MODULE NINE

___ paralysis is seen in dairy cows with milk fever

flaccid

this mineral is involved in the conversion of T4 to T3, which are both thyroid hormones

selenium

this protein is responsible for preventing calcium from causing cell death

calbindin

this is the primary site for absorption of copper in the body

small intestine

uric acid formation contributes to this condition

gout

a mineral required in the diet at a level of < 100 mg per day

micromineral

this is where most of the calcium and phosphorus is stored in the body

bone

this metal is known to reduce molybdenum absorption

tungsten

if at toxic levels, selenium can be eliminated via the

lungs

copper toxicity is especially a concern for this species

sheep

a calcium or phosphorus deficiency could cause this in young animals

rickets

limestone would be considered a good source of this mineral

calcium

this results from the combination of molybdenum and sulfates

thiomolybdates

this mechanism describes how sodium is exchanged between venous blood and arterial blood in order to keep sodium at the tip of the villi

counter current

a genetically caused deficiency in this mineral could lead to neurodevelopment disorders

molybdenum

copper is a component of this protein that is responsible for converting iron into transferrin for iron transport in the blood

ceruloplasm

wilson’s disease results in copper accumulation, which ultimately results in ____ failure

liver

this condition could arise if cattle graze early spring forage

grass tetany

tight curly brown hair on black Angus cattle could be a result of this mineral deficiency

copper

a copper deficiency could result in a functional ___ deficiency

iron

Which of the following are considered **macrominerals**? Please select all that apply.

chloride (Cl)

sodium (Na)

calcium (Ca)

Which of the following would be good sources of both calcium and phosphorus? Please select all that apply. 

dairy products

bone meal

Explain the mechanism of active calcium absorption.

During periods of low serum Ca++ the active transport of calcium is activated by calcitriol and becomes the primary mode of Ca++ absorption from the GI tract lumen.

**Calcitriol** elicits its effect on GI tract enterocytes by activating gene transcription *to upregulate three proteins in the enterocyte*.  

* **The first protein** is the **calcium channel** facing the lumen (apical membrane of enterocyte) of the GI tract. This channel allows for the flow of calcium into the cell.  
* **The second protein** is **calbindin**. Calbindin is responsible for binding free calcium in the cell and transporting it to the blood side of the enterocyte.  In this, it serves a secondary function in that it helps keep free calcium levels low in the cell which helps with the influx of calcium from the lumen, *but perhaps more importantly it keeps calcium from triggering cell death (apoptosis).*  
* **The third protein** facing the blood (basolateral membrane of enterocyte) is an active transport protein – **Calcium ATPase** – which uses ATP to power the pumping of calcium out of the cell.

When is active transport of calcium most important?

When serum calcium levels are low.

Explain passive absorption of calcium.

The passive transport of Ca++ *requires no metabolic energy* as calcium is flowing down its concentration gradient following water that is *pulled from the lumen to the blood by high sodium levels at the absorptive tip of the luminal villi.*  T

he villus tip **high sodium levels are created** by actively pumping sodium across the enterocytes and then maintaining the sodium levels at the tip of villi by a *counter current mechanism*. 

The **counter current mechanism** is an *exchange of sodium between venous (returning blood) and arterial (new blood entering the villi) blood* (i.e., moving down its concentration gradient in the exchange). This means that arterial blood is continually pushing sodium back to the tip of the villi.  *The flow of calcium with water is between the enterocytes (paracellular) - not through the cell as seen with active transport.*

Which of the following, when deficient, results in a condition known as "Milk Fever" with characteristic flaccid paralysis in high producing dairy cows ?

calcium

Which of the following, when deficient, results in a condition known as "Grass Tetany" with characteristic rigid paralysis in cattle grazing early spring forage?

magnesium

Which of the following should a nutritionist consider to avoid milk fever? Please select all that apply.

calcium in the diet

phosphorus level in diet

calcium:phosphorus ratio

phosphorus requirement of the animal

calcium requirement of the animal

Which of the following are microminerals (trace)? Please select all that apply. 

copper

zinc

iron

component of thiomolybdate

molybdenum

component of ceruloplasmin

copper

fatty acid peroxide repair

selenium

conversion of T4 to T3

selenium

purine catabolism

molybdenum

needed for iron transport and absorption

copper

sulfite dextoxification

molybdenum

Wilson's disease

copper toxicity

**abnormal movement/posture**

selenium toxicity

**loss of hair pigmentation**

copper deficiency

**dislocation of the lens of the eye**

molybdenum deficiency

microcytic hypochromic anemia

copper deficiency

long bone joint erosions

selenium toxicity

The __________________ is the primary excretory path for copper.

feces via bile

Why does a molybdenum toxicity lead to symptomology that is similar to that of copper deficiency? 

It forms thiomolybdates that tie up copper (e.g., reducing its absorption)

Which of the following forms of selenium have good absorption profiles and thus, would be good selenium supplements for animals? Please select all that apply.

Se-methinonine (selenomethionine)

Selenate (Se6+)

Selenite (Se4+)

Selenide (Se2-)

The primary excretory form of selenium is _______________________.

trimethyl selenium

Which of the following vitamins/minerals are involved in the protection the cell membrane? Please select all that apply.

sulfur

vitamin E

niacin

selenium

vitamin C

riboflavin

What is a mineral?

An inorganic element required for maintenance and production within living systems.

Macromineral defined

*A mineral required in the diet at a level of* __***>100 mg/day***__***.***

The Macrominerals

* *Calcium (Ca)*
* *Phosphorus (P)*
* *Magnesium (Mg)*
* Sodium (Na)
* Chloride (Cl)
* Potassium (K)
* Sulfur (S)

Calcium

Calcium is found in solution as a cation, Ca++ and as a component of ***hydroxyapatite in bone, Ca10(PO4)6(OH)2****.*

Calcium Feed/Food Sources

* Bone meal (top left, back)
*  \~2:1 Ca:P ratio
* Limestone (top left, front)
* Grass hay (top right)
* Moderate levels (0.31-0.36% of DM)
* Legume hay (bottom left)
* High levels (1.2-1.7% of DM)
* Dairy products

Calcium absorption

Calcium absorption occurs in the duodenum, jejunum and ileum of the small intestine and large intestine/cecum of an animal’s gastrointestinal tract.  It is transported via one of two primary transport mechanisms, __**Active or Passive Transport**__. (separate)

Sites of absorption and contribution of calcium

Passive and active transport are generally available in all these parts of the GI tract

* Duodenum
* greatest contribution when serum calcium is low (>50%)
* Jejunum/ileum
* most of the calcium absorption when calcium is adequate in diet and serum (>80%)
* Large intestine/cecum
* \~11% of calcium when serum calcium and dietary levels are adequate
* \~7% of total active transport of calcium

Storage of Calcium

* 98-99% in bone
* Storage is long term, months to years

Elimination of Calcium

* ***Primarily fecal***
* Dietary calcium
* Endogenous calcium from GI tract cell turnover
* Urinary
* Primarily only seen under *hypercalcemiaLinks to an external site.*
* 99% of filtered calcium normally reabsorbed by the kidney during periods of normal to low serum calcium levels

Functions of Calcium

* Structural component of bone
* Hydroxyapatite
* Activation of some hydrolytic enzymes, e.g.:
* Blood clotting proteins
* In fact, the purple top blood tubes (for collecting plasma) contain a compound called EDTALinks to an external site. that chelates Ca++ (i.e., binds it) in the collected blood thereby blocking interaction of Ca++ with blood clotting enzymes and subsequent clotting of blood.
* As a component of calcium binding proteins, affect:
* Cellular movement
* Movement of secretory vesicles/organelles
* Second messenger system component, affect:
* Hormone secretion
* Muscle contraction
* Phagocytosis
* Cell division

Calcium levels cause … and Deficiency

* __***Low serum calcium***__ results in increases of parathyroid hormone and calcitriol levels to increase blood calcium from bone and kidney and GI tract absorption.
* __***High serum calcium***__ results in stimulation of calcitonin that works to reduce blood calcium levels.

Phosphorus

Phosphorus is located in many places within the body and has a broad range of functions.  It is a component of hydroxyapatite in bone, functions as a buffer (e.g., saliva), functions as a regulator of some protein functions and is a component of ATP, RNA and DNA, just to name a few.

Sources of Phosphorus

* bone meal
* 2:1 ratio
* grains
* seed byproducts
* pelleted phosphorite
* guano phosphate
* dairy products

Phosphorus Absorption by tract region

* Duodenum
* Sodium dependent active transport, via co-transport with sodium
* Jejunum/Ileum
* Primarily passive transport (see description under calcium for general passive transport mechanism)

Storage of Phosphorus

* 85% in bone – long term storage
* 14% in cells
* 1% in extracellular fluid

Phosphorus Functions

* Structural component of bone
* Structural component of cell membranes
* Structural component of RNA/DNA
* Regulation of metabolism
* ATP production
* Buffering
* Cellular
* Renal
* Ruminant saliva

Phosphorus deficiency

* Anorexia
* Rickets (also seen in vitamin D and Ca++ deficiencies)
* Osteomalacia (also seen in vitamin D and Ca++ deficiencies)
* Muscle weakness
* Malaise

Calcium to Phosphorus Ratio – An Important Concept

Calcium to phosphorus ratio is an important concept to understand as it is important to balance not just the levels fed, but also their ratios to meet specific physiological functional needs.  Please note, we do not want to overfeed one in relation to the need of the other as regulation of the serum level and function of these minerals is linked.

For example, a high producing milking dairy cow would need a calcium to phosphorus ration of nearly 7:1 to adequately balance calcium against phosphorus, whereas a dry beef cow would only need a 1:1 ratio as her calcium needs would not be as high, but phosphorus would remain the same.  Likewise, a laying hen would need a ratio of 10:1. Both the dairy cow and the layer are losing calcium from the body to a product (milk and eggs, respectively).

Consequence of an incorrect ratio (Ca: P)

* Soft eggshells (loss of product)
* ***Milk fever*** (not only loss of product, but also loss of cow)
* Occurs in high producing dairy cow post parturition
* Hypocalcemia (low blood calcium) is the complication
* Can’t adequately meet calcium from bone stores and diet
* Causes a ***flaccid paralysis (no muscle contraction)***
* ***Treatment*** generally calcium chloride/calcium lactate injections or infusions
* ***Prevention*** pay close attention to calcium to phosphorus levels and ratios in the diet to insure meeting the animal’s needs (not exceeding or creating deficiency)

Magnesium

Magnesium is involved in cellular regulation and is required to stabilize ATP (see figure below) for utilization by metabolic pathways.

Sources of Magnesium

* Meat
* Most plants
* Season and rate of growth may affect plant levels
* Potential issue – Grass tetany
* Milk is not a good source for the most species
* Human milk is an exception – it is high in magnesium

Absorption of magnesium

* Sites of absorption
* Distal parts of ileum
* Descending colon
* Modes of absorption
* Active transport
* energy expended by Na+/K+ ATPase (coupled to transport)
* counter transport with Na+
* used to fine tune Mg++ levels
* may share Ca++ transporter
* Passive transport
* Paracellular transport similar to that of calcium

Storage of magnesium

* 60-65% in bone (20-30% freely exchangeable)
* 27% in muscle
* 6% in other cells
* 1% in extracellular fluids

Elimination of magnesium

* Fecal – unabsorbed dietary Mg
* Urinary
* 60-80% of Mg filtered is reabsorbed by the kidney

Functions of magnesium

* Required by ATP using enzymes
* Carbohydrate and lipid metabolism
* Cytoskeletal integrity
* Insertion of proteins in cell membrane
* Activation of adenylate cyclase
* DNA, RNA, protein synthesis
* Stabilize structure of DNA and ATP

Deficiency (rarely seen; see figure below) (magnesium)

Grass tetany

*a condition that may present in cattle grazing early spring lush forage*.  Forage early in the spring, when temperatures are cool and moisture tends to be widely available, grow rapidly and produce large amounts of feed.  However, the rapid growth limits the uptake of some minerals (e.g., magnesium) resulting in potential deficiencies in the grazing animal (primarily cattle).  In the case of magnesium deficiency, cattle may exhibit the condition.

* a "rigid paralysis (i.e., tetany)" of muscle in the animal (including the diaphragm) leading to death of the animal.  Generally there is no chance to treat the condition as the animal is usually found dead.  As a precaution, most producers provide a mineral supplement high in magnesium during the spring months.  As the plant matures and does a better job of taking up magnesium, the use of a high magnesium mineral supplement may decrease.

The Microminerals

* Boron (B)
* Cobalt (Co)
* Chromium (Cr)
* ***Copper (Cu)***
* Iodine (I)
* Fluorine (F)
* Iron (Fe)
* Manganese (Mn)
* ***Molybdenum (Mo)***
* ***Selenium (Se)***
* Silicon (Si)
* Zinc (Zn)

Copper

Copper is a micromineral that exhibits two oxidation states (see figure below), allowing it to participate in oxidation-reduction reactions.  For example, copper is a central element in electron transfer in cellular respirationleading to ATP production. (Cu+, Cu++)

Sources of Cu

* Seafood
* Meat
* Nuts
* Grains
* Dairy products
* Vegetables
* Fruits

Functions of Cu

* Oxidation/reduction reactions (e.g., electron transport chain – cellular respirationLinks to an external site.)
* Ceruloplasmin
* Oxidation of Fe++ to Fe+++ for incorporation into transferrin for iron transport in blood
* Cytoplasmic superoxide dismutase
* Functions to convert oxygen superoxideLinks to an external site. to peroxide
* Antioxidant function

Absorption of Cu

* Sites of absorption include the abomasum/stomach and the __***entire small instestine.***__ The small intestine is the most important as absorption via the abomasum/stomach is quantitatively limited.
* Copper is absorbed via both active and passive transport mechanisms.
* Rat research data indicates that absorption of copper by active transport is most important in mature animals, whereas the passive transport is most important for the neonate.
* Rate of absorption is affected by copper levels in the body
* Low levels increase copper absorption (to about 70% of available copper)
* High levels decrease copper absorption (to about 12% of available copper)
* Copper absorption can be antagonized by a number of interacting elements
* __*Thiomolybdates*__(molybdenum and sulfur complex)
* Phytates (phosphorus containing compound)
* Zinc, selenium, cadmium and copper
* Induce metallothionein
* Sequesters some metals (minerals) to protect animal against high levels (toxicity) – in the enterocyte this reduces absorption of these elements
* Copper least effective at inducing metallothionein, but its ability to induce metallothionein is one mechanism by which high copper reduces its absorption

Elimination of Cu

* Primarily via bile
* Recall enterohepatic circulation– reabsorption of bile eliminated compounds by the small intestine – considered a conservation process, but may lead to toxicity in the case of copper (and other compounds that are marginally high in the diet and eliminated by the bile)
* Minor losses via urine, sweat and pancreatic and intestinal secretions

Deficiency of Cu

* Anemia (*microcytic, hypochromic anemia*– creates a functional iron deficiency)
* Ceruloplasmin (copper containing) required for iron transport and absorption
* Without copper, iron absorption and transport is limited - leading to a functional iron deficiency anemia
* Bone disorders
* Reproductive failure
* Nerve disorders
* Cardiovascular disorders
* Loss of hair pigmentation (on Black Angus cattle this shows as tight curly light brown hair)
* Poor growth & appetite

Toxicity of Cu

* Dullness and weakness
* Hemolytic anemia (oxidative stress induced by excess copper leads to lysis of red blood cells)
* Respiratory distress
* Pulmonary edema
* Jaundice
* Hemoglobinuria
* Anorexia

Copper toxicity in sheep

In the figure below, you can see what some of the effects of copper toxicity would look like in sheep.  The urine is dark (black) due to hemoglobin from lysed red blood cells being eliminated by the kidney.  The kidney (from an intoxicated sheep) that is black and dark red (due to hemoglobin) stands in contrast to healthy kidneys that are light pink in nature.  The center photos show a jaundiced (yellowish) liver (due to bilirubin – breakdown product of red blood cells) in comparison to a dark red healthy liver being held in hand.  The pictures of the sheep lip and body show yellow of epithelial layers (due to jaundice).

Wilson’s disease

Genetic disorder of humans that results in buildup of copper (toxic levels) in the body and eventually death (see figure below).

Molybdenum

Molybdenum is a micromineral that exhibits eight oxidation states contributing to its broad range of interactions with other elements.  Of particular importance to nutrition are its interactions with sulfur to form thiomolybdates and its function in “molybdenum cofactor” (see figure below).

Sources of Molybdenum

* Meats
* Liver
* Nuts
* Seeds
* Vegetables
* Grains
* Forages (especially high molybdenum soils)

Molybdenum cofactor functions

Molybdenum’s primary metabolic functions are as an enzyme cofactor (molybdenum cofactor) in purine catabolism (breakdown of purine bases found in DNA/RNA).  One of the enzymes in this process, xanthine oxidase is involved in uric acid formation which contributes to the condition of gout(a type of arthritis).  The other enzyme system utilizing molybdenum cofactor is sulfite oxidase, which is responsible for detoxification of sulfite (an oxidant that would otherwise be toxic; see figure).

Absorption of Molybdenum

* As free molybdenum, absorption is rapid
* As a complex with sulfur, i.e., thiomolybdates – slow
* Tungsten is known to reduce absorption

Elimination of Molybdenum

Is via both the bile and urine and is generally rapid.

Deficiency of Molybdenum

* Actual cases are extremely rare (molybdenum ubiquitous in food)
* Deficiency symptoms/outcomes are "inferred" from genetic mutation in the enzymes requiring molybdenum cofactor
* Neurological damage
* Neurodevelopmental disorders
* Humans – reduced intellectual disability
* Dislocation of lens of the eye
* Death

Toxicity of Molybdenum

Primarily tied to the effects of thiomolybdates and/or sulfites generated by excess molybdenum.  With the exception of the superoxide radicals due to sulfite toxicity, the toxicity symptoms are primarily related to the induced copper deficiency caused by the thiomolybdates.

* *Microcytic hypochromic anemia* (induced copper deficiency leading to functional iron deficiency)
* Reduced growth
* Diarrhea
* Low reproduction
* Cardiac failure
* Generation of superoxide radicals
* Lipid peroxidation
* Protein oxidation

Thiomolybdates

### ***Thiomolybdates in the gut***

Thiomolybdates are the product of sulfide interactions with molybdenum and a bigger concern for ruminants than nonruminants due to microbial conversion of sulfate to sulfide in the rumen.  These negatively charged thiomolybdates readily react with positively charged copper ions in the lumen of the GI tract to form highly insoluble copper thiomolybdate and thus, prevent copper absorption.

### ***Thiomolybdates as copper toxicity treatment***

The interaction of thiomolybdate with copper led to speculation that the thiomolybdates might function as a treatment of copper toxicity.

Two routes of treatment have been tested, but neither proved very effective.


1. ***The sequestration of copper in the blood with thiomolybdates*** required large amounts of thiomolybdate to affect a reduction in copper availability and ceruloplasmin with limited effects on copper elimination.
2. ***Sequestration of copper in the lumen of the digestive tract,*** based on thiomolybdates effect on normal copper absorption, was proposed to limit enterohepatic circulation of copper and thereby decrease copper reabsorption in intoxicate animals. However, the approached appeared to have little effect on body copper stores.

Selenium

Selenium is a micromineral with at least 4 oxidation states, and is required for only two known enzymatic functions in the body.  It serves as a structural cofactor for both ***glutathione peroxidase*** and ***5’iodothyroinine deiodinase***.

* __***Glutathione peroxidase***__ is responsible for repairing fatty acid peroxides in cell membranes (antioxidant activity) and we will revisit it in a moment.
* __***5’iodothyroinine deiodinase***__ is the enzyme necessary for conversion of thyroxine (T4) to triiodothyronine (T3), both thyroid hormones containing iodine and involved in regulating body basal metabolism.

Sources of selenium

* Shrimp
* Meat
* Milk products
* Grains
* Accumulator plants (these are a toxicity concern as they concentrate selenium in their tissue)
* *Astragulus bisulcatus*
* *A. pectinatus*
* *A. grayi*
* *A. praelongus*

Absorption (see figure)

Selenium forms with positive charges or those that are organic (e.g., Se-methionine) are more easily absorbed.  In addition, the organic forms elicit almost no toxicity issues.

Selenium is for the most part absorbed in the duodenum and lower portions of the small intestine.

Selenium *intake levels of less than 0.05 ppm results in deficiency* for many livestock species, while *levels in the hundreds of ppm lead to acute toxicity*.  *Intake levels of 5 to 50 ppm for an extended period* of consumption leads to “*Chronic Alkali Disease*” (a chronic selenium toxicity).

For humans, the safe upper limit is 200 mg/day, whereas less than 20 mg/day would lead to deficiency.

Storage of selenium

* Is short term and found in many cell types
* Liver
* Kidney
* Skeletal muscle
* Bone
* Red blood cells
* Glutathione peroxidase levels in these cells serve as a good clinical measure of Se status.

Elimination of selenium

* Primarily via urine as trimethyl selenium (methylation occurs in the liver)
* At toxic levels of selenium, trimethyl selenium may be eliminated via the lungs
* Fecal excretion is a result of complexing with other metals such as, Cu, Ag, Cd, Hg, As, Fe and Zn

Acute toxicity symptoms/effects of selenium

* Abnormal movement/posture
* Difficulty breathing
* Diarrhea
* Rapid death
* Often associated with accumulator plants

Chronic Alkali Disease (Se)

* Emaciation
* Malaise
* Cardiac atrophy
* Long bone joint erosions
* Hepatic cirrhosis
* anemia

Glutathione peroxidase and cell membrane protection

There are several vitamins and minerals involved in protecting the cell membrane (see figure below).

* Niacin (NADPH,H+)
* Riboflavin (FAD cofactor)
* Vitamin C
* Vitamin E
* Sulfur (glutathione)
* Selenium (glutathione peroxidase)

Each of these, work together to pass electrons and hydrogens to oxidized fatty acids (PLOO•-and PLOOH; phospolipid containing fatty acid radicals) in the membrane to neutralize them to fatty acid alcohols for removal/repair so that there is not a propagation of oxidation in the lipids of the cell membrane.  The latter would lead to necrotic cell death.

\
Figure key:

* Circles denote where vitamins and minerals play a role in this process.
* *Vitamin/vitamin containing components*
* __**NADPH,H+**__ = Nicotinamide adenine dinucleotide phosphate (contains niacin) – provides electrons to power the antioxidant process (i.e., generally to allow recovery of the antioxidants – glutathione, vitamin E and vitamin C after neutralization of an oxidant)
* __**ASC**__ = ascorbate (vitamin C) – shown in figure above in various states of oxidation
* __**TOC**__ = vitamin E - shown in figure above in various states of oxidation
* *Mineral containing components*
* GSH = glutathione – contains sulfur
* GSSG = dimerized oxidized glutathione - contains sulfur
* *Enzymes*
* GR-SS/GR-\[SH\] 2 = glutathione reductase (oxidized/reduced) enzyme
* Cofactor FAD (flavin adenine dinucleotide) = riboflavin (vitamin) containing
* Plase = phospholipase – clips damaged fatty acid from the membrane
* GPX = glutathione peroxidase (selenium containing)
* GRO-SS/GRO-\[SH\]2 = glutathione oxidoreductase
* Cofactor FAD (flavin adenine dinucleotide) = riboflavin (vitamin) containing