
22. When soap is dissolved in water, the nonpolar tails don’t want to interact with the solvent. They can do this if hundreds of soap ions aggregate with the nonpolar tails gathered in the middle and the – carboxyl groups outside interacting with the water. This view is a cross-section of this structure, which is called a micelle.
23. However, from the outside, you can’t see the nonpolar tails at all, since they are buried, and thus not exposed to the water.
24. Dirt, represented by the gray blobs, is basically nonpolar stuff, that clings to dishes or clothing, and is not easily rinsed away by water. This is because they are not soluble in water, or in other words dirt is typically hydrophobic.
25. When you heat and agitate a soapy solution the micelle disperses. Now the dirt particles can cling to the nonpolar tails of the soap ions rather than the dishes or clothes.
26. When you stop the mechanical agitation, the micelles spontaneously reassemble. However, now the dirt is in the center of the micelle, buried from the water. Since the micelle as a whole is suspended in water (this is referred to as a colloidal dispersion as opposed to a true solution) it can be rinsed away, taking the dirt with it. You should understand the structure of a micelle and why ions of fatty acids form this structure.
27. In a triglyceride, there are three fatty acid esters to glycerol ….
28. In a phosphoglyceride, one of these is replaced by a phosphate ester to phosphoric acid, which in turn forms a 2nd ester to an amino alcohol.
29. This forms a structure with two nonpolar tails and an polar head group containing both a (+) charge on the amino end and a (-) charge on the phosphate group.
30. When placed in water these molecules associate so that the nonpolar tails are not exposed to the water. Unlike soap ions, the phosphoglycerides are a bit too bulky to form a micelle, and prefer a two-layer structure called the lipid bilayer. We owe our lives to this tendency.
31. The lipid bilayer is not linear, rather it forms a 3D sphere called a vesicle or liposome. In this spherical structure the lipid bilayer, separates the external and internal aqueous solutions. It acts as a barrier to prevent total freedom of movement of molecules between the two aqueous solutions. It is this feature that allows cells and organelles to exist, and maintain the nonequilibrium state required for life. The tails of the phosphoglycerides are a liquid rather than solid structure, forming a nonpolar solvent area in all of our cells. The inside of the lipid bilayer and the triglyceride ‘droplets’ in our adipose form the major nonpolar solvents in our bodies where molecules such as fat soluble vitamins reside.
32. Biological Lipids are made of lipid bilayers with embedded proteins that serve as channels to allow the passage of specific molecules/ions through the membrane, or receptors, that recognize signal molecules such as hromones and neurotransmitters. Thus, both material and ‘messages’ can pass through the membrane.
33. Steroids are another class of lipid. Steroids are easy to recognize by the characteristic cluster of four rings (3, 6-membered rings and 1, 5-membered ring) each sharing a side. Cholesterol is among the most well known (infamous) steroids. About 10% of the membrane content of animal cell membranes are made of cholesterol instead of phosphoglycerides. The hydroxyl group on cholesterol is the polar head group, and the fused rings are similar to two nonpolar tails. Cholesterol regulates the fluidity of animal cell membranes. It is not needed or found in plant cell membranes, which regulate their fluidity by varying the proportion of saturated to polyunsaturated fatty acids produced. Cholesterol is ‘bad’ only if we make too much of it. Excess cholesterol is the major material found in atherosclerotic plaque, and gall stones. Diets high in saturated fats (and thus usually cholesterol also) do lead to a tendency to overproduce cholesterol in the body. It is important to realize that this is over and above the simple addition of cholesterol in your diet.
34. Some lipids function as chemical signals in our bodies. They act to turn on (or off) various metabolic processes. These signal molecules include some lipids, e.g. steroid hormones and prostaglandins. The difference between the two is that hormones are ‘external’ signals and originate in a tissue other than the cell type where they have their effect. Prostaglandins are typically made and act within the same type of tissue cells.
35. For example cortisone is a much-used, natural, anti-inflammatory steroid. If you’ve ever had a rotator cuff injury, you may have received cortisone injections. Note the basic steroid ring structure.
36. Prostaglandins have a much simpler, 5-membered ring structure, shown here. You should be able to recognize the structures of triglycerides, phosphoglycerides, steroids, and prostaglandins. In addition you should understand the relation between their structures and functions in the body.
37. Cortisone prevents the synthesis of arachadonic acid. which is a lipid made from membrane phosphoglycerides, and used as a raw material to make prostaglandins. Prostaglandins mediate the inflammation response that leads to tissue swelling in injuries, arthritis, etc. If you prevent arachadonic acid synthesis you also prevent prostaglandin synthesis and inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs), like aspirin and ibuprofen, also prevent prostaglandin sytnthesis, but at a later step. Thus they allow leukotriene synthesis which can heighten the allergic response in some individuals.
38. Androstenedione, is another steroid that is often taken to boost testosterone levels in the body by weight lifters or body builders. Although banned by most sports organizations its use is still legal in major league baseball. It is most likely metabolized in the body to form testosterone (or destroyed in the liver), which as you can see has a very similar structure. One of the dangers of taking anabolic steroids is that your liver must work overtime to eliminate the excess steroids, and this can rapidly lead to liver damage and in some case renal failure. This is similar to cirrhosis, which can be caused by excess alcohol consumption. You only have one liver – treat it well.
Am J Clin Nutr 1997 Oct;66(4 Suppl):1006S-1010S
Health effects of trans fatty acids.
Ascherio A, Willett WC
Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, USA.
trans Fatty acids are formed during the process of partial hydrogenation in which liquid vegetable oils are converted to margarine and vegetable shortening. Concern has existed that this process may have adverse consequences because natural essential fatty acids are destroyed and the new artificial isomers are structurally similar to saturated fats, lack the essential metabolic activity of the parent compounds, and inhibit the enzymatic desaturation of linoleic and linolenic acid. In the past 5 y a series of metabolic studies has provided unequivocal evidence that trans fatty acids increase plasma concentrations of low-density-lipoprotein cholesterol and reduce concentrations of high-density-lipoprotein (HDL) cholesterol relative to the parent natural fat. In these same studies, trans fatty acids increased the plasma ratio of total to HDL cholesterol nearly twofold compared with saturated fats. On the basis of these metabolic effects and the known relation of blood lipid concentrations to risk of coronary artery disease, we estimate conservatively that 30,000 premature deaths/y in the United States are attributable to consumption of trans fatty acids. Epidemiologic studies, although not conclusive on their own, are consistent with adverse effects of this magnitude or even larger. Because there are no known nutritional benefits of trans fatty acids and clear adverse metabolic consequences exist, prudent public policy would dictate that their consumption be minimized and that information on the trans fatty acid content of foods be available to consumers.
DISCLAIMER: More recent studies have suggested the health impact of trans fatty acids listed above are not conservative, but rather exaggerated. This is not a resolved issue. The main point that most researchers agree on, is that the benefits of cholesterol free, partially hydrogenated vegetable oils as a replacement for saturated fats have been overestimated. Whether they are more detrimental is debatable.
07 & 08 Enzymes
1. Living organisms take in a limited number of chemicals, primarily carbohydrates, lipids, and proteins. They rely on chemical reactions to change this set of chemicals into other forms needed by the organism. Understanding life requires understanding the chemical reactions that support life. This involves some symbolism. Reactants, called substrate(s) (S) are converted in product(s), P. Potentially all cellular reactions are reversible, so P can be returned to form S.
2. When considering a chemical reaction, there are two important questions to ask. 1) How far will the reaction proceed? All reactions proceed until an equilibrium state is reached. At this point the concentration of substrates, [S], and the concentration of product(s), [P], remain constant because the forward and reverse reaction rates are the same. This is called a dynamic equilibrium. At this point the ratio [P]/[S] is called the equilibrium constant, Keq. 2) How fast will the reaction proceed? The chemical concentrations in a cell are always changing, so often equilibrium is never reached. In such cases the important question to understand what goes on in a cell is how fast are the reactions proceeding? Most cellular reactions are too slow to be allowed to proceed unaided. It is enzymes, proteins made in the cell that catalyze or speed cellular reactions toward their equilibrium state, even though this state is often never attained. As an example of the importance of rate, I’ll look at the oxidation of H2 with O2 to form H2O. The equilibrium of this reaction vastly favors the product state, yet the reaction is so slow at room temperature, you probably wouldn’t notice it happening…
3. …this is because the activation energy, Eact, is very large. Eact is the free energy difference between the substrate(s), and the transition state. For simplicity, let me define the transition state, S#, as a ‘distorted form of S that is the highest energy form that must be attained to make P. So even if P is at a lower energy than S (exergonic or exothermic reaction), energy must be added to produce P. Eact, is thus an an energy barrier that must be overcome to make P. The higher the barrier (Eact), the slower the reaction. However, the equilibrium state, and thus [P]/[S], is solely determined by the free energy difference between S and P, referred to as ΔG((. Thus Eact gives the answer to the question, how fast, while ΔG(( answers the question, how far!
4. A large Eact, as in the oxidation of H2, can be overcome by raising the temperature and increasing the concentrations of reactants. However, this option is rarely available to cells.
5. Thus, for a cell to make a reaction go faster, its only option is to lower Eact. This is what enzymes do! They do not affect the equilibrium distribution of products and substrates, [P]/[S], or ΔG((.
6. Enzymes are proteins that function as biological catalysts. Some biological catalysts are RNA molecules. These are called ribozymes.
7. Enzyme names usually (there are numerous exceptions) contain 3 parts. The 1st is the name of the substrate. For example pyruvate in pyruvate dehydrogenase. The 2nd is the type of reaction. For example a dehydrogenase will always remove two H atoms from its substrate and transfer them to a coenzyme (either NAD+ or FAD). The 3rd is the ending ‘ase’. For hexokinase, the substrate is a hexose (a 6-carbon sugar like glucose), the type of reaction, kinase, tells you the reaction is a phosphoryl transfer in which a Phosphate group is removed from ATP (the other substrate) and added to the hexose.
8. Chemical reactions in cells are required for two major reasons. 1) Generate the energy needed to sustain life and 2) produce the molecules the cell needs for its numerous structures and functions. Virtually all cellular reactions are too slow to sustain the cell’s life without the assistance of enzymes.
9. Enzymes are like the cell’s tool box. This slide lists just a few of the things they accomplish.
10. I’ll use tyrosinase as an example. It provides a good visual example of enzyme function, but also illustrates some of the complexitieis of metabolism brought about by the varitey of outcomes a single enzyme can produce. Tyrosinase is a copper containing enzyme that converts tyrosine (the substrate) to DOPA, (the product but also the second substrate) and can also oxidize this product further to produce dopaquinone (the 2nd product). On the surface this enzyme is necessary to produce molecules that other enzymes will convert into the pigment melanin. Melanin is the major pigment of skin and hair in humans and many other animals. Mutations in tyrosinase can lead to albinism in a variety of animals. In fruits and vegetables, it is responsible for the the yellow color that occurs when they are cut open and exposed to air. This coloring often makes the fruit less appealing. Restaurants used to spray salad bars with sulfites that inhibit tyrosinase and prevent this unsightly yellowing. However, many individuals had serious reactions to sulfites and the practice was terminated. Pictures show the effects of albinism, often due to the absence of functaional tyrosinase in the appropriate cells; in these cases melanocytes, or hair follicle cells.
11. (Not shown in handout) An albino snake.
12. (Not shown in handout) The same snake shown next to its natural colored relative.
13. (Not shown in handout) An albino penguin. You are looking at it’s back not it’s chest.
14. (Not shown in handout) An albino human.
15. Tyrosinase is most obviously active in hair and skin cells, or what are known as pigment cells. However, the same or similar enzymes (encoded by a separate gene) may find use in other types of cells. For example, the pathway that produces norepinephrine, a neurotransmitter, that along with serotonin, mediates much of our behavioral characteristics; utilizes the same starting chemical reactions used to make melanin. In addition, the norepinephrine can also be converted into epinephrine in adrenal glands. This hormone stimulates muscle glycogenolysis in response to emotional stimuli. Additional enzymes in brain neurons and adrenal cells will produce neurotransmitters or hormones. These cells do not make (express) the additional enzymes needed to make melanin.
16. The Michaelis-Menten model illustrates how an enzyme interacts with the substrate during an enzyme reaction. You should be familiar with the equation E + S ( ES ( E + P as a representation of the process by which enzymes work. Note that the transition state could also be included in this model, but for the sake of simplicity, it is easier to leave it out.
17. It is simplest to think of enzyme function as a two step process. In the first step, the Enzyme binds to the substrate. Once bound the second step involves the chemical change of S into P, a process that takes it through the transition state, S#. Eventually, the product, P, is released and the Enzyme is free to bind to another substrate. One requirement for any catalyst, is that it must be reusable.
18. The binding step is often explained by an analogy with a lock and key. This analogy emphasizes that the enzyme contains a substrate-binding site, called the active site, that has a complementary shape to the substrate. Active sites viewed from crystal structures of enzymes, are literally depressions in the surface of the enzyme that have many possible shapes. However, they must be similar to the shape of the substrate. The lock and key model emphasizes the specificity of the enzyme for a particular substrate, just as not any key will open the lock on your door (hopefully). Some enzymes will accept a variety of similar substrates, just as some locks will accept a variety of related keys.
19. However, shape alone is not enough to cause the bonding between the enzyme and its substrate. Weak chemical interactions, H-bonds, salt bridges, and hydrophobic interactions, must be present to achieve binding. For example, even if the shape is right, two negative charged groups will repel.
20. In some cases the enzyme’s active site does not appear to have a complementary shape with the substrate until the substrate is added. Then the enzyme, reforms its active site to make it fit the substrate. In these cases the conformational rearrangement of the enzyme is referred to as induced fit. Hexokinase, and enzyme that binds glucose (and ATP) and makes glucose-6-phosphate, is an example of an induced fit model. Glucose binding induces a structural change in the enzyme to conform more closely to the structure of glucose.
21. (Not shown in handout) The enzyme, hexokinase, is an example of induced fit. When glucose binds the enzyme folds over the molecule like a baseball glove folds over a baseball. This prevents the substrate, glucose, from leaving until the reaction has been completed (in this case the transfer of a phosphate group from ATP).
22. (Not shown in handout) This slide illustrates the subtle differences between the lock-and-key enzymes vs. induced-fit enzymes.
23. (Not shown in handout – same as slide #17) The second step of enzyme action involves the conversion of S into P. This requires that some covalent bonds of the substrate must break. Bonds are held together by sharing electrons. When S binds to E the enzyme attracts some of the electron density of susceptible bonds away from the substrate bond and thus weakens the bond. It now takes less energy input to ‘finish off’ the bond, and thus Eact is lowered.
24. Essentially S in its weakened bond state is S#, the transition state. Such weakened bonds are typically stretched or lengthened. However, once the bond is broken, the enzyme also serves to position atoms nearby that are required to form the new bonds to be found in P.
25. One way to study enzyme behavior is to plot the rate of the reaction (v) vs. the amount of substrate available ([S]). I’ll refer to this as a v vs. [S] plot, or a saturation plot. The shape of the curve for this plot is usually hyperbolic. It levels off when the enzyme is saturated with substrate. v at this point is the maximum velocity or Vmax. Adding additional S will not make the reaction go faster because all active sites are occupied already. Another way of looking at enzyme function is to ask how much substrate is required to give half of the maximal velocity. The name for this is KM (or the Michaelis constant). KM tells you something about the binding step (smaller is tighter). Vmax tells you about the 2nd or catalytic step.
26. Many chemicals that we may breathe or ingest are toxic. Often the reason for their toxicity is that they function as enzyme inhibitors. These are molecules that slow down the activity of enzymes (lower v). Inhibitors come in a number of types, but the most common are competitive and noncompetitive. This slide lists some features of these two types.
27. A competitive inhibitor functions by binding to the active site of the enzyme, and thus not allowing the substrate to bind. In order to do this it must be structurally similar to the substrate. Compared to S, it must have both a similar shape and similar chemical groups to interact with the active site of the enzyme. It affects only the binding step in enzyme action and thus increases KM (makes binding to S weaker) without changing Vmax.
28. A noncompetitive inhibitor can bind to the enzyme simultaneously with substrate. Thus it does not affect binding (KM), but rather decreases Vmax, or affects the catalytic step.
29. Sulfa drugs are antibiotics. This means that they kill bacteria. They do this by ‘mimicking’ the substrate (PABA or para amino benzoic acid) for a bacterial enzyme (DHPS). As such, sulfa drugs are competitive inhibitors.
30. They do not affect human metabolism, because humans do not have the same enzyme that bacteria do to make folic acid. In fact, for us, folic acid is a vitamin, and must be present in out diets. Thus if you ingest folic acid, but prevent the bacteria residing in you from making folic acid from PABA, you can kill the bacteria without harming your own cells.
31. This slide emphasizes the structural (and shape) similarity between a substrate and a competitive inhibitor for an enzyme. In this case sulfa drugs are very similar to PABA. Drug companies use this knowledge to provide a starting point for making new drugs. If we understand the reaction catalyzed by an enzyme, and the substrate, that provides the necessary starting point to develop molecules that will inhibit the action of the enzyme.
32. Penicillin is another antibiotic that is made by a fungus to prevent bacteria from sharing its food source. It was discovered on bread mold. It inhibits the enzyme responsible for synthesizing the final stage in the construction of the bacterial cell wall, which is made up of a molecule called a peptidoglycan (meaning that it contains both sugar groups and peptide groups).
33. The enzyme typically adds itself to the carboxy end of a peptide ending in AA. Then it cuts the last D-Ala from this peptide …
34. … and connects it to a pentaglycine peptide. This pentaG peptide links different peptidoglycan units conferring strength to the cell wall.
35. Penicillin binds to the enzyme in place of the peptide that end in AA. It attaches itself to the enzyme, but then it cannot be linked to the pentaG linking peptide, thus preventing the final construction of the bacterial cell wall. This effectively kills the bacteria. Penicillin resistant bacteria produce an enzyme called penicillinase (or β-lactamase) that destroys the penicillin before it can inhibit the enzyme. Bacteria are constantly evolving to develop resistance to the toxic inhibitors humans develop and often overuse.
36. AZT, is one of the drugs used in the anti-AIDs cocktail. It is a nucleotide analog. Nucleotides are the substrates that the enzyme Reverse Transcriptase uses to make DNA from the RNA genetic material found in retroviruses. As such AZT is a competitive inhibitor of this enzyme.
37. In a final example, I will use to indicate that not all drugs function by killing bacteria or viruses; tacrine is a competitive inhibitor of acetylcholine esterase. This enzyme is responsible for destroying the neurotransmitter, acetylcholine. We need it to recycle our never impulse signals. However, Alzheimer patients have too little acetylcholine in their brains. This apparently diminishes brain cell communication, which is required for memory and thinking. Thus, inhibiting acetylcholine esterase, the enzyme that destroys this neurotransmitter, can restore some cognitive function to Alzheimer’s patients. This is definitely treating the symptoms, not curing the disease.
38. Drugs have to go through many levels of testing before they appear on the market, a process that typically takes more than 10 years. Some of process was partly developed in response to the problems with thalidomide. The increased use of molecular modeling using computers to design molecules that will mimic the substrate and bind at the active site of the enzyme has helped limit the number of compounds to be tested. If a compound shows promise in animal testing it must go through four phases of testing on humans. Such studies seek to find out both the effectiveness and toxicity of the drug. What doses are needed and what the side effects of those will be. Phase IV testing occurs after the drug has been approved to monitor long-term effects that may not have shown up in initial tests.
09 THALIDOMIDE:
1. Glucose and Fructose are sugars that are major components of our dietary carbohydrates. They are also good examples of compounds that contain a chiral carbon. A chiral carbon is any carbon in a molecule that has four different ‘groups’ (not atoms) attached to it. Glucose and fructose both have the same formula. This makes them isomers of each other. Because they have an entirely different bonding arrangement, glucose is an aldohexose while fructose is a ketohexose, they are structural isomers. The term isomer always explains a relationship of one molecule to another. This is similar to using the words sibling, or cousin in describing the relationship between individuals. However, molecules with a chiral carbon may also have optical isomers. Optical isomers have the same formula and the same bonding arrangement of atoms, but they have a different orientation in space. This can be very important when considering the effectiveness and toxicity of a drug. We’ll investigate this using thalidomide as an example.
2. Thalidomide is a synthetic drug that was synthesized in West Germany in 1954 and gained widespread use in Europe in 1957 as an over-the-counter soporific (induces sleep) and sedative. Note that the carbon in red is chiral, which means it has four different groups attached. This is the only carbon on the molecule that is chiral. You should be able to identify chiral carbons on any structure.
3. This slide indicates some landmark dates in relation to thalidomide use.
4. Any molecule that has a chiral carbon can exist in two distinct forms that are called optical isomers. Such molecules have all the same atoms bonded in the same way. The only difference is that they have a different spatial (3D) orientation. If there is only a single chiral carbon on a molecule, the optical isomer of that molecule is called its enantiomer. This is a term describing a relationship between two molecules, much like the terms sibling, or cousin define family relationships.
5. This simple ball and stick model represents a chiral center in a molecule. The gray ball represents a chiral carbon, whereas the colored balls represent distinct chemical atoms or groups, e.g H, CH3, OH, or even long chains such as –CH2-CH2-CH2-NH3+. The cube around the molecule helps you to understand the tetrahedral arrangement of 4 groups attached to a central carbon. They have typical bond angles of about 109(.
6. Enantiomers can be defined as optical isomers that are mirror images of each other. Imagine the dotted line shown as a mirror. Optical isomers seem to look the same but they are not superimposable. This means there is not orientation where all the groups in each molecule will overlap each other.
7. (not shown in handout) This can be tested by rotating the molecule at the right along a horizontal axis going into the screen.
8. (not shown in handout) Rotating about a vertical axis doesn’t make them superimposable either.
9. This slide represents ball & stick models of D-glyceraldehyde, and L-glyceraldehyde. If the principle functional group is oriented to the top back of the structure relative to the chiral carbon, and the rest of the chain is lower back, then the designation of molecules as D or L is determined by the relative position of the principle functional group on the chiral carbon, which is –OH in this molecule. In either case it will be in the foreground relative to the chiral carbon, but if it points to the left it is L, and to the right it is D.
10. But are such molecules really different? In addition to being non-superimposable, they rotate the plane of plane polarized light in opposite directions. In fact this is how the D & L designations originated (now (+) & (-) is used to indicate a clockwise, or counterclockwise rotation of plane polarized light). More importantly, however, they can react differently in any chemical environment that is also chiral. This includes most interactions with proteins in living systems.
11. All that is need for enantiomers to interact differently with proteins, is that the binding protein surface has at least 3 contact points for the molecule (ligand).
12. If these models represent two enantiomers that bind to a protein….
13. One will simultaneously contact all three binding points on the protein’s surface….
14. .... The other contacts only one. No matter how you turn the ‘incorrect’ enantiomer, it will never match all three groups with the correct binding site on the enzyme.
15. This is why living cells often distinguish between molecules differing only by being mirror images of each other. Thus enantiomers usually don’t react the same in living systems (they would in a situation where the enzyme only contains two contact points rather than three).
16. Glucose has four chiral carbons in its open form. This means there there is not only 2 optical isomers in the family but 24 = 16 optical isomers. However, only one, L-Glucose, is the mirror image of D-glucose. The last one is used to determine the D or L designation. The structure ahown is ‘D’. Nature only makes D-sugars like glucose and fructose. Eating food composed of L-sugars would lead to starvation, because your body could not utilize them as fuel.
17. For amino acids, its just the opposite, nature only makes ‘L’ forms, and all the proteins in your body are polymers of only L amino acids. This is why the react with specific enantiomers of ligands if they contain 3 or more binding points.
18. This slide shows a ball & stick model of L-alanine.
19. In this view of thalidomide the orientation is not designated. You cannot tell without seeing the 3D structure or using the porper 2D formality for drawing the structure whether it is D or L. However, laboratory synthesis of thalidomide produced a racemic mixture of the enantiomers. This means that equal amounts of D & L are present.
20. This slide gives a better 2D representation of the differences between D & L thalidomide. Since 1961 it has been determined that the L-form is the teratogen, while the D-form has the soporific effects. Now the two can be separated. Currently the technology to separate enantiomers exists (it didn’t in 1961), and all drugs must be labeled as to enantiomeric purity. Many drugs are still racemic mixtures if safety tests show the alternate enantiomeric form is harmless, but usually it is still useless as well. Generic drugs sometimes don’t take the added expense of removing such enantiomers.
10 ENZYME REGULATION
1. Cells must regulate Enzyme catalyzed reactions to be able to adjust their priorities when environmental changes make it prudent to do so. For example if I lose a large chunk of my income, I might give up going to movies before I give up eating, because eating is a higher priority in lean times. I might also prepare my own food instead of going to restaurants. How we behave depends on available resources.
2. Cells can regulate enzyme reactions by increasing the amount of enzyme available. This is really gene regulation not enzyme regulation, and we will leave this for later in the course. Some reactions are regulated by changing the rate at which an enzyme molecule works. Here the amount of the enzyme remains the same, but its activity level either increases or decreases. This is the aspect of regulation we are concerned with. There are three varieties on this theme, allosteric enzymes, covalent modification, and proenzymes. You should be able to distinguish between these different types of enzyme regulation both in terms of how it occurs and why one way is more advantageous in certain situations.
3. In allosteric regulation, the enzyme actually has two folded forms. One is active and the other inactive. These exist in equilibrium with each other. Allosteric enzymes possess an allosteric site (for binding regulatory molecules) in addition to the active site (for binding substrate).
4. They also typically have a sigmoidal v vs. [S] plot rather than a hyperbolic one. You should be able to distinguish between these two types of plot.
5. A negative effector, binds only to the inactive form of the enzyme. This increases the amount of inactive form relative to the active form by shifting the equilibrium between the two.
6. This shifts the sigmoidal v vs. [S] curve to the right on the graph. This means that given the same amount of S, the enzyme will produce P at a lower rate.
7. Positive effectors bind only to the active form of the enzyme, and increase the equilibrium amount of this form.
8. This shifts the sigmoidal v vs. [S] plot to the left. In other words, the same amount of S will give a higher rate of P formation (or v).
9. What are the negative and/or positive effectors that regulate allosteric enzyme activity? Usually, a negative effector is the product of a metabolic pathway or pathways. For example one of the primary goals of Glucose oxidation (via Glycolysis) is to make ATP, the energy currency of the cell. If the cell already has enough ATP, why should it make more? Thus ATP become the signal molecule (- effector) that tells an early enzyme of the glycolysis pathway to slow down. However, partial activity of the pathway may remain to accomplish the other goals of the pathway. In this case the partial activity of glycolysis in the presence of ATP allows the cell to make fatty acids. Incremental changes in activity is one of the characteristics of allosteric regulation.
10. Another form of enzyme regulation is covalent modification. Like allosteric regulation the enzyme has an active and an inactive form. However, instead of a conformational change, the form is changed by adding a covalently bonded phosphate group to a side chain (usually a hydroxyl side chain like Serine or tyrosine). This requires an enzyme called a ‘protein kinase’, which transfers a phosphate from ATP to the enzyme. Another enzyme called a phosphatase will cut off the phosphate and reverse the activation/inactivation.
11. In this example the circle represents a protein being activated by phosphorylation. Usually almost all of the protein copies are activated at the same time, switching the enzyme 100% on. When the activation is no longer needed, a phosphatase cleaves the phosphate group to deactivate the enzyme so that it is essentially 0% on. I’ll refer to this as ‘all-or-none’ activation, a characteristic of covalent modification.
12. Some enzymes are made in a preliminary form, called a proenzyme. Proenzymes have no enzymatic activity. They are activated when another proteolytic enzyme cuts a specific peptide bond(s) of the proenzyme. Once activated the enzyme never reverts back to its proenzyme or inactive form. This is how this type of regulation differs from allosteric and covalent modification; it is not reversible. However, like covalent modification it is essentially an ‘all-or-none’ process.
13. Trypsinogen is an example of a proenzyme. It becomes trypsin only when the propeptide, an octapeptide following the signal sequence in this case, is removed.
14. This is accomplished by another enzyme, enterokinase. Although trypsinogen is made in the pancreas it is never activated until it is secreted into the intestines.
15. This is because trypsin is a general proteolytic enzyme used to digest other proteins. If it were active in the pancreas it would merrily digest away at the very proteins that make up the pancreas, thus destroying the organ. This would not be a wise move. Nature has kindly developed trypsinogen in a proenzyme form only to activated where its function is desired, during digestion in the intestinal cavity. Most proteolytic enzymes, and blood clotting enzymes are synthesized as proenzymes, but other examples are numerous.
16. This table summarizes some of the similarities and differences with the three types of enzyme regulation with respect to 1) reversiblilty, 2) “all-or-none’ activation, & 3) the need for another enzyme in the activation sequence.
11 & 12 Metabolism 1 – General Aspects of Metabolism – Metabolic Mainstreet
1. Metabolism is the sum of all the chemical reactions and processes that take place within a living organism. This includes reactions inside the cell, and in any other extracellular fluid of the organism (e.g. blood). Most reactions are catalyzed by enzymes, so the understanding the enzymes (+ channels, receptors, etc.) made by a cell are the keys to understanding their metabolism.
2. Cells need to do two basic things. 1) Maintain a constant energy level. Cells require a chemical energy supply. ATP is the key energy ‘currency’ of the cell, but other nucleotides, UTP and GTP are utilized in certain types of reactions. Catabolic pathways convert our food into energy in the form of ATP. 2) Cells must synthesize all structural and functional molecules required for metabolism. This includes the macromolecules; proteins, polysaccharides and lipids. Those that synthesize ‘parts’ are anabolic. As we study individual pathways you should be able to recognize whether they are catabolic or anabolic and explain why.
3. A pathway is a series of enzyme catalyzed reactions. The reactions of a pathway will be located in the same location of the cell (e.g. cytoplasm), and the net outcome has a collective purpose that is understandable without knowing the intermediates of the pathway. Molecules that are made during one reaction of a pathway and then used up in another reaction are called metabolic intermediates (in the context of the pathway) or metabolites (in the context of the source food molecule).
4. The net outcome of a pathway can be best represented by the net reaction. This is obtained by adding all of the individual steps of the pathway, canceling any intermediate that is a product of one step and then a reactant in a later step.
5. Catabolic pathways, lead to the production of ATP, breakdown fuel molecules to smaller metabolites, and oxidize ‘fuel’ molecules. If the # of C atoms in the reactant are greater than in the product(s) of a pathway it is catabolic. Anabolic pathways consume a cell’s energy supply (use up ATP), produce molecules with more C’s than the reactant (building block), and do so by a chemical reduction of these reactants. Sugars, amino acids, fatty acids, and their metabolites serve as the fuel molecules of catabolic pathways. However, they also serve as the building blocks for anabolic processes. A molecule’s ‘fate’ in the cell will determine whether I refer to it as a fuel or a building block.
6. Anabolic processes are producing products at a higher free energy level than the building blocks. Catabolic processes eventually oxidize C-containing compounds down into CO2, removing all their H atoms. ATP can be produced because these products are at a lower free energy value than the original fuel molecule.
7. For example, glycolysis is a sum of 10 different enzyme-catalyzed reactions. We will be more concerned with the overall outcome than with learning each intermediate and enzyme.
8. ATP is a nucleotide. It contains the base, adenine, and the sugar, ribose. What we are interested in are the phosphate groups added to the sugar in a series. ATP has three phosphates; ADP – two; and AMP – 1.
9. Removing the phosphates provides energy. This is what allows the cell to perform anabolic processes. The hydrolysis of ATP to ADP provides energy. The energy contained in this molecule (7.3 Kcal/mole) is produced by the hydrolysis of either the last or last two phosphate esters on the molecules. Adding phosphates (phosphorylation) requires energy from the oxidation of fuels that occurs during catabolic pathways.
10. When anabolic pathways consume ATP, the Adenine nucleotide is still present, but in its diphosphate form, ADP (or sometimes AMP). To retain a constant, immediately useable, energy level in the cell, catabolic pathways must phosphorylate ADP (or AMP) to replace used ATP. You should understand the difference between the hydrolysis of ATP and the phosphorylation of ADP to make ATP, and the importance of these two reactions in the cell.
11. The energy charge of a cell can be indicated by determining what fraction of the adenine ribonucleotides are found in the high energy ATP form, or the part energy ADP form compared to the sum of all adenine ribonucleotides, including AMP.
12. What does the organism use ATP for? In addition to providing the energy to run anabolic reactions, ATP is also required for maintaining gradients across cell membranes, and mechanical motion, like muscle contraction.
13. An organism derives energy from food intake in three distinct stages. The first is the digestion of food macromolecules (or the hydrolysis of macromolecular stores in the body). Second is the oxidation of fuel molecules. This occurs by the action of enzymes called dehydrogenases, that remove hydrogen atoms from the energy rich fuels. They transfer these hydrogen atoms to either NAD+ or FAD (oxidized coenzymes) to make NADH, or FADH2 (reduced coenzymes). In the final stage of energy production, the oxidative phosphorylation pathway oxidizes thse NADH or FADH2 and uses the derived energy to phosphorylate ADP into ATP.
14. Thus fuels must constantly be taken in by living organisms to maintain a constant work output. Even when you sleep your cells are working and consuming fuel. The amounts of ATP vs. ADP and NAD+ vs. NADH constantly cycle, but the total amount of each pair remains essentially constant.
15. There are four pathways involved in the complete oxidation of glucose in a cell.
16. I’ll refer to this set of pathways as ‘Metabolic Mainstreet’. Only 40% of the energy contained in glucose is converted in the useable, chemical form of ATP. The remainder is converted into heat, and although not totally wasted (particularly in warm-blooded mammals), is essentially useless in terms of your metabolism.
17. This metabolic chart indicates the key intermediates of Metabolic Mainstreet. These include pyruvate, acetylCoA, NADH, and FADH2. Pyruvate and acetylCoA are also two of the major branching points for connecting to other metabolic pathways we will study later.
18. When studying a pathway we will focus on the 4 w’s. What it does – usually indicated by knowing the net reaction. Why it does it – usually indicated by understanding the usefulness of the products of the net reaction. Where it occurs – indicated by knowing what organisms use the pathway, in which specialized cell types it occurs within an organism, and in what organelle within the organism the enzymes are located. We will mostly focus on human cells, and thus will be most concerned with what cell type (live, muscle, etc…) and/or what location/organelle (cytoplasm, mitochondria, etc….). When it occurs – this requires understanding how the pathway is regulated. Under what cellular conditions, fed state vs. fasting, resting state vs. exercise, etc.. will the pathway be needed the most and thus be ‘turned on’. You should know the 4 w’s for each pathway in metabolic mainstreet.
19. This slide shows the net reaction of glycolysis. Glucose (C6H12O6) is oxidized into 2 pyruvate (C3H4O3), while NAD+ is reduced to NADH, and ADP is phosphorylated into ATP.
20. The complete list of intermediates produced during this pathway indicates that the process is more complicated than the net reaction might indicate. Nonetheless, if you understand the net reaction, you should have clues into its primary purpose.
21. Why? The primary purpose is to generate 2 ATP molecules, that are immediately useable, but possibly more importantly, the pyruvate and NADH produced can be oxidized by other pathways to eventually make 34 more ATP. However, some of these additional ATPs can be ‘sacrificed’ if glycolysis intermediates or pyruvate are used for anabolic processes. This however, will be considered a ‘secondary’ objective.
22. Where? Glycolysis, and all the other pathways of Metabolic Mainstreet are universal. They occur in all living cells. Within humans they also occur in all tissue types. The location is in the cytoplasm, which is significant to understanding the anaerobic nature of the 2 ATPs produced. We will discuss this later.
23. When? Basically if a cell needs energy glycolysis will be turned on at a high rate. High amounts of ATP signal a decreased need for cellular energy and slow down the pathway. This occurs because ATP is a negative effector of a key pathway enzyme.
24. (Not shown in handout – same as slide #17) The Bridging Reaction converts pyruvate into acetylCoA.
25. This slide shows the net reaction of the Bridging Reaction. Pyruvate is oxidized into an acetyl group and NAD+ is once again reduced to NADH.
26. Once again the potential to further oxidize acetylCoA and NADH can lead to significant amounts of ATP. This is the primary goal of the pathway. However, acetylCoA is also a key intermediate branching point for making fatty acids, cholesterol and other steroids, etc. This is the secondary purpose.
27. Pyruvate produced by glycolysis must enter the mitochondria, which is the location of the bridging reaction, Krebs Cycle, and Oxidative Phosphorylation Pathway. As with glycolysis, a low energy charge is the main stimulator of the pathway, while a high energy charge will slow it down. This is a key irreversible step in metabolism and is highly regulated by allosteric enzymes, covalent modification, and product inhibition.
28. In the Krebs cycle, the acetyl group from acetylCoA is oxidized to CO2. Four different dehydrogenases reduce 3NAD+, and 1 FAD into 3NADH and FADH2. Also 1 GDP is phosphorylated to GTP, the energy equivalent of ATP.
29. The reasons are very similar to glycolysis and the bridging reaction. The reduced coenzymes formed can eventually be converted into ATP by the Oxidative Phosphorylation pathway. Also Krebs intermediates can be converted into glucose, amino acids, or heme groups.
30. The full Krebs cycle with intermediates shown. The key dehydrogenase steps produce NADH or FADH2. The regulatory steps are indicated by the molecules in blue, which are inhibitors or allosteric effectors. Once again, high ATP levels (a high energy charge) is the primary negative effector. High AMP or low EC will accelerate the pathway. The steps that can be diverted for anabolic synthesis reactions are shown with the products in orange.
31. (Not shown in handout – same as slide #17). All the NADH and/or FADH2 produced by glycolysis, the bridging reaction, and the Krebs cycle can yield additional ATP if they are oxidized by the Oxidative Phosphorylation Pathway (also called the Respiratory Chain).
13 Metabolism2 – Oxidative Phosphorylation
1. The three stages of catabolism are: 1) Digestion/hydrolysis of macromolecules into their corresponding monomers (fatty acids, amino acids, or sugars). 2) Oxidation of these ‘fuel’ molecules by many pathways causing the reduction of NAD+/FAD into NADH/FADH2. 3) The oxidation of NADH/FADH2 by the Oxidative Phosphorylation pathway that provides the energy to convert ADP into ATP.
2. In oxidiative phosphorylation, the NADH produced by glycolysis, the bridging reaction, the Krebs Cycle, or other catabolic pathways are oxidized. Oxygen is the oxidizing agent and gets reduced to form water. This allows for the conversion of ~ 89% of the energy contained in a glucose molecule into ATP.
3. The net reaction for the oxidation of NADH by the Oxidative Phosphorylation pathway includes the oxidation of NADH/H+ to NAD+ with the reduction of O2 to H2O. The benefit of this is that 3 ADP are phosphorylated to make 3 ATP. This is how the cell replenishes its spent energy currency. The combination of the oxidation/reduction part only is called electron transport.
4. If FADH2 is oxidized instead of FAD, then there are only 2 rather than 3 ATP molecules formed.
5. The enzyme complexes for electron transport and the ATP Synthase complex are both found in the inner membrane of the mitochondria. The inner membrane is highly folded to increase its surface area, which allows a larger number of the electron transport and ATP synthase complexes. The aqueous fluid between the two membrane areas is called the inner membrane space (ims), while the aqueous fluid at the center is called the matrix.
6. This slide lists the names of the electron transport complexes. You need not memorize these. However, the name indicates what is oxidized and what gets reduced. For example in complex I, NADH is oxidized (to NAD+), and CoQ (coenzyme Q) is reduced (to CoQH2).
7. During electron transport, protons (H+ ions), are pumped by each of the transmembrane complexes of Electron Transport (I(, succinate dehydrogenase is not a transmembrane complex) across the inner membrane of the mitochondria. That means that there are more H+ ions out in the inner membrane space, than in the matrix. The difference in ion concentration across a membrane is called a gradient.
8. Each of the three transmembrane complexes of the electron transport chain, form enough of a gradient to produce 1ATP (per NADH oxidized). Each complex involves an oxidation/reduction process with the final reduced product moving on to the next complex to be oxidized while allowing more protons to be pumped out of the matrix. The final oxidizing agent is oxygen. This is why we breathe, and why Hemoglobin must transport oxygen to every cell.
9. The net effect of electron transport is to produce the H+ or pH gradient across the inner membrane. As a result the ims has a pH about 1.4 units lower than the matrix.
10. Just as a dam holding back water flow produces a potential energy difference that can be converted into useable energy (electricity), the proton gradient is a form of potential energy to the cell that can be converted into useable energy (ATP).
11. ATP Synthase contains a transmembrane channel that brings protons back into the matrix. It also contains a catalytic subunit that phosphorylates ADP to make ATP. The energy required to drive this reaction comes from the proton flow through the channel portion, just like the energy required to generate electricity comes from water flow through the dam.Another transmembrane protein allows the ATP produced in the matrix to flow out of the mitochondria into the cytosol, and brings ADP into the matrix to be reconverted into ATP. This allows the cell to match the rate of ATP production to the rate of work output. In other words the more energy the cell consumes, the faster oxidative phosphorylation will run.
12. Because the succinate dehydrogenase complex (I() is not a proton channel, this complex does not contribute to the proton gradient when FADH2 is oxidized. However, it still produces CoQH2 that is sent to the 2nd electron transport complex. This essentially skips the first complex and the oxidative phosphorylation pathway will only make 2 ATP rather than 3.
13. If the protons in the ims can enter the matrix without passing through the ATP synthase channel, they will not produce any ATP. They will however, produce heat. If this happens the electron transport system is said to be ‘uncoupled’ from ATP production. This can be accomplished by adding chemical uncouplers such as 2,4–dinitrophenol. In large enough amounts this compound is very toxic since it prevents cellular ATP production. Transmembrane proteins that are passive H+ transporters such as the Uncoupler Protein (UCP) also produce the same effect.
14. Hibernating animals, and newborns have more UCP in their brown adipose tissue because they need warmth more than the extra ATP. Essentially, your body can adjust your basal metabolic rate (like the idle on a car), by producing more or less UCP. This may help to partially explain those skinny people you know who eat everything in sight and never seem to gain any weight!
15. The NADH and FADH2 oxidized by the Oxidative Phosphorylation pathway can be made from glucose, amino acids, or fatty acids. The pathways producing these reduced coenzymes include glycolysis, the bridging reaction and the Krebs Cycle, which we have already seen. Later we will add two more catabolic pathways, β-oxidation, and oxidative deamination, that allow the oxidation of fatty acids and amino acids to make ATP. However, both of theses still require the production of NADH/FADH2 and the oxidative phosphorylation pathway.
14 & 15 Metabolism 3: Glycogen, Anaerobic Exercise, and Fasting
1. Nature contains numerous polysaccharides. Polysaccharides are carbohydrates made up by the connection (glycosidic bond) between many sugar units. Two common functions of polysaccharides are as structural support or energy (glucose) reserve molecules. Cellulose, starch, and glycogen are all polymers of glucose. However, because they are put together differently, they have different functions. Cellulose is a structural material used in forming plant cell walls (animal cells don’t have cell walls). Both starch (plants) and glycogen (animals) are glucose storage polysaccharides.
2. The pathway that makes glycogen from glucose in liver and muscle cells is called glycogenesis (make glycogen). Later when the cell needs a supply of glucose it retrieves it from stored glycogen by the pathway called glycogenolysis (break down glycogen).
3. The glucose units in glycogen (and the amylopectin of starch) are connected by α-1,4 & occasionally α -1,6 glycosidic bonds. The α-1,6 glycosidic bonds in glycogen cause a branching of the molecule. This allows more dense packing. Glycogen in the liver and muscle are present as “soluble” granules in the cytosol. You should be able to recognize the difference between these glycosidic bonds.
4. A schematic representation of glycogen where each circle represents a glucose unit. Horizontal lines represent α-1,4 glycosidic bonds and vertical lines represent α -1,6 glycosidic bonds.
5. Glycogen is stored for different purposes in liver and muscle cells. In liver, glucose stores are used to provide the blood with glucose during times of fasting, when the blood sugar levels are low. In muscle the glucose stores are used to meet the muscle’s ATP demands, particularly during anaerobic exercise. You should understand the different roles of glycogenolyis in liver and muscle and their connection to fasting and anaerobic exercise.
6. The Glycolytic intermediate, glucose-6-P, is the point where glycolysis can be abandoned in favor of glycogen storage. The enzyme glycogen synthetase is the major enzyme involved in making glycogen.
7. Glycogenolysis is the pathway that removes glucose one unit at a time from glycogen. This is done by the enzyme phosphorylase, that also add a phosphate group. It produces Glucose-1-P, which is eventually converted into Glucose-6-P, where it reenters the glycolysis (or gluconeogenesis) pathway.
8. This schematic diagram illustrates the function of phosphorylase.
9. In liver cells this glucose-6-P is converted into glucose via the gluconeogenesis pathway (to be introduced later). Muscle cells will simply convert glucose-6-P into pyruvate/lactate via glycolysis to produce ATP.
10. Glucose-6-Phosphate cannot pass through the cell membrane. The liver (but not muscle) contains glucose-6-phosphatase to cleave off the phosphate group. This allows glucose to be transported into the blood through the glucose channel. The glucose channel in the liver is an un-gated membrane protein that allows glucose to enter or leave the cell. During fasting conditions, when phosphorylase is activated, there will be more glucose in the cell than in the blood. This means that the net flow of glucose is outward into the blood.
11. Internal regulation of cell processes use allosteric enzymes or covalent modification. However, global regulation and cooperation among different cell types requires hormonal signals. This allows different cell types to perform specific functions in sync with different functions in other cell types. Hormones typically activate/inactivate internal regulation processes in their target cell types.
12. Epinephrine (adrenalin) is secreted in response to heightened emotional states. It works on muscle cells where it increases the glucose available for anaerobic muscle activity. In essence the emotional state is a cue for the body to prepare for anaerobic muscle activity that would be needed for fight or flight.
13. Epinephrine (or glucagon on liver) works via a cascade process that involves examples of allosteric stimulation as well as covalent modification.
14. A review of the net reaction of glycolysis as it would exist under aerobic conditions.
15. In anaerobic activity in muscle cells, there is insufficient oxygen delivered to the muscle to meet all of its ATP demands for exercise via the oxidative phosphorylation pathway. If the NADH produced in glycolysis could be reoxidized back into NAD+ without using oxygen, then glycolysis could continue to produce 2 ATP per glucose over and above the ATP produced by aerobic means. This supplemental ATP production is important during anaerobic exercise.
16. The means to convert the NADH back into NAD+ without using the oxidative phosphorylation pathway is the enzyme lactate dehydrogenase. Lactate is a byproduct of this reaction and has no value by itself. Later you can recycle the lactate back into glucose using gluconeogenesis in the liver. What is important here is to convert NADH back into NAD+ without consuming oxygen.
17. With an oxygen free method of reoxidizing NADH back into NAD+, the glycolysis pathway can now continue to produce ATP beyond the normal limit imposed by oxygen availability. The downside is that you consume much more glucose since you’re only getting 2ATP instead of 36. Because of this your muscle will run out of glycogen much faster during anaerobic activities compared to aerobic ones. Elite marathon runners usually work efficiently enough to minimize anaeorbic muscle activity while retaining a faster pace. They typically do not run out of muscle glycogen, a key contributor to ‘hitting the wall’.
18. Under anaerobic conditions the net reaction of glycolysis is slightly different, with no NADH available for the oxidative phosphorylation pathway and lactate as the final product rather than pyruvate.
19. A metabolic disorder is due to the absence of an enzyme (or some other protein). It can be congenital, which means it was due to a defect in your genes that you inherited from your parents. Or it may be acquired due to disease or failure of organs.
20. An example of a congenital metabolic disorder is McArdle’s disease. An individual who inherits this disorder does not produce sufficient muscle phosphorylase, the enzyme required for glycogenolysis. Since muscle glycogen is mainly present to support anaerobic exercise, an individual with McArdle’s disease is incapable of prolonged anaerobic activity. They can live normal lives by restricting muscle activity to aerobic levels.
21. Insulin is a hormone secreted in response to elevated glucose levels in the blood. It signals the Fed state. It acts on muscle cells to increase the number of glucose channels. This causes a decrease in blood glucose levels, since much of that glucose can now enter the muscle. There it can be stored as glycogen.
22. A plot of the glucose concentration level in blood over time is used to distinguish the fed vs. fasting states. After a meal the amount of glucose in the blood increases for a short period of time. This is the fed state. Insulin is secreted. The elevated amount of glucose in the blood is brief because the insulin causes the muscle cells to absorb the glucose from the blood thus decreasing the concentration. As it nears the normal level, glucagon is secreted and insulin is destroyed. This marks the beginning of the fasting state. You should be able to understand this plot and its connection to Fed vs. fasting states and metabolic activity.
23. Your metabolsim is set up to maintain a glucose concentration of about 80 – 100 mg/dL (a deciliter, dL, = 100 ml) of glucose. Having either too much or too little glucose in the blood can be harmful.
24. A diabetic who does not produce insulin will maintain high concentrations of glucose in their blood for a prolonged period of time, if they are not taking insulin injections. This produces the condition called hyperglycemia (hyper always means too much while hypo means too little).
25. Glucagon is the hormone secreted in response to lower glucose levels in the blood. It binds to a liver receptor and activates the glycogenolysis pathway. This pathway converts stored glycogen into glucose that the liver exports into the blood. Just like insulin, glucagon tends to reverse the condition that signaled its release. Insulin decreasing the glucose concentration in the blood while glucagon tends to increase it, or at least prevent it from dropping below normal levels. You should understand the difference between epinephrine, glucagon, and insulin and how they affect metabolsim. Later we see that they have other effects on different cell types.
26. During the early fasting stage, glucose levels are maintained in the blood by liver glycogenolysis. However, liver glycogen will only last about 6 – 12 hours after your last meal. When it is gone, you enter the late fasting stage.
27. If you have a meal that is too rich in simple sugars and low in protein and fat, you can develop a temporary condition called hypoglycemia (too little glucose in your blood). Eventually glucagon will remedy this situation, but your cognitive functions may be depressed as the brain is getting less glucose. In extreme cases an individual can pass out due to hypoglycemia.
28. (Not included in handout – same as #26) Review of the plot of glucose in the blood.
29. But what happens when you have used up all of your liver glycogen? How are the blood glucose levels maintained under these conditions (referred to as the late fast)?
30. This slide extends the x-axis of the previous slide well into the late fasting period which can last for about 3-6 days. Fortunately, the liver is capable of making glucose by a pathway called gluconeogenesis. In the late fasting stage, gluconeogenesis in the liver ensures that glucose levels in the blood are maintained.
31. Gluconeogenesis converts pyruvate (or the Krebs intermediate oxaloacetate) into glucose. This requires energy because it is an anabolic (3 carbons going to 6) pathway.
32. Gluconeogenesis uses glucose-6-phosphatase (just like glycogenolysis) in order to export glucose into the blood. Essentially, it is the reverse of glycolysis, with a few added steps to bypass the one-way reactions of the glycolytic pathway.
33. This slide places gluconeogenesis on the metabolic chart with glycolysis, the Krebs Cycle and Oxidative Phosphorylation. Gluconeogenesis is represented by the dotted arrow.
34. The pyruvate/oxaloacetate used to make glucose comes predominantly from the oxidation of amino acids. Since this means you are in a fasting state, you will also be hydrolyzing your triglyceride reserves to make fatty acids. This releases glycerol, which can also make glucose in the liver.
35. The amino acids used to make glucose come from the hydrolysis of your body protein. Thus beginning at the late fasting stage, your body will sacrifice its protein to maintain its glucose.
36. (Not included in handout – same as #30).
37. Pyruvate and oxaloacetate are α-keto acids. This means they have a keto group (-C=O) adjacent to a carboxyl group (-COO-).
38. Amino acids have the α-amino group adjacent to a carboxyl group. Note the similarity between alanine and pyruvate and between Aspartic acid and oxaloacetate. You should be able to recognize the structural difference between an amino acid and an α-keto acid.
39. To convert an amino acid into an α−keto acid one only has to remove the amino group and add an oxygen atom. Thus alanine can be made into pyruvate.
40. The reaction that accomplishes this is called oxidative deamination. In this reaction an amino acid is oxidized. NAD+ is reduced to NADH, which can be used for energy in the oxidative phosphorylation pathway. However, we are concerned with the remaining α−keto acid, which can be used to make glucose, if necessary.
41. The oxidative deamination of all 20 amino acids eventually results in the production of either pyruvate, oxaloacetate or acetylCoA. We will not go into the reactions that accomplish this, but you should understand the basic process of an oxidative deamination.
42. Only pyruvate and oxaloacetate can be converted into glucose, so an amino acid that is deaminated to produce either of these α−keto acids is called a glucogenic amino acid. If an amino acid is deaminated to produce acetylCoA it is called a ketogenic amino acid. You do not need to know the classification for each amino aicd, but you should understand what this classification means.
43. (Not included in handout – same as #34).
44. Liver gluconeogenesis is also utilized to recycle lactate produced by anaerobic exercise in the muscle, back into glucose. This process is called the Cori Cycle and is unrelated to fasting.
45. Both Hers’ disease and Von Gierke’s disease reduce an individuals ability to respond to fasting conditions. Hers’ disease only affects glycogenolysis. It is much more severe than McArdle’s disease which is due to the same problem (no phosphorylase) only in muscle rather than liver. However, Von Gierke’s disease, due to the absence of Glucose-6-Phosphatase, prevents both glycogenolysis and glucoeneogenesis responses during fasting. In its severest form it will be fatal.
Metabolic Diseases
Generic term for diseases caused by an abnormal metabolic process. It can be congenital due to inherited enzyme abnormality or acquired due to disease of an endocrine organ or failure of a metabolically important organ such as the liver. (Stedman, 26th ed)
Glycogen Storage Disease Type V (McArdle’s Disease)
Glycogenosis due to muscle phosphorylase deficiency. Characterized by painful cramps following sustained exercise.
16 Metabolism4: Fat Metabolism
1. Before we saw that insulin is the signal of the fed state. In addition to increasing the number of glucose channels in muscle cells, it also stimulates fatty acid synthesis in liver and adipose cells.
2. Fatty acids are produced from acetylCoA. The acetylCoA can come from glucose via glycolysis, and the Bridging Reaction, or from amino acids via oxidative deamination. The fatty acids are stored as triglycerides in adipose tissue.
3. Fatty acid synthesis is an anabolic process that consumes ATP. The hydrogen atoms necessary for this reduction process are donated by the coenzyme, NADPH, a counterpart to NADH (the P is a phosphate group). The purpose is to store energy as fat. It can occur in adipose tissue, but fatty acids are also made in the liver and sent to the adipose for conversion to fat. It makes sense that energy reserves will be bolstered during the fed state. It is the effects of insulin on fatty acids synthesis that accomplish this.
4. Fat is an excellent fuel reserve, because fatty acids contain > than 2x the energy per gram compared to carbohydrate or protein. In addition, fat is stored in an anhydrous state, where glycogen must be hydrated. It takes 2g of water to store 1g of glycogen. This provides an extra 3x benefit in terms of energy/per gram of fuel storage. Thus, fat is 6x more efficient than carbohydrate (or protein) for energy storage. Note that the only reason to have glycogen at all is to provide anaerobic capabilities in muscle, and for glucose export capabilities in liver. The latter is necessary because the brain can’t consume fatty acids. You should understand the chemical reasons that fat is a more efficient fuel source than carbohydrate or protein.
5. Glucagon, the signal of the fasting state, activates lipases in adipose.
6. Lipases hydrolyze the ester bonds of triglycerides. This provides fatty acids that are exported to the blood, where they provide a fuel source for many cells. In addition glycerol can also be oxidized for energy or converted into glucose in the liver (via gluconeogenesis). Insulin inhibits the lipase activity, so after you ingest a meal and your ‘fast’ is over, you will no longer release fatty acids from adipose tissue. You should understand the function of lipases and the conditions leading to their activation/inactivation.
7. Fatty acids are nonpolar, and thus insoluble in water. They must be transported in the blood by serum albumin. Primary consumers of fatty acids include either liver or muscle cells. However, fatty acids do not readily cross the blood/brain barrier, and thus fatty acids are not available to brain cells as a fuel source. This is the reason for the brain’s dependence on glucose for a fuel.
8. Once at their destination, fatty acids are oxidized by the β-oxidation pathway.
9. This pathway converts the 16 carbon fatty acid, palmitic acid, into 8 acetylCoA molecules. In addition the hydrogen atoms removed from the fatty acids are sufficient to reduce 7NAD+, and 7FAD coenzymes to 7NADH & 7FADH2. This is another example of the use of dehydrogenases. The AcetylCoA can be further oxidized by the Krebs Cycle. This produces yet more NADH/FADH2 which can be oxidized by the Oxidative Phosphorylation pathway to generate ATP. Fats require aerobic conditions for ATP production.
10. The primary consumers of fatty acids are liver and muscle. It occurs during the fasting state, since glucagon is the hormone that signals the adipose to release fatty acids.
11. However, this presents some unusual problems for the liver. During the fasting state the liver is also running gluconeogenesis, which robs the Krebs cycle of its starting molecule. Thus, much of the acetylCoA produced by β-oxidation goes un-oxidized, particularly in late and long-term fasting stages.
12. Instead of wasting this excess of acetylCoA, the liver converts them into ketone bodies. These are also exported into the blood along with the glucose from glycogenolysis/gluconeogenesis. The ketone bodies provide an alternate fuel source that can be used by heart muscle cells, and eventually by brain cells after they have adapted to absorb this fuel. This adaptation process takes about 3 days. This is what determines the transition from late fast to the long-term fasting state.
13. There are three ketone bodies, acetone, β-hydroxy butyrate, and acetoacetate. They are all in equilibrium, but acetoacetate is the main export product that can be picked up as a fuel by other tissues.
14. The brain can adapt to using acetoacetate as a fuel over time. It is the ability of ketone bodies to enter the brain across the blood-brain barrier that produces the change. Brain cells then convert the ketone bodies back into acetylCoA to be oxidized by the Krebs cycle. They still needs some glucose which will continue to come from amino acids via oxidative deamination and gluconeogenesis.
15. The adaptation to obtaining 75% of their energy from ketone bodies is beneficial because it reduces the need to oxidize amino acids to provide glucose for the brain. Thus it reduces protein breakdown in the body, conserving protein for synthesizing proteins. You should understand the relationship between the various fasting conditions and ketone body formation.
16. In a diabetic, liver gluconeogenesis continues during the fed state, because insulin is not available to turn it off in liver. Thus ketone bodies are exported at a time the brain has plenty of glucose and no need to use them. This results in a condition known as ketosis. Since 2 of the ketone bodies are acidic, this may result in serious acidosis (lowering of blood pH). This is often referred to as keto-acidosis, and can be fatal.
17. Leptin is a peptide hormone that aids in the regulation of body weight. It is secreted by subcutaneous fat tissue. Higher amounts of fat leads to higher [leptin] secretion. The hypothalmus reponds to high [leptin] by suppressing appetite, and possibly by turning up the bodies ‘idle’ metabolic rate (more UCP in the mitochondrial membrane). Low levels of leptin stimulate appetite, thus leading to weight gain. It has been suggested that leptin may be a possible obesity drug. However, obese mice have high levels of leptin, suggesting the obesity may be caused by an insensitivity to leptin. This could be caused by absent or defective leptin receptors in the hypothalamus of the brain. If this is the case, providing extra leptin would not be helpful.
17 Metabolism Review: (See handout with 4 w’s for each pathway)
1. Metabolic Main Street – Glycolysis, Bridging Reaction, Krebs Cycle, & Oxidative Phosphorylation. Anaerobic muscle activity and glycolysis.
2. Glycogenesis and Glycogenolysis.
3. Fatty Acid Metabolism – Lipase activity, β-oxidation, and ketone body formation.
4. Gluconeogenesis and the oxidative deamination of amino acids.
5. Specialization of the metabolism of brain, adipose, muscle, and liver cells.
6. Brain metabolism in the Fed state and early and late fasting states.
7. Brain metabolism in the long term fasting state.
8. Adipose metabolism in the fed state.
9. Adipose metabolism in all stages of fasting.
10. Muscle metabolism in the fed state following insulin secretion.
11. Muscles metabolism at rest.
12. Muscle metabolism during anaerobic exercise.
13. McArdle’s disease prevents anaerobic exercise in affected individuals.
14. Liver metabolism during the fed state.
15. Liver metabolism during the early fasting state.
16. Liver metabolism during the late fasting state.
17. Continuation of liver metabolism during the late fasting state.
18. Liver metabolism during the long-term fasting state.
19. (Not present in handout) Diabetics continue to run gluconeogenesis and export glucose during the fed state. This is because insulin is the original signal causing the shutdown of liver gluconeogenesis.
18 Nucleotides
1. Proteins, whether they be enzymes that catalyze reactions of key pathways, or act as channels to transport molecules into the cell, etc. are critical to an organisms function. But recall that their function depends on their structure, which in turn is predetermined by their amino acid sequence. Now we turn to the 4th class of macromolecules, nucleic acids. Nucleic Acids contain the directions that determine the sequence of an organism’s proteins.
2. There are two types of nucleic acids, DNA and RNA (RiboNucleic Acid) , which each play a role in specifying a protein’s sequence. However, it is the DNA that contains the entire set of directions within each cell. To understand how, we need to understand the structure of nucleotides, the building block units that make up nucleic acids.
3. Amino acids needed to make proteins generally come from our diet. Both amino acids, and the sugar ribose, which is made from glucose, are required to build nucleotides. These then can be polymerized to make either DNA or RNA. Once again metabolic pathways are critical to all aspects of cellular performance. We will not cover the pathways by which nucleotides are made.
4. Nucleotides have three components. A base, a sugar (a form of ribose), and 1 to 3 phosphate groups. A nucleoside is the combination of a base and sugar, with no phosphates.
5. Bases can be divided into two categories, pyrimidines and purines. Note the name ending changes slightly when the bases are connected to the sugar to form a nucleoside or nucleotides. Nucleotide names like Adenosinetriphosphate (ATP) use the name of the nucleoside not the base.
6. Pyrimidines contain a single 6-membered heterocyclic ring (this means there are two different types of atoms, C & N, in the ring). Purines contain a fused-ring structure where a 6-membered hetercycle shares a side with a 5-membered heterocycle. You should be able to recognize and classify a nucleotide into one of these two categories
7. The information found in DNA & RNA is contained within the bases structures. Specifically, it depends on the groups attached to the heterocyclic rings, and their ability to either donate or accept hydrogen bonds. This slide illustrates the structures and H-bonding patterns of the pyrimidines. You should be able to recognize the hydrogen-bonding pattern of a base given its structure, and understand how this influences the information content of nucleotides and nucleic acids.
8. This slide illustrates the structures and H-bonding patterns of the purines.
9. Ribose is a pentose (a 5-carbon sugar). The ring is numbered clockwise from right to left in this view of the molecule. ‘Primes’ are used to distinguish the numbering system from that of the purine/pyrimidine rings, which we did not cover. You should know the sugar numbering system since it is needed to understand terminology regarding the connection of nucleotides into nucleic acids. If the 2( Hydroxyl group (OH) is missing the sugar is called 2(-deoxyribose. The term deoxy accounts for the ‘D’ in DNA (DeoxyriboNucleic Acid)
10. In a nucleotide, the base is connected to the 1(-carbon of the ribose. The phosphate is attached by a phosphate ester bond to the 5(-carbon. Any additional phosphates are connected to the previous phosphate. Abbreviations are typically used to denote nucleotides, like dCDP in this case. The lower case ‘d’ denotes the deoxy form.
11. Nucleic acids are formed by connecting numerous nucleotides (always starting with triphosphate nucleotides for free energy reasons) into a long chain.
12. Two phosphates are removed from the 2nd nucleotide. The remaining phosphate is connected to the 5( carbon of one sugar by a phosphate ester bond, and to the 3( carbon of the other sugar, also by a phosphate ester bond. This is referred to as a 3(-5( phosphodiester bond (although it is really two bonds).
13. The sugar & phosphate groups make up the backbone of a nucleic acid strand. The bases are to a nucleic acid strand what the amino acid side chains are to a polypeptide. The order of bases from one end to the other is called the sequence. The information contained in a nucleic acid is determined by its sequence. You should understand the chemistry behind the covalent bond that connects nucleotides within a DNA or RNA strand.
14. In 1950, Erwin Chargaff determined that all sources of DNA contain equal numbers of A & T nucleotides and equal numbers of G & C nucleotides.Watson and Crick determined that this was due to the double helix structure in which every A on one strand will be paired (by hydrogen bonding) to a T on the other strand. The same is true for G and C. In the double helix the complementary strands always run in opposite directions relative to the 3’ and 5’ ends.
15. The information in the sequence is contained in the H-bonding capabilities of the bases. In order to see how the base-pairing occurs, we have to flip and turn one of the bases. Now you can see that Adenine will form 2 H-bonds to Thymine.
16. Note that in the same orientation, A cannot form H-bonds to C.
17. However, G and C can form 3 H-bonds. Thus we can have G(C or A=T pairs, called base-pairs, but not AC or GT pairs (more on this later).
18. Thus the base pairing between G(C & A=T pairs is what allows DNA to form a double helix. You should be able to distinguish between the hydrogen bonding that combines two DNA strands into a double helix and the covalent bonding (phosphodiester bind) that connects nucleotides into a strand.
19. The structure of the double helix.
20. This is the shorthand representation of a double-helix sequence. Note you must specify the ends of at least one of the strands, but the other is always opposite. Also if you know the sequence of one strand you should be able to indicate the sequence of the other.
21. Few things in chemistry are absolute. All of the bases actually exist in 2 structural forms. In the case of Thymine one is referred to as the keto form and the other the enol form. These two structures are in equilibrium with each other, much the same as the α & β forms of glucose or the active and inactive forms of an allosteric enzyme. However, by a ratio of 10,000:1 (or 104:1) the keto form predominates. Nonetheless, this provides the foundation for mis-pairing that leads to mutations. These mutations arise from an ‘honest mistake’, not environmental stresses like radiation, uv rays, chemicals, etc., which will increase the baseline mutation frequency even more. This is a case where a low mutation rate is good, since it is required for the adaptation of organisms to environmental changes. However, more is definitely not better, and a higher mutation rate due to extreme environmental challenges is almost always detrimental to the organism.
22. In its ‘enol’ form, T can no longer pair with A …..
23. … but it can pair with G. Each base has a similar ‘improper’ form.
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