Novel uses of Hydroxyproline compositions

All patent and non-patent references cited in the present application are hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to novel uses of Hydroxyproline in a pharmaceutical or dietary food supplement composition for treating, ameliorating, or curing certain disorders in an individual in need thereof such as a human being, in particular disorders in collagen-rich tissues and organs, such as muscle and/or connective tissue disorders. The invention further relates to a food composition comprising hydroxyproline for feeding animals, such as fish, resulting in increased muscle strength, texture and firmness.

Background of invention

Essential amino acids are designated according to classic nutritional studies as being either essential or non-essential. While non-essential amino acids are able to be synthesised by the organism, essential amino acids must be supplied via the diet. Which amino acids are essential or non-essential respectively, varies according the biochemical make-up of the organism in question.

To ensure the optimal growth of an organism, it is generally accepted that the amino acids essential to that organism should be provided in sufficient amounts in the food or feed. This can be achieved either by choosing a source rich in those amino acids, or by supplementing the diet with the essential amino acids. Concurrently, it is also accepted that those amino acids established as non-essential amino acids do not necessarily need to be provided via diet.

Hydroxyproline is regarded as a non-essential amino acid. Hydroxyproline can be synthesized in the organism, and the metabolic capacity is expected to meet the amount required by the organism. Because it is formed by post-transcriptional hydroxylation of Proline, dietary supplementation of Hydroxyproline has not been expected to influence processes in the organisms that require Hydroxyproline. Instead, efforts to influence the amount of Hydroxyproline available to an organism have been concerned with either supplementing the diet with Proline, precursor to Hydroxyproline, and/or stimulating the hydroxylation process, most notably by increasing availability of the hydroxylase co-factor, vitamin C.

The texture of the muscle tissue of fish, i.e. the fish fillet, is important for both the initial handling of the fish during slaughter, the appearance of the product to be sold and for the taste and mouthfeel of the final product. This is also true for the texture of the meat of animals other than fish.

A number of different biological, environmental and dietary factors are reported to influence the texture of fish fillet including season, exercise, stress, chemical composition, size, slaughtering method and storing temperature and time. In addition, structural factors such as muscle fibre diameter distribution, collagen and collagen cross-links also influences texture.

A serious problem related to texture and the appearance of the fillet is a phenomena called "gaping". Gaping can be seen as splits and tears in a fillet where the myocommata fails to hold together the myotomal muscle, which is affected by handling, storage temperature and nutritional value. The knowledge of the mechanisms behind the phenomena are limited, but gaping is believed to be caused by a degradation of the myotendious junctions (the connection between ECM and the myofibres) caused by enzymatic degradation during storage. It has been shown previously that muscle fibre density has a significant influence on the presence of gaping. It was concluded that in salmon (Salmo salar) having a fibre density of > 95 fibres/mm, little or no gaping was present.

The cross-linking process is the final step in the collagen fibril formation, and the copper dependent enzyme lysyl oxidase is the only enzyme known to be directly involved and plays a crucial role in the side-by-side cross-linking of tropocollagens. However, the number of cross-links per tropocollagen is limited by the number of binding sites. In collagen type I four cross-link binding sites have been identified, one of which is located in each of the N-terminal and C-terminal telopeptide and two within the helical region. The functionality and tensile strength of the collagen fibril is primary due to the intermolecular cross-link formation. Cross-link formation can take place either via an enzymatic step or a step called glycation. The enzyme mediated lysine-aldehyde cross-link in collagen type I has two major pathways:

1 ) The allysine pathway which produces aldimine cross-links formed from aldehydes.

2) The hydroxyallysine pathway which produces ketoamine cross-links formed from hydroxyaldehydes.

The aldimine and ketoamine -cross-links in muscle tissue are both divalent reducible crosslinks capable of binding two tropocollagens together and takes place between the telopeptide and the helical region. In time these intermediate products are to a large extent replaced by mature non-reducible trivalent cross-links.

One example of a mature cross-link that accumulates in muscle tissue with age is the mature divalent hydroxylysyl pyridinoline (PYD). The formation of mature cross-links increases the strength of the collagen fibres significantly, since PYD is capable of binding three tropocollagen molecules together. The PYD crosslink is formed via the hydroxyllysine pathway together with another mature cross-link, lysyl pyridinoline, a dominant cross-link in bone. The formation of PYD takes place via a condensation reaction of two ketoamine cross-links. The final divalent cross-linking formation is mediated by fibre associated collagens and proteoglycans, in which proteoglycans can bind to collagens via their glycosaminoglycan chains.

Summary of invention

The present invention relates to novel uses of Hydroxyproline in a pharmaceutical or dietary food supplement composition for treating, ameliorating, or curing disorders in tissues and organs in an individual in need thereof such as a human being, in particular disorders of muscle and/or connective tissue, said tissues having high levels of collagen and/or elastin.

The invention further relates to a food or feed composition comprising hydroxyproline for feeding animals, such as fish, resulting in increased muscle strength, texture and firmness.

Definitions a-i collagen is total alkaline-insoluble collagen, a-i collagen can be measured as a-i hydroxyproline. a-s collagen is total alkaline-soluble collagen, a-s collagen can be measured as a-s hydroxyproline.

Cartilage is a type of dense connective tissue. It is composed of specialized cells called chondrocytes that produce a large amount of extracellular matrix composed of collagen fibers, abundant ground substance rich in proteoglycan, and elastin fibers. Cartilage is classified in three types, elastic cartilage, hyaline cartilage and fibrocartilage, which differ in the relative amounts of these three main components.

Cardiovascular disease is any of a number of specific diseases that affect the heart itself and/or the blood vessel system, especially the veins and arteries leading to and from the heart.

Connective tissue is derived from the mesodermal germ layer and refers to any type of biological tissue with an extensive extracellular matrix that supports, binds together, and protects organs. These tissues form a framework, or matrix, for the body, and are composed of two major structural protein molecules, collagen and elastin.

A connective tissue disease is any disease that has the connective tissues of the body as a target of pathology.

Edema is an abnormal accumulation of fluid beneath the skin, or in one or more cavities of the body. Generally, the amount of interstitial fluid is determined by the balance of fluid homeostasis, and increased secretion of fluid into the interstitium or impaired removal of this fluid may cause oedema.

Feed or food: Used herein these terms include feed or food additives, feed or food supplements, feed or food premixes.

Hydroxyproline (Hyp) is an amino acid produced by hydroxylation of the amino acid Proline Hydroxyproline differs from Proline by the presence of a hydroxyl (OH) group attached to the C (gamma) atom. Other forms of Hydroxyproline also exist in nature, notably 2,3-cis 3,4-trans-diHydroxyproline, as well as L- and D-isomers. The term hydroxyproline as used herein refers to all the above mentioned forms. Incontinence refers to involuntary discharge of urine or feces.

Diseases of the joints are any of the diseases or injuries that affect e.g. human joints.

A ligament is a tough band of connective tissue that connects various structures such as two bones.

The Mesoderm is a primary germ cell layer found in all higher animals (from flatworms to humans) in the very early embryo. Higher animals are thus triploblastic, possessing a mesoderm in addition to the two germ layers found in Diploblasts (the endoderm and the ectoderm). Triploblastic animals develop recognisable organs. The mesoderm forms: skeletal muscle, the skeleton, cartilage, peritoneum, the dermis of skin, connective tissue, the urogenital system, the heart and major blood vessels, blood (lymph cells), and the spleen. These tissues and organs are thus derived from the mesoderm and are considered mesodermal in origin.

Muscle is the contractile tissue of the body and is derived from the mesodermal layer of embryonic germ cells. Muscle as used herein refers to any type of muscle including skeletal muscle, smooth muscle and heart muscle.

A muscle disease is any disease that affects the function, quality, strength and/or firmness of one or more muscles.

PYD is short for pyridinoline. PYD cross-links are mature cross-links in muscle tissue and can be evaluated by hydroxylysyl pyridinoline cross-link analysis. The formation of mature cross-links increases the strength of the collagen fibres significantly, since PYD is capable of binding three tropocollagen molecules together.

Tendons are like ligaments in being tough, flexible cords. But tendons differ from ligaments in that tendons extend from muscle to bone whereas ligaments go from bone to bone at a joint. Tendons are considered a form of connective tissue. Brief description of Drawings

Figure 1 shows the mean SGR and FCR.

Figure 2 shows the pH in salmon muscle during storage for 5, 9 and 15 days.

Figure 3 shows the muscle firmness/texture measured as "work" of salmon muscle during storage for 5, 9 and 15 days.

Figure 4 shows the muscle firmness/texture measured as "shear force" of salmon muscle during storage for 5, 9 and 15 days.

Figure 5 shows insoluble collagen measured as acid-insoluble Hyp (a-i Hyp) in salmon muscle during storage at 4 ⁰ C for 5 and 9 days.

Figure 6. Shows soluble collagen measured as acid-soluble Hyp (a-s Hyp) in salmon muscle during storage at 4 ⁰ C for 5 and 9 days.

Figure 7. Free Hyp and GIy in salmon plasma.

Figure 8. Hyp in urine (A) and muscle tissue (B).

Figure 9. Shows PYD cross-links in salmon muscle during storage at 4 ⁰ C for 5, 9 and 15 days.

Figure 10 depicts the synthesis of hydroxyproline from proline.

Figure 1 1 depicts ketoamide cross-links of collagen.

Fig. 12 depicts hydroxylysyl pyridinoline (PYD) cross-linking of collagen.

Figure 13 depicts fish muscle structure (top and middle) and collagen arrangement (bottom).

Figure 14. (A) shows a flow diagram of collagen analysis. (B) shows a flow diagram of the PYD cross-link assay. Detailed description of the invention

Hydroxyproline is a major component of the protein collagen. Hydroxyproline and proline play key roles for collagen stability. Defects in collagen synthesis lead to easy bruising, internal bleeding, breakdown of connective tissue of the ligaments and tendons, and increased risk to blood vessel damage. Increased spill of hydroxyproline in the urine is generally associated with breakdown of connective tissue due to disease process and may also be a manifestation of vitamin C deficiency. Deficiency of hydroxyproline is only known to occur if there is a deficiency of vitamin C. Hydroxyproline has no known therapeutic use.

The only other mammalian protein which includes hydroxyproline is elastin. Elastin serves an important function in arteries and is particularly abundant in large elastic blood vessels such as the aorta. Elastin is also very important in the lungs, elastic ligaments, the skin, the bladder, elastic cartilage, and the intervertebral disc above the sacroiliac. It is present in all vertebrates above the jawless fish

Hydroxyproline is present in natural protein sources at varying levels.

Hydroxyproline is an amino acid produced by hydroxylation of the amino acid Proline Hydroxyproline differs from Proline by the presence of a hydroxyl (OH) group attached to the C (gamma) atom. Other Hydroxyprolines also exist in nature, notably 2,3-cis 3,4- trans-diHydroxyproline, as well as L- and D-isomers. The term hydroxyproline as used herein refers to all the above mentioned forms.

The present invention may comprise free hydroxyproline, or hydroxyprolyl residues such as those part of an oligopeptide, a polypeptide or a protein.

The invention concerns a composition comprising Hydroxyproline in the range of 0.1 % (w/w) to 100 % (w/w) of protein or a composition comprising a derivative of Hydroxyproline in the range of 0.1 % (w/w) to 100 % (w/w) of protein. For pharmaceutical applications pure Hydroxyproline could be applied.

The invention relates to all forms of hydroxyproline. The natural form of Hydroxyproline is present in blood plasma. Ionic forms and other derivatives of Hydroxyproline might be transformed to the pure form during digestion and/or intestinal absorption. The invention further relates to derivatives including a chelated Hydroxyproline compound (e.g. for slow release) as well as novel and inventive formulations of e.g. acylated derivatives of Hydroxyproline. An example is "biodegradable microsphere" comprising Hydroxyproline.

Muscle and connective tissue are derived from the mesoderm and comprise high levels of collagen and/or elastin. As hydroxyproline plays an important role in collagen and/or elastin function, the present invention contemplates the use of hydroxyproline for improving and/or increasing the quality of tissues derived from the mesodermal germ cell layer, in particular the mesodermally derived tissues having high levels of collagen and/or elastin, such as muscle and/connective tissue.

In one embodiment, the present invention thus relates to administration of a hydroxyproline composition to an individual in need thereof to improve and/or increase tissue quality, tone, texture, elasticity, firmness and strength, wherein said tissue is a mesodermally derived tissue, such as muscle and/or connective tissue with high levels of collagen and/or elastin.

The improved tissue quality may be measured by a number of methods known by a person of skill, such as those mentioned below. The mentioned indicators of improved tissue quality are exemplary only and are not to be construed as limiting for the scope of the invention:

• Increase in total amount of amino acids in tissues • Increase in concentration of acid-soluble and -insoluble hydroxyproline in tissues

• Increase in the level of PYD cross-linking in tissues

In one embodiment the hydroxyproline composition can be administered to an individual in need thereof to tighten and/or tone one or more muscles of said individual, where said muscles can comprise, but are not limited to muscles of e.g. buttocks, breasts, stomach, thighs and upper arms. Such treatment may be for cosmetic reasons only. In one embodiment the hydroxyproline composition can be administered to an individual in need thereof, including but not limited to athletes, bodybuilders and weight lifters, to obtain an improvement in muscle quality, tone, texture, firmness and strength.

In one embodiment the hydroxyproline composition can be administered to an individual in need thereof to tighten and/or tone subcutaneous connective tissue to obtain a cosmetic toning effect of the body.

Effect on muscles Muscle tissue is derived from the mesoderm and refers to any type of muscle including skeletal muscle, smooth muscle and heart muscle.

In one embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to affect e.g. the texture and/or strength of one or more muscles. This administration of the hydroxyproline composition can result in improved muscle quality, tone, firmness and/or strength.

In one embodiment the hydroxyproline composition can be administered to an individual to tighten one or more muscles of said individual. The treatment may be for health reasons or for cosmetic reasons only.

Another embodiment of the present invention relates to administration of the hydroxyproline composition to an animal to improve the meat quality of said animal. This administration can result in increased muscle firmness and/or improved taste and mouthfeel of the meat. The animal could in one embodiment be a fish, chicken, turkey, goose, duck, cow, horse, pig or sheep

In one embodiment, the present invention relates to a hydroxyproline administration to animals, such as fish for improving shelf-life of the final meat product.

The hydroxyproline composition can also be used for treatment of one or more muscle related diseases. Such muscle related diseases can be a muscle disease related to skeletal muscle, cardiac muscle, smooth muscle and/or the heart muscle. The muscle disease can be a muscle disease listed herein below or muscle weakness in general.

- Myopathy: Myopathies are diseases of skeletal muscle which are not caused by nerve disorders. These diseases cause the skeletal or voluntary muscles to become weak or wasted. Myopathies are usually degenerative, but they are sometimes caused by drug side effects, chemical poisoning, or a chronic disorder of the immune system.

- Chronic fatigue syndrome: Chronic fatigue syndrome (CFS) is an illness characterized by prolonged, debilitating fatigue and multiple nonspecific symptoms such as headaches, recurrent sore throats, muscle and joint pains, memory and concentration difficulties. Chronic fatigue syndrome is a debilitating and complex disorder characterized by profound fatigue of six months.

- Fibromyalgia: Fibromyalgia is a debilitating chronic illness characterized by diffuse pain, fatigue, and a wide range of other symptoms. It is a syndrome, not a disease. It is not contagious, and is probably genetic. It affects more women than men, mostly between ages 20 and 50. It is seen in 3-10% of the general population.

- Muscular dystrophy: Muscular dystrophy (MD) is a broad term that describes a genetic (inherited) disorder of the muscles. Muscular dystrophy causes the muscles in the body to become very weak. The muscles break down and are replaced with fatty deposits over time. Muscular dystrophy is a genetic disorder that is characterized by muscle wasting and weakening.

- Dermatomyositis: Dermatomyositis is one of a group of inflammatory muscle diseases. It is a subtype of inflammatory muscle disease. Dermatomyositis may affect people of any race, age or sex, although it is twice as common in women than in men. Dermatomyositis belongs to a group of conditions called inflammatory myopathies.

- Polymyositis: Polymyositis is a systemic connective tissue disorder characterized by inflammatory and degenerative changes in the muscles, leading to symmetric weakness and some degree of muscle atrophy. Polymyositis is slightly more common in females. It affects all age groups, although its onset is most common in middle childhood and in the twenties. - Rhabdomyolysis: Rhabdomyolysis is the breakdown of muscle fibers resulting in the release of muscle fiber contents into the circulation. Some of these are toxic to the kidney and frequenty result in kidney damage. Many clinical features of rhabdomyolysis are nonspecific, and the course of the syndrome varies depending on the underlying condition.

- Compartment syndrome: Compartment syndrome involves the compression of nerves and blood vessels within an enclosed space. Compartment syndrome is a condition in which there is swelling and an increase in pressure within a limited space (a compartment) that presses on and compromises blood vessels, nerves, and/or tendons that run through that compartment.

- Metabolic Diseases of Muscle: Metabolic diseases of muscle is caused by different genetic defects that impair the body's metabolism. Metabolic diseases of muscle include a) acid maltase deficiency (Pompe's disease), b) carnitine deficiency, c) carnitine palmityl transferase deficiency, d) debrancher enzyme deficiency (Cori's or Forbes' disease), e) lactate dehydrogenase deficiency, f) myoadenylate deaminase deficiency, g) phosphofructokinase deficiency (Tarui's disease), h) phosphoglycerate kinase deficiency , i) phosphoglycerate mutase deficiency and j) phosphorylase deficiency (McArdle's disease).

- Muscle diseases related to the nerve system: Muscle weakness may be due to problems with the nerve supply, neuromuscular disease such as myasthenia gravis or problems with muscle itself. These diseases include polymyositis, Amyotrophic lateral sclerosis, Botulism, Centronuclear myopathy, Myotubular myopathy, Dysautonomia, Charcot-Marie-Tooth, Hypokalemia, Lou Gehrig's disease, Motor neurone disease, Muscular dystrophy, Myotonic dystrophy, Myasthenia gravis, Progressive muscular atrophy, Spinal muscular atrophy, Cerebral palsy, Infectious mononucleosis, Herpes zoster, Vitamin D deficiency, Fibromyalgia, Celiac disease, Hypercortisolism (Cushing's syndrome), Hypocortisolism (Addison's disease), Primary hyperaldosteronism (Conn's syndrome), Ehlers-Danlos syndrome, Diarrhea, McArdle's Disease, Ross River Fever, Barmah Forest Fever and Conn's Syndrome. - degenerative skeletal muscle diseases including muscular dystrophies, myophathies and neurogenic diseases.

Effect on connective tissue

Connective tissue is derived from the mesoderm and the term as used herein refers to any type of connective tissue including bones, cartilage, tendons and ligaments.

In one embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on one or more connective tissues. The administration of the hydroxyproline composition can result in improved and/or increased connective tissue quality, connective tissue firmness, connective tissue elasticity and/or connective tissue strength.

In one embodiment the hydroxyproline composition can be administered to an individual to tighten and/or tone one or more connective tissues of said individual.

The hydroxyproline composition can also be used for treatment of one or more connective tissue related diseases or disorders to obtain a palliative, curative or preventive effect.

The connective tissue disease can be a connective tissue disease listed herein below or connective tissue weakness in general.

A connective tissue disease is any disease that has the connective tissues of the body as a target of pathology. Connective tissue diseases can have strong or weak inheritance risks, and can also be caused by environmental factors. Connective tissue diseases include the ones listed herein below.

1 ) Heritable Connective Tissue Disorders such as

- Marfan syndrome - a genetic disease causing abnormal fibrillin.

- Ehlers-Danlos syndrome - causes progressive deterioration of collagens, with different EDS types affecting different sites in the body, such as joints, heart valves, organ walls, arterial walls - Osteogenesis imperfecta (brittle bone disease) - caused by insufficient production of good quality collagen to produce healthy, strong bones.

- Stickler syndrome - affects collagen, and may result in a distinctive facial appearance, eye abnormalities, hearing loss, and joint problems.

2) Autoimmune Connective Tissue Disorders

These are also referred to as systemic autoimmune diseases. The autoimmune CTDs may have both genetic and environmental causes. Genetic factors may create a predisposition towards developing these autoimmune diseases. They are characterized as a group by the presence of spontaneous overactivity of the immune system that results in the production of extra antibodies into the circulation. The classic collagen vascular diseases have a "classic" presentation with typical findings that doctors can recognize during an examination. The classic collagen vascular diseases include:

- Systemic Lupus Erythematosus (SLE) - An inflammation of the connective tissues, SLE can afflict every organ system. It is up to nine times more common in women than men and strikes black women three times as often as white women. The condition is aggravated by sunlight.

- Rheumatoid Arthritis - Rheumatoid arthritis is a systemic disorder in which immune cells attack and inflame the membrane around joints. It also can affect the heart, lungs, and eyes. Of the estimated 2.1 million Americans with rheumatoid arthritis, approximately 1 .5 million (71 percent) are women.

- Scleroderma - an activation of immune cells that produces scar tissue in the skin, internal organs, and small blood vessels. It affects women three times more often than men overall, but increases to a rate 15 times greater for women during childbearing years, and appears to be more common among black women.

- Sjogren's syndrome - also called Sjogren's disease, is a chronic, slowly progressing inability to secrete saliva and tears. It can occur alone or with rheumatoid arthritis, scleroderma, or systemic lupus erythematosus. Nine out of 10 cases occur in women, most often at or around mid-life. - Mixed Connective Tissue Disease - Mixed connective-tissue disease (MCTD) is a disorder in which features of various connective-tissue diseases (CTDs) such as systemic lupus erythematosus (SLE); systemic sclerosis (SSc); dermatomyositis (DM); polymyositis (PM); and, occasionally, Sjogren syndrome can coexist and overlap. The course of the disease is chronic and usually milder than other CTDs. In most cases,

MCTD is considered an intermediate stage of a disease that eventually becomes either SLE or Scleroderma.

Effects on joints, ligaments and tendons

In one embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on one or more joints and/or ligaments and/or tendons, where said effect can be palliative, curative or preventive.

The present invention further relates to a method for treatment of one or more joint diseases comprising administration of the hydroxyproline composition. Joint diseases include but are not limited to Ankylosis, Arthralgia, Arthritis, Arthrogryposis, Arthropathy Neurogenic, Bursitis, Chondromatosis Synovial, Contracture, Hallux Rigidus, Hemarthrosis, Hip Dislocation Congenital, Joint Instability, Metatarsalgia, Nail-Patella Syndrome, Osteoarthropathy Primary Hypertrophic, Osteoarthropathy Secondary Hypertrophic, Patellofemoral Pain Syndrome, Shoulder Impingement Syndrome, Synovitis, Temporomandibular Joint Disorders, Spondylitis Ankylosing, Arthralgia, Shoulder Pain, Arthritis Rheumatoid, Gout, Osteoarthritis Periarthritis, Bursitis, Dupuytren's Contracture, Hip Dislocation Congenital, Joint Deformities Acquired, Osteoarthropathy Primary Hypertrophic, Shoulder Impingement Syndrome, Temporomandibular Joint Disorders / Temporomandibular Joint Dysfunction Syndrome, Arthritis in the knee, Osteoarthritis of the knee, Isolated femropatellar osteoarthritis (kneecap osteoarthritis), Rheumatoid arthritis in the knee, Lupus, Osteonecrosis of the knee. Joint disorders further comprises Symphysis Pubis Dysfunction (SPD), which is most commonly associated with pregnancy and childbirth. It is a condition that causes excessive movement of the symphysis pubis, either anterior or lateral, as well as associated pain, possibly because of a misalignment of the pelvis. SPD is a dysfunction that is associated with pelvic girdle pain and the names are often used interchangeably.

Tendon disorders include the following: • lateral epicondylitis (commonly known as tennis elbow) - a condition characterized by pain in the back side of the elbow and forearm, along the thumb side when the arm is alongside the body with the thumb turned away. The pain is caused by damage to the tendons that bend the wrist backward away from the palm.

• medial epicondylitis (commonly known as golfer's or baseball elbow) - a condition characterized by pain from the elbow to the wrist on the palm side of the forearm. The pain is caused by damage to the tendons that bend the wrist toward the palm. • rotator cuff tendonitis - a shoulder disorder characterized by the inflammation of the shoulder capsule and related tendons.

• DeQuervain's tenosynovitis - the most common type of tenosynovitis disorder characterized by the tendon sheath swelling in the tendons of the thumb.

• trigger finger/trigger thumb - a tenosynovitis condition in which the tendon sheath becomes inflamed and thickened, thus preventing the smooth extension or flexion of the finger/thumb. The finger/thumb may lock or "trigger" suddenly.

The term ligament is used to denote fibrous tissue that connects bones to other bones. They are sometimes called "articular ligaments", "fibrous ligaments", or "true ligaments". Ligament-related disorders include the following:

• Ligament pain in the knees originating from: Anterior cruciate ligament (ACL), lateral collateral ligament (LCL), posterior cruciate ligament (PCL), and medial collateral ligament (MCL).

• Chronic instability of knee defined by slipping knee, pain and recurrent hydrarthrosis • Other spontaneous disruption of ligament(s) of knee

• Other internal derangements of knee

• Ligament laxity -arthralgia, or symptoms such as frequent sprained ankles, shoulder dislocations, knee effusions and back problems are common among individuals with ligament laxity. • Laxity of ligament of knee

• Snapping knee - This type of mechanical symptom is often a sign of a meniscus tear or a loose piece of cartilage within the joint. The torn meniscus or loose cartilage may catch in the knee as it moves back and forth causing a popping sensation. Crepitus is the word used to describe a crunching sensation as the knee bends back and forth. Crepitus can be seen in patients with cartilage irritation, as is the case in chondromalacia, or in patients with cartilage wear, such as knee arthritis. Unlike a mechanical popping where there is a sensation of something getting caught in the knee, the sensation of crepitus is a more constant problem.

• Internal derangement of knee - a chronic disorder of the knee due to a torn, ruptured or deranged meniscus of the knee, or a partial or complete cruciate rupture, with or without injury to the capsular ligament of the knee, resulting in ongoing or intermittent signs and symptoms such as pain, instability, or abnormal mobility of that knee.

• Pregnancy- related ligament pain.

• Broad ligament laceration syndrome - Damage to muscle layers in the pelvis which allows the abnormally increased movement of the cervix. It often occurs after a traumatic surgical birth, induced abortion or excessive vaginal packing. • Instability secondary to old ligament injury

• Familial ligamentous laxity - also known as or related to hypermobility syndrome association, hypermobility syndrome (disorder), hypermobility syndrome, hms, double-jointed (hypermobility)

Effects on cartilage In one embodiment, the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on cartilage, where said effect can be palliative, curative or preventive.

The present invention further relates to a method for treatment of one or more cartilage diseases or disorders comprising administration of the hydroxyproline composition. Cartilage diseases and disorders include but are not limited to:

• Osteoarthritis: The cartilage covering bones (articular cartilage) is thinned, eventually completely worn out, resulting in a "bone against bone" joint, reduced motion and pain. Osteoarthritis is very common, affects the joints exposed to high stress and is therefore considered the result of "wear and tear" rather than a true disease. It is treated by Arthroplasty, the replacement of the joint by a synthetic joint made of titanium and teflon. Chondroitin sulfate, a monomer of the polysaccharide portion of proteoglycan, has been shown to reduce the symptoms of osteoarthritis, possibly by increasing the synthesis of the extracellular matrix. • Achondroplasia: Reduced proliferation of chondrocytes in the epiphyseal plate of long bones during infancy and childhood, resulting in dwarfism.

• Costochondritis: Inflammation of cartilage in the ribs, causing chest pain.

• Spinal disc herniation: Asymmetrical compression of an intervertebral disc ruptures the sac-like disc, causing a herniation of its soft content. The hernia compresses the adjacent nerves and causes back pain.

• Relapsing polychondritis: a destruction, probably autoimmune, of cartilage, especially of the nose and ears, causing disfiguration. Death occurs by suffocation as the larynx loses its rigidity and collapses. • Cartilage tumors

• Osteochondrosis - is a family of orthopedic diseases of the joint that occur in children and in rapidly growing animals, particularly pigs, horses, and dogs. They are characterized by interruption of the blood supply of a bone, in particular to the epiphysis, followed by localized bony necrosis, and later, regrowth of the bone. This disorder is defined as a focal disturbance of enchondral ossification

• Chondrocostal junction syndrome

• Relapsing polychondritis - is an uncommon, chronic disorder of the cartilage that is characterized by recurrent episodes of inflammation of the cartilage of various tissues of the body. Tissues containing cartilage that can become inflamed include the ears, nose, joints, spine, and windpipe (trachea). Tissues that have a biochemical makeup similar to that of cartilage such as the eyes, heart, and blood vessels, can also be affected.

• Chondromalacia patellae (also known as CMP, Patello-femoral Pain Syndrome) is a term that goes back eighty years. It originally meant "soft cartilage under the knee cap," a presumed cause of pain at the front of the knee. It has gradually come to mean pain at the front of the knee from just about any cause. Pain at the front of the knee is common in young adults. The condition may result from acute injury to the patella or from chronic friction between the patella and the groove in the femur through which it passes during motion of the knee. • Chondrolysis - The disappearance of articular cartilage as the result of lysis or dissolution of the cartilage matrix and cells.

• Chondrocalcinosis or calcium pyrophosphate dihydrate disease (CPPD) is a rheumatologic disorder with varied clinical manifestations due to precipitation of calcium pyrophosphate dihydrate crystals in the connective tissues.

Effects on cardiovascular disease o

The heart comprises heart muscle cells and connective tissue. Blood vessels comprise smooth muscle and connective tissue.

In one embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain a cardiovascular effect, where said effect can be palliative, curative or preventive by way of improved muscle and/or connective tissue quality, tone, texture, elasticity, firmness and/or strength.

Cardiovascular disease is any of a number of specific diseases that affect the heart itself and/or the blood vessel system, especially the veins and arteries leading to and from the heart.

The present invention further relates to a method for treatment of one or more cardiovascular diseases comprising administration of the hydroxyproline composition.

Cardiovascular diseases include but are not limited to:

• Coronary heart disease • Coronary artery disease is a disease of the artery caused by the accumulation of atheromatous plaques within the walls of the arteries that supply the myocardium. Angina pectoris (chest pain) and myocardial infarction (heart attack) are symptoms of and conditions caused by coronary heart disease.

• Cardiomyopathy - It is the deterioration of the function of the myocardium (i.e., the actual heart muscle) for any reason. People with cardiomyopathy are often at risk of arrhythmia and/or sudden cardiac death.

• Extrinsic cardiomyopathies - cardiomyopathies where the primary pathology is outside the myocardium itself. Most cardiomyopathies are extrinsic, because by far the most common cause of a cardiomyopathy is ischemia. Extrinsic cardiomyopathies include but are not limited to: Alcoholic cardiomyopathy,

Coronary artery disease, Congenital heart disease, nutritional diseases affecting the heart, Ischemic (or ischaemic) cardiomyopathy, Hypertensive cardiomyopathy, Valvular cardiomyopathy, Inflammatory cardiomyopathy, Cardiomyopathy secondary to a systemic metabolic disease • Intrinsic cardiomyopathies - weakness in the muscle of the heart that is not due to an identifiable external cause, including but not limited to: Dilated cardiomyopathy (DCM) - most common form, and one of the leading indications for heart transplantation. In DCM the heart (especially the left ventricle) is enlarged and the pumping function is diminished, Hypertrophic cardiomyopathy (HCM or HOCM) - genetic disorder caused by various mutations in genes encoding sarcomeric proteins. In HCM the heart muscle is thickened, which can obstruct blood flow and prevent the heart from functioning properly, Arrhythmogenic right ventricular cardiomyopathy (ARVC) - arises from an electrical disturbance of the heart in which heart muscle is replaced by fibrous scar tissue. The right ventricle is generally most affected, Restrictive cardiomyopathy (RCM) - least common cardiomyopathy. The walls of the ventricles are stiff, but may not be thickened, and resist the normal filling of the heart with blood. • Atherosclerosis

• lschaemic heart disease -characterized by reduced blood supply to the organs.

• Heart failure, also called congestive heart failure (or CHF), and congestive cardiac failure (CCF), is a condition that can result from any structural or functional cardiac disorder that impairs the ability of the heart to fill with or pump a sufficient amount of blood throughout the body. Therefore leading to the heart and body's failure.

• Cor pulmonale, a failure of the right side of the heart.

• Hypertensive heart disease is heart disease caused by high blood pressure, especially localised high blood pressure. Conditions that can be caused by hypertensive heart disease include: Left ventricular hypertrophy, Coronary heart disease, (Congestive) heart failure, Hypertensive cardiomyopathy, Cardiac arrhythmias

• Inflammatory heart disease -involves inflammation of the heart muscle and/or the tissue surrounding it.

• Endocarditis - inflammation of the inner layer of the heart, the endocardium. The most common structures involved are the heart valves.

• Inflammatory cardiomegaly

• Myocarditis - inflammation of the myocardium, the muscular part of the heart.

• Valvular heart disease is disease process that affects one or more valves of the heart. The valves in the right side of the heart are the tricuspid valve and the pulmonic valve. The valves in the left side of the heart are the mitral valve and the aortic valve.

• Aortic valve stenosis

• Mitral valve prolapse

• Valvular cardiomyopathy • Aorta pathologies including: • Aneurysm of sinus of Valsalva

• Aortic aneurysm - myotic, bacterial (e.g. syphilis), senile, genetic, associated with valvular heart disease

• Aortic coarctation - pre-ductal, post-ductal • Atherosclerosis

• Marfan syndrome

• Ehlers-Danlos syndrome

• Aortic stenosis

• Trauma, such as traumatic aortic rupture, most often thoracic and distal to the left subclavian artery and frequently quickly fatal.

Effects on venous insufficiency

Veins comprise abundant smooth muscle and connective tissue. The present invention contemplates the use of hydroxyproline to improve and/or increase venous smooth muscle and/or connective tissue quality, tone, texture, elasticity, firmness and/or strength.

In one embodiment the present invention thus relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on venous insufficiency.

The present invention further relates to a method for treatment of varicose veins to obtain a palliative or ameliorating, and/or curative effect on varicose veins. The present invention also relates to a method for the prevention of varicose veins.

Varicose veins are veins that have become enlarged and tortuous. The term commonly refers to the veins on the leg, although varicose veins can occur elsewhere. Veins have leaflet valves to prevent blood from flowing backwards (retrograde). Leg muscles pump the veins to return blood to the heart, against the effects of gravity. When veins become varicose, the leaflets of the valves no longer meet properly, and the valves don't work. This allows blood to flow backwards and they enlarge even more. Varicose veins are most common in the superficial veins of the legs, which are subject to high pressure when standing. Besides cosmetic problems, varicose veins are often painful, especially when standing or walking. They often itch, and scratching them can cause ulcers. Serious complications are rare. Non-surgical treatments include sclerotherapy, elastic stockings, elevating the legs, and exercise. The traditional surgical treatment has been vein stripping to remove the affected veins. Newer, less invasive treatments, such as radiofrequency ablation and endovenous laser treatment, are slowly replacing traditional surgical treatments. Because most of the blood in the legs is returned by the deep veins, the superficial veins, which return only about 10 per cent of the total blood of the legs, can usually be removed or ablated without serious harm.

The present invention further relates to a method for treatment of reticular veins to obtain a palliative or ameliorating, and/or curative effect on reticular veins. The present invention also relates to a method for the prevention of reticular veins. Reticular veins, also known as feeder veins, are the dilated blue and green veins beneath the skin surface.

The present invention further relates to a method for treatment of telangiectasias (spider veins) to obtain a palliative or ameliorating, and/or curative effect on telangiectasias. The present invention also relates to a method for the prevention of telangiectasias. Telangiectasias are small dilated blood vessels near the surface of the skin or mucous membranes.

In another embodiment, the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on weak blood vessels. The present invention further relates to a method for treatment of weak blood vessels to obtain a palliative or ameliorating, and/or curative effect comprising administration of the hydroxyproline composition. The present invention also relates to a method for the prevention of weak blood vessels.

Effects on respiratory diseases

The respiratory system includes airways, lungs, and the respiratory muscles.

Connective tissue is important part of the respiratory system and necessary for proper respiratory function.

In one embodiment, the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on respiratory diseases. This administration of the hydroxyproline composition can result in increased respiratory muscle and/or connective tissue quality, strength and/or elasticity and can be used to treat, ameliorate or prevent one or more respiratory diseases. Respiratory Disease is the term for diseases of the respiratory system. These include diseases of the lung, pleural cavity, bronchial tubes, trachea, upper respiratory tract and of the nerves and muscles of breathing. Respiratory diseases range from mild and self-limiting such as the common cold to life-threatening such as bacterial pneumonia or pulmonary embolism.

Lung diseases include, but are not limited to, lung diseases selected from the group consisting of Acute Bronchitis, Acute Respiratory Distress Syndrome (ARDS), Alpha-1 Antitrypsin Deficiency Emphysema, Chronic Obstructive Pulmonary Disease, Asbestosis, Asthma, Asthma in Children, Asthma in Adults, Avian Influenza (Bird Flu), HIV/AIDS realted Lung Disease, influenza, Obstructive Sleep Apnea (Sleep-Disordered Breathing), Occupational or Work-Related Asthma, Severe Acute Respiratory Syndrome, Bronchiectasis, Bronchiolitis, Bronchopulmonary Dysplasia (BPD), Byssinosis, Respiratory Distress Syndrome and Bronchopulmonary Dysplasia, Chronic Bronchitis, Coal Workers' Pneumoconiosis, Coccidioidomycosis, Cystic Fibrosis (CF), Lung Cancer, Occupational Lung Cancer, Emphysema, Hypersensitivity Pneumonitis, Hay Fever, Hantavirus pulmonary syndrome, Histoplasmosis, Tuberculosis, Opportunistic lung Infection, Human Metapneumovirus (HMPV), Hypersensitivity Pneumonitis, Pneumonia, Interstitial Lung Disease, Primary Ciliary Dyskinesia, Lymphangioleiomyomatosis (LAM), Lymphangiomatosis, Mesothelioma, Multidrug- Resistant Tuberculosis, Coal Workers' Pneumoconiosis, Hantavirus pulmonary syndrome, Hypersensitivity Pneumonitis, Pulmonary Fibrosis, Pandemic Influenza, Pediatric Tuberculosis, Pertussis (Whooping Cough), Pleurisy, Primary Ciliary Dyskinesia, Primary Pulmonary Hypertension (PPH), Pulmonary Arterial Hypertension (PAH), Spontaneous Pneumothorax, Acute Respiratory Distress Syndrome (ARDS), Respiratory Distress Syndrome and Bronchopulmonary Dysplasia (RDS & BPD),

Respiratory Distress Syndrome of the Newborn, Respiratory Syncytial Virus, Severe Acute Respiratory Syndrome, Sarcoidosis, Sick Building Syndrome, Silicosis, Spontaneous Pneumothorax, Anthrax, Sudden Infant Death Syndrome (SIDS), Extensively drug-resistant Tuberculosis (XDR TB), Pediatric Tuberculosis.

Respiratory diseases further include:

• Obstructive lung diseases -are diseases of the lung where the bronchial tubes become narrowed making it hard to move air in and especially out of the lung. • Restrictive lung diseases -also known as interstitial lung diseases are a category of respiratory disease characterized by a loss of lung compliance, causing incomplete lung expansion and increased lung stiffness. E.g. in infant respiratory distress syndrome (IRDS)

• Respiratory tract infections -Infections can affect any part of the respiratory system. They are traditionally divided into upper respiratory tract infections and lower respiratory tract infections. The most common upper respiratory tract infection is the common cold however infections of specific organs of the upper respiratory tract such as sinusitis, tonsillitis, otitis media, pharyngitis and laryngitis are also considered upper respiratory tract infections.

• Pleural cavity diseases -include empyema and mesothelioma. A collection of fluid in the pleural cavity is known as a pleural effusion. This may be due to fluid shifting from the bloodstream into the pleural cavity due to conditions such as congestive heart failure and cirrhosis. It may also be due to inflammation of the pleura itself as can occur with infection, pulmonary embolus, tuberculosis, mesothelioma and other conditions. A pneumothorax is a hole in the pleura covering the lung allowing air in the lung to escape into the pleural cavity. The affected lung "collapses" like a deflated balloon. A tension pneumothorax is a particularly severe form of this condition where the air in the pleural cavity cannot escape, so the pneumothorax keeps getting bigger until it compresses the heart and blood vessels, leading to a life threatening situation.

• Pulmonary vascular diseases -are conditions that affect the pulmonary circulation. Examples of these conditions are

• Pulmonary embolism, a blood clot that forms in a vein, breaks free, travels through the heart and lodges in the lungs (thromboembolism). Large pulmonary emboli are fatal, causing sudden death. A number of other substances can also embolise to the lungs but they are much more rare: fat embolism (particularly after bony injury), amniotic fluid embolism (with complications of labour and delivery), air embolism (iatrogenic). • Pulmonary arterial hypertension, elevated pressure in the pulmonary arteries. It can be idiopathic or due to the effects of another disease, particularly COPD. This can lead to strain on the right side of the heart, a condition known as cor pulmonale.

• Pulmonary edema, leakage of fluid from capillaries of the lung into the alveoli (or air spaces). It is usually due to congestive heart failure. • Pulmonary hemorrhage, inflammation and damage to capillaries in the lung resulting in blood leaking into the alveoli. This may cause blood to be coughed up. Pulmonary hemorrhage can be due to auto-immune disorders such as Wegener's Granulomatosis and Goodpasture's syndrome.

• Disorders of breathing mechanics. The brain co-ordinates breathing and sends messages via nerves to the muscles of respiration. The muscles produce the movements of breathing. Disorders of the brain's control of breathing, the nerves or the muscles of respiration can affect the respiratory system. Common disorders of breathing mechanics are:

• Obstructive sleep apnea • Central sleep apnea

• Amyotrophic lateral sclerosis

• Guillan-Barre syndrome

• Myasthenia gravis

Effects on incontinence Incontinence, involuntary discharge of urine or feces, may refer to fecal incontinence, the inability to control one's bowels and urinary incontinence, the involuntary excretion of urine. The quality and strength of muscle and connective tissues of the lower abdomen and pelvic region play an important role for the ability to control discharge of urine or feces.

In one embodiment, the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on incontinence.

The present invention further relates to a method for treatment of incontinence to obtain a palliative or ameliorating, and/or curative effect comprising administration of the hydroxyproline composition. The present invention also relates to a method for the prevention of incontinence. Fecal incontinence is the loss of regular control of the bowels. Involuntary excretion and leaking are common occurrences for those affected. Subjects relating to defecation are often socially unacceptable, thus those affected are often beset by feelings of shame and humiliation. Some refuse to seek medical help, and instead attempt to self- manage the problem. This can lead to social withdrawal and isolation, which can turn into cases of agoraphobia. Such effects may be reduced by undergoing prescribed treatment, taking prescribed medicine and making dietary changes.

Urinary incontinence is any involuntary leakage of urine. It is a common and distressing problem, which may have a profound impact on quality of life. Urinary incontinence almost always results from an underlying treatable medical condition. There are several types of urinary incontinence including:

• Stress incontinence, also known as effort incontinence, is due essentially to insufficient strength of the pelvic floor muscles. It is the loss of small amounts of urine associated with coughing, laughing, sneezing, exercising or other movements that increase intra-abdominal pressure and thus increase pressure on the bladder. The urethra is supported by fascia of the pelvic floor. If this support is insufficient, the urethra can move downward at times of increased abdominal pressure, allowing urine to pass. In men, stress incontinence is common following a prostatectomy. It is the most common form of incontinence in men. In women, physical changes resulting from pregnancy, childbirth, and menopause often contribute to stress incontinence. Stress incontinence can worsen during the week before the menstrual period. At that time, lowered estrogen levels may lead to lower muscular pressure around the urethra, increasing chances of leakage. The incidence of stress incontinence increases following menopause, similarly because of lowered estrogen levels. In female high-level athletes, effort incontinence occurs in all sports involving abrupt repeated increases in intra-abdominal pressure that may exceed perineal floor resistance.

• Urge incontinence is involuntary loss of urine occurring for no apparent reason while suddenly feeling the need or urge to urinate. The most common cause of urge incontinence is involuntary and inappropriate detrusor muscle contractions. Idiopathic Detrusor Overactivity - Local or surrounding infection, inflammation or irritation of the bladder. Neurogenic Detrusor Overactivity - Defective CNS inhibitory response. Medical professionals describe such a bladder as "unstable", "spastic", or "overactive". Urge incontinence may also be called "reflex incontinence" if it results from overactive nerves controlling the bladder. Patients with urge incontinence can suffer incontinence during sleep, after drinking a small amount of water, or when they touch water or hear it running (as when washing dishes or hearing someone else taking a shower). Involuntary actions of bladder muscles can occur because of damage to the nerves of the bladder, to the nervous system (spinal cord and brain), or to the muscles themselves. Multiple sclerosis, Parkinson's disease, Alzheimer's Disease, stroke, and injury-including injury that occurs during surgery-can all harm bladder nerves or muscles.

• Functional incontinence occurs when a person recognizes the need to urinate, but cannot physically make it to the bathroom in time due to limited mobility. The urine loss may be large. Causes of functional incontinence include confusion, dementia, poor eyesight, poor mobility, poor dexterity, unwillingness to toilet because of depression, anxiety or anger, or being in a situation in which it is impossible to reach a toilet. People with functional incontinence may have problems thinking, moving, or communicating that prevent them from reaching a toilet. A person with

Alzheimer's Disease, for example, may not think well enough to plan a timely trip to a restroom. A person in a wheelchair may be blocked from getting to a toilet in time. Conditions such as these are often associated with age and account for some of the incontinence of elderly women and men in nursing homes. Disease or biology is not necessarily the cause of functional incontinence. For example, someone on a road trip may be between rest stops and on the highway; also, there may be problems with the restrooms in the vicinity of a person.

• Overflow incontinence. Sometimes people find that they cannot stop their bladders from constantly dribbling, or continuing to dribble for some time after they have passed urine. It is as if their bladders were like a constantly overflowing pan, hence the general name overflow incontinence. Overflow incontinence occurs when the patient's bladder is always full so that it frequently leaks urine. Weak bladder muscles, resulting in incomplete emptying of the bladder, or a blocked urethra can cause this type of incontinence. Autonomic neuropathy from diabetes or other diseases (e.g Multiple sclerosis) can decrease neural signals from the bladder

(allowing for overfilling) and may also decrease the expulsion of urine by the detrusor muscle (allowing for urinary retention). Additionally, tumors and kidney stones can block the urethra. Spinal cord injuries or nervous system disorders are additional causes of overflow incontinence. In men, benign prostatic hyperplasia (BPH) may also restrict the flow of urine. Overflow incontinence is rare in women, although sometimes it is caused by fibroid or ovarian tumors. Also overflow incontinence can be from increased outlet resistance from advanced vaginal prolapse causing a "kink" in the urethra or after an anti-incontinence procedure which has overcorrected the problem. Early symptoms include a hesitant or slow stream of urine during voluntary urination. Anticholinergic medications may worsen overflow incontinence.

• Structural incontinence. Rarely, structural problems can cause incontinence, usually diagnosed in childhood, for example an ectopic ureter.

• Bedwetting (enuresis). Bedwetting is episodic Ul while asleep. It is normal in young children.

• Other types of incontinence. Stress and urge incontinence often occur together in women. Combinations of incontinence — and this combination in particular — are sometimes referred to as "mixed incontinence." "Transient incontinence" is a temporary version of incontinence. It can be triggered by medications, urinary tract infections, mental impairment, restricted mobility, and stool impaction (severe constipation), which can push against the urinary tract and obstruct outflow.

Common causes of incontinence are:

• Constipation is the most common cause of fecal incontinence. Constipation causes prolonged muscle stretching and leads to weakness of the intestinal muscles. After a certain point, the rectum will no longer close tightly enough to prevent stool loss, resulting in incontinence.

• Muscle damage. Fecal incontinence can be caused by injury to one or both of the ring-like muscles at the end of the rectum called the internal and external anal sphincters. During normal function, these sphincters help retain stool. In women, damage can occur during childbirth. The risk of injury is greatest when the birth attendant uses forceps to help the delivery or does an episiotomy. Hemorrhoid surgery can damage the sphincters as well. A pelvic tumor that grows in or becomes attached to the rectum or anus also can cause muscle damage, as can surgery to remove the tumor.

• Nerve damage. Fecal incontinence can also be caused by damage to the nerves that control the anal sphincters or to the nerves that detect stool in the rectum. Damage to the nerves controlling the sphincter muscles may render the muscles unable to work effectively. If the sensory nerves are damaged, detection of stool in the rectum is disabled, and one will not feel the need to defecate until too late. Nerve damage can be caused by childbirth, long-term constipation, stroke, and diseases that cause nerve degeneration, such as diabetes and multiple sclerosis.

• Loss of storage capacity. Normally, the rectum stretches to hold stool until it is voluntarily released. But rectal surgery, radiation treatment, and inflammatory bowel disease can cause scarring, which may result in the walls of the rectum becoming stiff and less elastic. The rectum walls are unable to stretch as much and are unable to accommodate as much stool. Inflammatory bowel disease also can make rectal walls very irritated and thereby unable to contain stool.

• Diarrhea. Diarrhea, or loose stool, is more difficult to control than solid stool that is formed. Where diarrhea is caused by temporary problems such as mild infections or food reactions, incontinence tends to last for a period of days. Chronic conditions, such as Irritable Bowel Syndrome, or Crohn's disease can cause severe diarrhea lasting for weeks or months until successful treatment can be found.

• Pelvic floor dysfunction. Abnormalities of the pelvic floor can lead to incontinence. Examples of some abnormalities are decreased perception of rectal sensation, decreased anal canal pressures, decreased squeeze pressure of the anal canal, impaired anal sensation, a dropping down of the rectum (rectal prolapse), protrusion of the rectum through the vagina (rectocele), and generalized weakness and sagging of the pelvic floor as for example after childbirth. Pelvic floor dysfunction is a group of clinical conditions that includes urinary incontinence, fecal incontinence, pelvic organ prolapse, sensory and emptying abnormalities of the lower urinary tract, defecatory dysfunction, sexual dysfunction and several chronic pain syndromes, including vulvodynia.

• Other causes. Fecal incontinence can have other causes including one or a combination of the following: Excretory problems, Fecal impaction, Diseases, drugs, and indigestible dietary fats that interfere with the intestineal absorption, Lateral internal sphincterotomy (Surgical procedure for helping Anal fissures heal), Seizure.

Effects on edemas

Edema is an abnormal accumulation of fluid beneath the skin or in one or more cavities of the body. Generally, the amount of interstitial fluid is determined by the balance of fluid homeostasis, and increased secretion of fluid into the interstitium or impaired removal of this fluid may cause edema. The quality and strength of muscle and/or connective tissues is important for the formation of edemas. In one embodiment, the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to obtain an effect on edema by way of improving and/or increasing muscle and/or connective tissue quality, strength and elasticity.

The present invention further relates to a method for treatment of edema to obtain a palliative or ameliorating, and/or curative effect comprising administration of the hydroxyproline composition. The present invention also relates to a method for the prevention of edemas.

Five factors can contribute to the formation of edema: 1 ) It may be facilitated by increased hydrostatic pressure or, 2) reduced, oncotic pressure within blood vessels; 3) by increased blood vessel wall permeability as in inflammation; 4) by obstruction of fluid clearance via the lymphatic; or, 5) by changes in the water retaining properties of the tissues themselves. Raised hydrostatic pressure often reflects retention of water and sodium by the kidney.

In a particular embodiment the present invention relates to a method for treatment of peripheral edema to obtain a palliative or ameliorating, and/or curative effect comprising administration of the hydroxyproline composition. The present invention also relates to a method for the prevention of peripheral edemas. Peripheral edema is the swelling of tissues, usually in the lower limbs, due the accumulation of fluids. The condition is commonly associated with aging, but can be caused by many other conditions, including congestive heart failure, trauma, alcoholism, pregnancy, hypertension or merely long periods of time sitting or standing without moving. Some medicines (e.g. amlodipine, pregabalin) may also cause or worsen the condition.

Foot, leg, and ankle swelling is the most common form of peripheral edema. It is common with the following situations:

• Prolonged standing

• Long airplane flights or automobile rides

• Menstrual periods (for some women)

• Pregnancy - excessive swelling may be a sign of pre-eclampsia, a serious condition sometimes called toxemia, that includes high blood pressure and swelling • Being overweight

• Increased age

• Injury or trauma to your ankle or foot

• Swollen legs may be a sign of heart failure, kidney failure, or liver failure. In these conditions, there is too much fluid in the body.

Other conditions that can cause swelling to one or both legs include:

• Blood clot

• Leg infection

• Venous insufficiency (when the veins in your legs are unable to adequately pump blood back to the heart)

• Varicose veins

• Burns (including sunburn)

• Insect bite or sting

• Starvation or malnutrition • Surgery to your leg or foot

• Certain medications may also cause your legs to swell:

• Hormones like estrogen (in birth control pills or hormone replacement therapy) and testosterone

• A group of blood pressure lowering drugs called calcium channel blockers (such as nifedipine, amlodipine, diltiazem, felodipine, and verapamil)

• Steroids

• Antidepressants, including MAO inhibitors (such as phenelzine and tranylcypromine) and tricyclics (such as nortriptyline, desipramine, and amitriptyline)

Cosmetic applications of hvdroxyproline

In one embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof in relation to cosmetic surgery e.g. in the combination with administration of collagen.

In another embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to tighten subcutaneous connective tissue to obtain tightening and/or strengthening of the skin. Tightening and/or strengthening of the skin is relevant e.g. when skin is very stretchy or very loose. In another embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to treat cellulite. The present invention further relates to a method for treatment of cellulite to obtain a palliative or ameliorating, and/or curative effect on cellulite. The present invention also relates to a method for the prevention of cellulite. Cellulite describes a condition where the skin of the lower limbs, abdomen, and pelvic region becomes dimpled after puberty. The causes are poorly understood, and may involve changes in metabolism and physiology such as gender specific dimorphic skin architecture, alteration of connective tissue structure, vascular changes and inflammatory processes.

In another embodiment the present invention relates to administration of a hydroxyproline composition to an individual in need thereof to treat stretch marks. The present invention further relates to a method for treatment of stretch marks to obtain a palliative or ameliorating, and/or curative effect on stretch marks. The present invention also relates to a method for the prevention of stretch marks. Stretch marks or striae (singular stria), as they are called in dermatology, are a form of scarring on the skin with an off-color hue. They are caused by tearing of the dermis, and over time can diminish but not disappear completely. The dermis is a mesodermally derived tissue and comprises high levels of collagen and elastin. It is a common misconception that stretch marks are solely the result of the rapid stretching of the skin associated with rapid growth (common in puberty) or weight gain (e.g. pregnancy or muscle building) that overcomes the dermis's elasticity. Stretch marks are influenced by hormonal changes associated with puberty, pregnancy, muscle building etc. Although stretch marks are generally associated with pregnancy and obesity, they can also develop during rapid muscle growth. Medical terminology for these kinds of markings includes striae atrophicae, vergetures, stria distensae, striae cutis distensae, striae gravidarum (in cases where it is caused by pregnancy), lineae atrophicae, striae distensae, linea albicante, or simply striae.

Functional Food

The hydroxyproline composition of the present invention can be part of a food or feed composition for feeding a human being or an animal such as a fish, chicken, goose, turkey, duck, cow, horse or pig. In one embodiment of the present invention, the food or feed composition is a functional food suitable for human beings or animals. Said functional food comprises hydroxyproline. It is preferred that the functional food is suitable for at least weekly oral intake, such as for daily oral intake. Alternatively, said functional food product may be suitable for use in parenteral or enteral nutrition, preferably in combination with formulations comprising other nutrients known to one skilled in the art.

Products according to the invention may be used for promoting health of human beings or animals, for example for maintaining, strengthening or promoting bone, muscle, cardiovascular or connective tissue health. In one preferred embodiment of the present invention, the functional food can be used for the prevention or reduction of osteoporosis, a muscle disease, a connective tissue disease, a joint disease, a tendon disease, a ligament disease or any other disease or condition mentioned in this application. In another preferred embodiment of the present invention, regular consumption of said functional food, such as for example once a day, twice a day, or three times a day, leads to a reduction of the risk of diseases such as osteoporosis, a muscle disease, a connective tissue disease, a joint disease, a tendon disease or a ligament disease or any other disease or condition mentioned in this application and reduces tiredness and fatigue.

While the method of administration or consumption may vary, the functional food is preferably ingested by a human or an animal as an ingredient of his or her daily diet. Any of the agents described herein can be combined with a liquid vehicle, such as water, milk, vegetable oil, juice and the like, or with an ingestible solid or semi-solid foodstuff. For example, they may be mixed into foods such as milk shakes, milk shake mixes, breakfast drinks, juices, flavored drinks, flavored drink mixes, yogurts, puddings, ice creams, ice milks, frostings, frozen yogurts, cheesecake fillings, candy bars, including "health bars" such as granola and fruit bars, gums, hard candy, mayonnaise, pastry fillings such as fruit fillings or cream fillings, cereals, breads, stuffings, dressings and instant potato mixes. The present invention thus relates to a method of producing a functional food composition, comprising mixing of hydroxyproline with a foodstuff.

For example, said functional food product may be selected from the group of meal replacers, dietary supplements, ice-cream, sauces, dressing, spreads, bars, sweets, snacks, cereals and beverages. In another preferred embodiment, said functional food is a dietary supplement suitable for a human being or an animal such as a fish, chicken, goose, turkey, duck, cow, horse or pig, preferably suitable for ingestion in capsule, tablet or liquid form.

In one embodiment, products according to the invention are prepared whereby hydroxyproline is added to the food product such that the level of the agent is between 1 mg to 99 g per 100 g product, such as from 1 mg to 10 mg per 100 g product, for example from 10 mg to 100 mg per 100 g product, such as from 100 mg to 200 mg per 100 g product, for example from 200 mg to 300 mg per 100 g product, such as from 300 mg to 400 mg per 100 g product, for example from 400 mg to 500 mg per 100 g product, such as from 500 mg to 600 mg per 100 g product, for example from 600 mg to 700 mg per 100 g product, such as from 700 mg to 800 mg per 100 g product, for example from 800 mg to 900 mg per 100 g product, such as from 900 mg to 1 g per 100 g product, for example from 1 g to 5 g per 100 g product, such as from 5 g to 10 g per 100 g product, for example from 10 g to 15 g per 100 g product, such as from 15 g to 20 g per 100 g product, for example from 20 g to 25 g per 100 g product, such as from 25 g to 30 g per 100 g product, for example from 30 g to 35 g per 100 g product, such as from 35 g to 40 g per 100 g product, for example from 40 g to 45 g per 100 g product, such as from 45 g to 50 g per 100 g product, for example from 50 g to 55 g per 100 g product, such as from 55 g to 60 g per 100 g product, for example from 60 g to 65 g per 100 g product, such as from 65 g to 70 g per 100 g product, for example from 70 g to 75 g per 100 g product, such as from 75 g to 80 g per 100 g product, for example from 80 g to 85 g per 100 g product, such as from 85 g to 90 g per 100 g product, for example from 90 g to 95 g per 100 g product, and/ or such as from 95 g to 99 g per 100 g product.

Dairy product

In one preferred embodiment of the present invention, the functional food is a dairy product. Thus, said functional food may for example be selected from any of the following: cultured dairy products, yogurts, cottage cheese, cream cheese, dairy dips, sour cream, milkshakes, Butter, Margarine, Low-fat spreads, Cheese, Cottage cheese, Cheese spread, Cheese "strings" for children. Cheese slices, yoghurt, Yoghurt-based carbonated drinks, drinkable yoghurts, low-fat yoghurts, refrigerated dips, sour cream, Ice cream, Cream, Low-fat cream-replacement, Fermented milk such as kefir. In one preferred embodiment of the present invention, said dairy product is a cheese- based product, such as selected from low-fat cheese, hard cheese, soft cheese, cottage cheese, cheese spread, cheese "strings" for children or cheese slices suitable for sandwiches.

In another preferred embodiment of the present invention, said dairy product is a yoghurt-based product, such as selected from a set yoghurt, a runny or pourable yoghurt, a yoghurt-based carbonated drink, a drinking or drinkable yoghurt, a low-fat yoghurt. Said yoghurt-based product may for example be fermented with Lactobacillus bulgaricus and/or Streptococcus thermophilus.

In another preferred embodiment of the present invention, said dairy product is a cultured dairy product, such as a cultured fluid (for example drinkable yogurt/yogurt smoothies, kefir, probiotic shots); a non-drinkable yogurt (for example in a cup or tubes); and/or another non-pourable cultured dairy product (for example cottage cheese, cream cheese, dairy dips or sour cream).

In another preferred embodiment of the present invention, said dairy product is another type of dairy product, such as selected from the group consisting of: refrigerated dips and sour cream, ice cream, cream, low-fat cream-replacement, fermented milk such as kefir, fermented beverages, such as drinkable yoghurt and kefir.

Health drink In another preferred embodiment of the present invention, the functional food according to the present invention is a health drink. Said health drink is in one embodiment fruit juice-based, which may be concentrated as a "squash", to be diluted to taste. Said fruit juice or squash preferably comprises concentrated fruit juice. Preferred fruit juices include, but are not restricted to, citrus fruit juices such as orange, grapefruit, lemon or lime, or combinations thereof. In another preferred embodiment, said fruit juice or squash comprises (preferably concentrated) berry juice(s), such as from raspberries, strawberries, blackberries, loganberries, cranberries, redcurrants, blackcurrants, blueberries, or combinations thereof, and/or combinations with citrus fruit juices. In another preferred embodiment, said fruit juice or squash comprises juice(s) from one or more of Pineapple, Passion Fruit, Mango, apple, pear, apricot, Pomegranate, guava, tomato and/or combinations with any other types of fruit juices. Preferred juice bases can be selected from but is not limited to the following group apple, apricots, banana, blackberries, blueberries, carambola (Starfruit), cherries, dates, figs, fruit cocktail, grape, grapefruit, Kiwi Fruit, lemons, Mandarin Orange, Mangos, melon, nectarines, orange, papaya, peaches, pear, pineapple, plantain, plum, raspberries, strawberries, tangerines, watermelon or combinations thereof. Further preferred juice bases are selected from the following group apple, carrot, cranberry, grape, grapefruit (pink or white), lemon, lime, orange, pineapple, prune, tangerine, tomato or combinations thereof.

Said health drink may also be water-based, such as a mineral water-based product, such as flavoured mineral water-based products. Said flavouring is preferably from fruit juices and/or other natural products.

In one preferred embodiment of the present invention, said health drink is an energy shot comprising sugars and other energy-providing products.

In another preferred embodiment of the present invention, said health drink is an alcoholic beverage, such as a dairy-based alcoholic beverage.

In another preferred embodiment of the present invention, said health drink is a meal replacement drink.

It is envisaged that the health drink of the present invention may also be manufactured as a concentrate or premix, ready for making the drink at a later stage, preferably by the consumer.

Solid Functional Food

In one preferred embodiment of the present invention, the functional food is a solid functional food, such as selected from the group consisting of: Biscuits/crackers, breakfast cereal, soup, muesli, Chewing gum, Sweets (such as boiled sweets), fresh bakery products (fresh bread, cakes, muffins, waffles etc.), dry bakery products (crispbread, biscuits, crackers etc.), cereal products (breakfast cereals, fibre and sterol enriched flours, mueslis, cereal based and muesli bars, such bars possibly containing chocolate, pasta products, snacks etc.), bran products (granulated and/or toasted bran products, flavoured and/or sterol coated bran products and bran-bran mixes etc.).

In another preferred embodiment of the present invention, said solid functional food is a ready mix (preferably in powder form), either for baking (e.g. breads, cakes, muffins, waffles, pizzas, pancakes) or for cooking (e.g. soups, sauces, desserts, puddings) to be used in preparing or manufacturing of foods.

In another preferred embodiment of the present invention, said solid functional food is a meat product (sausages, meat-balls, cold cuts etc.)

In another preferred embodiment of the present invention, said solid functional food is a bread or morning product/bakery snack. Thus, said bread may be white, brown or wholemeal bread. In another preferred embodiment of the present invention, said bread may be selected from the following bread types: malted wheats, milk breads, bran-enriched and mixed grain breads. The bread may be any shape, such as e.g. cob, coburg, cottage, cholla, bloomer, barrel, batch, sandwich, tin, Vienna or farmhouse. In one preferred embodiment of the present invention, said bread is selected from any of the following bread types: wholemeal bread, brown bread, wheatgerm bread (bread containing added processed wheatgerm of no less than 10%), softgrain bread (made from white flour with additional grains of softened rye and wheat to increase the fibre content (preferably by 30%) compared with conventional white bread), granary breads and malt breads.

In another preferred embodiment of the present invention, said bread is selected from any of the following bread types: ciabatta, pitta, naan, cholla, focaccia, Soda Bread or brown soda bread (made using wholemeal flour), rye breads, baguette or French stick, croissants and bagel.

In another preferred embodiment of the present invention, said bread is a flat bread, such as selected from any of the following bread types: Chapattis, Paratas and Roti, Mexican tortilla, flat "wrap" or flour tortilla, pancakes.

In another preferred embodiment of the present invention, the functional food is a morning snack or bakery product. Said bakery product may be either sweet or savoury, for example savoury. Preferred bakery products include, but are not restricted to: rolls and baps, toasting products such as muffins, crumpets and pikelets, scones, teacakes, buns and other fruited products, hot plate products such as pancakes and griddle scones, waffles and potato cakes, hot cross buns, croissants, brioches, pain-au-chocolat, bagels, American sweet muffins and other semi-sweet bread products.

Vegetable oil-based product

In another preferred embodiment of the present invention, the functional food is a vegetable oil-based product (spreads, salad oils, mayonnaise etc.)

Frozen Confectionery Products

In another preferred embodiment of the present invention, the functional food is a frozen confectionary product. For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as ice-cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees.

Preferably the level of solids in the frozen confection (e.g. sugar, fat, flavouring etc) is more than 3 wt %, more preferred from 10 to 70 wt %, for example 40 to 70 wt %.

Ice-cream will typically comprise 2 to 20 wt % of fat, 0 to 20 wt % of sweeteners, 2 to 20 wt % of non-fat milk components and optional components such as emulsifiers, stabilisers, preservatives, flavouring ingredients, vitamins, minerals, etc, the balance being water. Typically ice-cream will be aerated e.g. to an overrun of 20 to 400 %, more general 40 to 200 % and frozen to a temperature of from -2 to -200 degrees. C, more general -10 to -30 degrees C. Ice-cream normally comprises calcium at a level of about 0.1 wt %.

A typical size of an average serving of frozen confectionery material is 66 g.

Hydroxyproline may be encapsulated or combined with emulsifiers, detergents or other agents to ensure solubilisation and stabilisation of the substance in the product.

Meal Replacers In another preferred embodiment of the present invention, the functional food is a meal o

replacer. Meal replacer drinks are typically based on a liquid base which may for example be thickened by means of gums or fibers and whereto a cocktail of minerals and vitamins are added. The drink can be flavoured to the desired taste e.g. fruit or choco flavour. A typical serving size may be 330 ml or 330 g. Hydroxyproline may be encapsulated or combined with emulsifiers, detergents or other agents to ensure solubilisation and stabilisation of the substance in the beverage.

Meal replacer snacks or bars often comprise a matrix of edible material wherein hydroxyproline can be incorporated. For example the matrix may be fat based (e.g. couverture or chocolate) or may be based on bakery products (bread, dough, cookies etc) or may be based on agglomerated particles (rice, grain, nuts, raisins, fruit particles). A typical size for a snack or meal replacement bar could be 20-200 g, generally from 40 to 100 g. Further ingredients may be added to the product e.g. flavouring materials, vitamins, minerals etc.

Combinations

In one aspect of the present invention, the functional food comprises hydroxyproline in combination with another survival enhancing agent, longevity enhancing agent, health enhancing agent and/or a modulator of muscle and/or connective tissue.

For example, one preferred embodiment of said functional food is a food comprising one or more of the agents according to the present invention and a probiotic, such as in a probiotic "shot". Another preferred embodiment of the functional food is a food comprising the compounds according to the present invention and a prebiotic, such as in a prebiotic "shot". Another preferred embodiment of the functional food is a food comprising the compounds according to the present invention and a symbiotic, such as in a symbiotic "shot". In one preferred embodiment of the present invention, preferred bacteria for use in the above-mentioned shots are any of the following: Lactobacillus sp., such as L. acidophilus, L. casei, L. fermentum, L. johnsonii, L. lactis, L. plantarum, L. reuteri, L. rhamnosus and/or L. salivarius. In another preferred embodiment of the present invention, preferred bacteria for use in the above-mentioned shots are any of the following: Bifidobacterium sp., such as B. bifidium, B. breve, B. lactis, and/or B. longum. In another preferred embodiment of the present invention, preferred bacteria for use in the above-mentioned shots are any of the following: Enterococcus faecalis. Escherichia coli, Saccharomyces boulardii, Saccharomyces cerevisiae and/or Streptococcus thermophilus.

Hydroxyproline may also be combined with other ingredients in a dietary supplement, such as e.g. botanical supplements and/or in a vitamin E capsules, or in a selenium pill. Further preferred combination in said dietary supplements may be with e.g. one or more of the following: antioxidant(s), vitamin C, vitamin E, beta-carotene.

The functional food of the invention can further encompass other healthy components such as for example vitamins like vitamin A, B, C, D, E, minerals such as calcium, potassium, magnesium, iron, copper, zinc, selenium and anti-oxidants such as tocopherols, polyphenols.

In a preferred embodiment, compositions of the invention may comprise further ingredients which are believed to reduce or prevent osteoporosis, a muscle disease, a connective tissue disease, a joint disease, a tendon disease, a ligament disease or any other disease or condition mentioned in this application. Examples of such ingredients are calcium, vitamin D, magnesium etc.

Thus, in one embodiment, the present invention is concerned with use of hydroxyproline in the manufacture of a functional food, such as any of the functional foods described herein.

In one embodiment, the present invention relates to a functional food comprising hydroxyproline, wherein said functional food, or any raw material used for its synthesis, has been treated by a method known to a person skilled in the art, to increase the availability of hydroxyproline in said functional food. In a particular embodiment, heat treatment of the functional food comprising hydroxyproline or the raw materials used in the synthesis of the functional food can be used to increase the availability of hydroxyproline in the feed composition.

Feed composition

The present invention relates in one embodiment to a feed composition comprising Hydroxyproline for animals such as fish, chicken, goose, turkey, duck, cow, horse or pig. The feed composition can be any feed composition comprising Hydroxyproline that affects the muscle and/or connective tissue quality in said animal. The present invention further relates to a method for improvement of the muscle quality in an animal comprising a step of feeding said animal with a composition comprising Hydroxyproline, said improvement in muscle quality resulting in improved quality and/or texture of the final meat product.

The texture of the muscle tissue of fish, i.e. the fish fillet, is important for both the initial handling of the fish during slaughter, the appearance of the product to be sold and for the taste and mouthfeel of the final product. This is also true for the texture of the meat of animals other than fish.

In one preferred embodiment the present invention relates to a fish feed composition comprising Hydroxyproline. The present invention further relates to a method for improvement of the muscle and/or connective tissue quality in fish comprising a step of feeding said fish with a feed composition comprising Hydroxyproline. The fish feed composition comprising Hydroxyproline can be, but is not limited to, the fish feed compositions disclosed in WO 2008/148873. WO 2008/148873 is incorporated by reference in its entirety in the present application. The fish feed composition comprising Hydroxyproline can be given to any type of fish such as the fish disclosed in WO 2008/148873.

In one embodiment, the present invention relates to a feed composition comprising Hydroxyproline, wherein said feed composition or any raw material used for its synthesis, has been treated by a method known to a person skilled in the art, to increase the availability of hydroxyproline in said feed composition. In a particular embodiment, heat treatment of the feed composition comprising hydroxyproline, or the raw materials used in the synthesis of the feed, can be used to increase the availability of hydroxyproline in the feed composition.

Administration

Administration routes

The Hydroxyproline composition of the invention can be formulated in any suitable way, i.a. depending on the route of administration, and the amount of active ingredient to be administered. The hydroxyproline composition according to the present invention can be administered to an individual in need thereof such as a human being by any route of administration including the one or more of the route(s) of administration listed herein below.

In particular, the therapeutic compositions of the invention can be formulated for parenteral administration, including intravenous, intramuscular, intraarticular, subcutaneous, intradermal, epicutantous/transdermal, and intra- peritoneal administration, for infusion, for oral administration, for nasal administration, for rectal administration, for vaginal administration, or for topic administration.

Any suitable route of administration may be employed for providing an animal or a human being, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, mixtures, liquids, ointments, aerosols, and the like.

In another embodiment the administration is by one or more injections such as systemic injections and/or local injections.

Other types of administration includes Jet-infusion (micro-drops, micro-spheres, micro- beads) through skin, drinking solution, suspension or gel, inhalation, nose-drops, eyedrops, ear-drops, skin application as ointment, gel or lotion, Vaginal application as ointment, gel, creme or washing, gastro-intestinal flushing and rectal washings or by use of suppositories.

The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.

Dosage regime

The hydroxyproline composition can be administered by a) single intake e.g. by a single injection or b) multiple intake such as by injection or application. The hydroxyproline composition can be administered once or over prolonged time as days, months, years. The dosage regime can be modified during the treatment. Encapsulation of the hydroxyproline composition

The hydroxyproline composition can be encapsulated, preferably inside a microcapsule. The microcapsule may be made from any suitable material such as a hydrophilic or a hydrophobic material. Likewise the microcapsule may be provided with a coating. This coating may be of a kind that prevents agglomeration or sticking of the microcapsules or prevents evaporation of the hydroxyproline and/or a solvent comprising the hydroxyproline inside the microcapsule. The invention also foresees the use of coatings providing the microcapsule with an affinity for specific cells or tissues. Such an affinity-coating may be in the form of specific amino-acid sequences or even anti-bodies or parts of antibodies having an affinity for specific proteins. Thereby the delivery of hydroxyproline can be targeted to exactly those cells (e.g. particular muscle cells, connective tissue cells) to which the hydroxyproline is intended. Likewise this makes it possible to use the microcapsules for diagnostic use and the hydroxyproline could in such cases be substituted by a compound suitable for labelling the targeted cells.

Agents for encapsulation include but are not limited to alginate, hydrocolloids such as gelatine, exudates such as gum arabic, tragacanth, gum karya, gum ghatti; extracts from seaweed such as agar, carrageenan and furcellaran; extracts from plants such as pectin and arabinogalactan; colloids; extracts from marine and terrestrial animals such as gelatines and other proteinaceous hydrocolloids; flours from seeds such as guar, locust bean, soya bean; proteins from seeds such as soya bean proteins; flours from cereals such as starches and microcrystalline cellulose; biosynthetic or fermentation derived hydrocolloids such as dextran, xanthan and curdlan; chemically modified hydrocolloids such as cellulose derivatives, including methyl cellulose and other derivatives, including modified starches and low methoxyl pectin; synthetic hydrocolloids such as polyvinylpyrrolidone, carboxyvinyl polymers etc.

Dosage In one embodiment, the hydroxyproline composition to be used for treatment of an individual in need thereof such as a human being is administered at a total daily dosage of from about 0.01 milligram to about 1000 milligram per kilogram of the body weight. The dosage regimen may be adjusted within this range or even outside of this range to provide the optimal therapeutic response. The hydroxyproline composition used according to the present invention is given in an effective amount to an individual in need thereof such as a human being. The amount of hydroxyproline composition according to the present invention in one preferred embodiment is in the range of from about 0.01 milligram per kg body weight per dose to about 1000 milligram per kg body weight per dose, such as from about 0.01 milligram per kg body weight per dose to about 0.025 milligram per kg body weight per dose, for example from about 0.025 milligram per kg body weight per dose to about 0.05 milligram per kg body weight per dose, such as from about 0.05 milligram per kg body weight per dose to about 0.075 milligram per kg body weight per dose, for example from about 0.075 milligram per kg body weight per dose to about 0.1 milligram per kg body weight per dose, such as from about 0.1 milligram per kg body weight per dose to about 0.25 milligram per kg body weight per dose, such as from about 0.25 milligram per kg body weight per dose to about 0.5 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 0.75 milligram per kg body weight per dose, such as from about 0.75 milligram per kg body weight per dose to about 1 .0 milligram per kg body weight per dose, for example from about 1.0 milligram per kg body weight per dose to about 2.5 milligram per kg body weight per dose, such as from about 2.5 milligram per kg body weight per dose to about 5 milligram per kg body weight per dose, for example from about 5 milligram per kg body weight per dose to about 7.5 milligram per kg body weight per dose, such as from about 7.5 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 10 milligram per kg body weight per dose to about 25 milligram per kg body weight per dose, such as from about 25 milligram per kg body weight per dose to about 50 milligram per kg body weight per dose, such as from about 50 milligram per kg body weight per dose to about 75 milligram per kg body weight per dose, for example from about 75 milligram per kg body weight per dose to about 100 milligram per kg body weight per dose, such as from about 100 milligram per kg body weight per dose to about 250 milligram per kg body weight per dose, for example from about 250 milligram per kg body weight per dose to about 500 milligram per kg body weight per dose, such as from about 500 milligram per kg body weight per dose to about 750 milligram per kg body weight per dose, for example from about 750 milligram per kg body weight per dose to about 1000 milligram per kg body weight per dose. The amount of hydroxyproline composition according to the present invention in another embodiment is in the range of from about 0.01 milligram per kg body weight per dose to about 20 milligram per kg body weight per dose, such as from about 0.02 milligram per kg body weight per dose to about 18 milligram per kg body weight per dose, for example from about 0.04 milligram per kg body weight per dose to about 16 milligram per kg body weight per dose, such as from about 0.06 milligram per kg body weight per dose to about 14 milligram per kg body weight per dose, for example from about 0.08 milligram per kg body weight per dose to about 12 milligram per kg body weight per dose, such as from about 0.1 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.3 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.7 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.9 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 1.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 1.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 1 .4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 1.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 1.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 2.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 2.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 2.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 2.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 2.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 3.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 3.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 3.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 3.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 3.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 4.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 4.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 4.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 4.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 4.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 5.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 5.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 5.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 5.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 5.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 6.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 6.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 6.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 6.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 6.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 7.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 7.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 7.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 7.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 7.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 8.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.3 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.7 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.9 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 1.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 1.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 1 .4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 1.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 1 .8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 2.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 2.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 2.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 2.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 2.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 3.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 3.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 3.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 3.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 3.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 4.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 4.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 4.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 4.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 4.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 5.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 5.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 5.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 5.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 5.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 6.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.3 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 0.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 0.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.7 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 0.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.9 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 1 .0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 1.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 1.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 1.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 1 .8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 2.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 2.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 2.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 2.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 2.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 3.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 3.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 3.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 3.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 3.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 4.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 4.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 4.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 4.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 4.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 5.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose.

A dose or dosage may be given as a single dose or in divided doses. A single dose occurs only once, with the hydroxyproline composition administered either as a bolus or by continuous infusion. Alternatively, the dose may be divided into multiple doses and given recurrently, such as twice, for example three times, such as four times, for example five times, such as six times, for example seven times, such as eight times, for example nine times, such as ten divided doses. Furthermore, the dose may be given repeatedly, i.e. more than once, such as twice, for example three times, such as four times, for example five times, such as six times, for example seven times, such as eight times, for example nine times, such as ten times a day.

It follows that the hydroxyproline may be given once or more daily, or alternatively may be given with intervals of 1 day, such as 2 days, for example 3 days, such as 4 days, such as 5 days, for example 6 days, such as 7 days, for example 8 days, such as 9 days, such as 10 days, for example 1 1 days, such as 12 days, for example 13 days, such as 14 days, such as 3 weeks, for example 4 weeks, such as 5 weeks, for example 6 weeks, such as 7 weeks, such as 8 weeks, for example 12 weeks.

When the dose is given as a continuous infusion, the dose may be expressed as milligram per kilo body weight per dosage, wherein the dosage is given over a prolonged period, such as an hour (expressed as mg/kg/hr). The doses according to the present invention may be infused over 15 minutes to 24 hours, such as 15 to 30 minutes, for example 30 to 60 minutes, such as 60 to 90 minutes, for example 90 to 120 minutes, such as 2 hour to 3 hours, for example 3 hour to 4 hours, such as 4 hour to 5 hours, for example 5 hours to 6 hours, such as 6 hours to 7 hours, for example 7 hours to 8 hours, such as 8 hours to 9 hours, for example 9 hours to 10 hours, such as 10 hours to 1 1 hours, for example 1 1 hours to 12 hours, such as 12 hours to 14 hours, for example 14 hours to 16 hours, such as 16 hours to 18 hours, for example 18 hours to 20 hours, such as 20 hours to 22 hours, for example 22 hours to 24 hours.

Patient groups

The Hydroxyproline composition of the invention can be administered to any individual in need thereof, where the individual can be, but is not limited to:

• An animal such a fish, chicken, goose, turkey, duck, cow, horse or pig • a human being

• a man

• a woman

• a post-menopausal women

• a pregnant woman • a lactating woman

• an infant

• a child

• an adult

• an individual of any age such as from newborn to 120 years old, for example from 0 to 6 months, such as from 6 to 12 months, for example from 1 to 5 years, such as from 5 to 10 years, for example from 10 to 15 years, such as from 15 to 20 years, for example from 20 to 25 years, such as from 25 to 30 years, for example from 30 to 35 years, such as from 35 to 40 years, for example from 40 to 45 years, such as from 45 to 50 years, for example from 50 to 60 years, such as from 60 to 70 years, for example from 70 to 80 years, such as from 80 to 90 years, for example from 90 to 100 years, such as from 100 to 1 10 years, for example from 1 10 to 120 years.

• An individual of any race such as a Caucasian, a black person, an East Asian person, a person of Mongoloid race, a person of Ethiopian race, a person of Negroid race, a person of American Indian race, or a person of Malayan race. • An individual that is healthy • An individual that is ill.

• An individual that is diagnosed with one or more diseases.

• An individual that has one or more symptoms of one or more diseases

• An individual that is asymptomatic • An individual that is genetically disposed for one or more diseases.

• An individual that is an athlete

• An individual that is a bodybuilder

• An individual that is a weightlifter

Detailed description of the drawings

Figure 1. Shows the specific growth rate (SGR, %) and feed conversion rate (FCR) in salmon fed a high plant protein control diet, and with Hyp added in free and bone- bound form. Values are given as means and SEM. No statistical significant differences were found between the three diets.

Figure 2. Muscle pH in salmon muscle during storage for 5, 9 and 15 days. Muscle pH increased slightly by 2.6 -3 % for all dietary groups during storage and a significant increase in muscle pH occurred at day 5 of storage in fish fed free Hyp, but not following 9 and 15 days of storage in fish fed Hyp in either free or bone-bound form, b indicates statistical significance (p < 0.05), ab indicates a non-significant difference.

Figure 3. Muscle firmness or texture (analysed as shear work) was non-significantly improved day 5, 9 and 15 in fish fed dietary Hyp in free form by 5, 10 and 6.5 %, respectively, relative to control fish when measured as shear work.

Figure 4. Muscle firmness or texture (analysed as shear force) of salmon muscle was non-significantly improved day 5, 9 and 15 in fish fed dietary Hyp in free form by 5, 1 1 and 9 %, respectively, relative to control fish when measured as shear force. In all dietary groups, muscle firmness decreased as expected during the storage trial due to autolysis.

Figure 5. Acid insoluble (a-i) Hyp, as indicator of insoluble collagen in the muscle tissue, was significantly increased in fish fed Hyp in free form after 5 days of storage (b, p < 0.05), but not after 9 days of storage (p > 0.05). In fish fed dietary Hyp in bone- bound form, a tendency towards improved collagen formation relative to the control fish also appeared at day 5, although the result was not statistical significant (ab, p > 0.05). Similar to Hyp in free form, no differences in a-i Hyp appeared following 9 days of storage.

Figure 6. Shows soluble collagen measured as acid-soluble Hyp (a-s Hyp) in salmon muscle during storage at 4 ⁰ C for 5 and 9 days. The a-s Hyp in muscle was significantly increased in fish fed Hyp in both free and bone-bound form following 5 and 9 days of storage (b, p < 0.05), as also reflected when measured as free amino acids in muscle

(Table 7) and in blood (Table 8) after 5 days of storage. The results suggest that more

Hyp in the free form is available for collagen synthesis in the tissue, possibly explaining the higher level of a-i Hyp as a measure of collagen formation after 5 days (Table 5 and Fig. 5).

Figure 7. Shows the amount of free Hyp and GIy in salmon plasma measured 24 hours post-feeding, b indicates statistical significance.

Figure 8. (A) Shows the amount of free Hyp in urine. The urinary excretion of Hyp is decreased significantly by dietary administration of Hyp in both diets (free Hyp and Bone Hyp). (B) shows total Hyp in muscle tissue. Hyp is increased in muscle by dietary administration of Hyp in free form, b indicates statistical significance.

Figure 9. PYD cross-links in salmon muscle during storage at 4 ⁰ C for 5, 9 and 15 days. PYD cross-links in the muscle tissue were increased by approximately 10, 18 and 6 % at day 5, 9 and 15, respectively, in fish fed free Hyp as compared to control fish. Dietary differences were significant after 9 days of storage (b, p < 0.05), but not after 5 and 15 days of storage. PYD cross-links were non-significantly decreased by 7, 4 and 4 % at day 5, 9 and 15, respectively, in fish fed bone-bound Hyp as compared to control fish (p > 0.05).

Figure 10. Depicts the synthesis of hydroxyproline from proline. Vitamin C and Fe are involved in the hydroxylation reaction catalysed by proline hydroxylase. In an analog reaction, vitamin C participate as a cofactor in the hydroxylation of lysine residues catalyzed by Cu-dependent Lysyl-hydroxylase. Hydroxylation of peptide bound proline and lysine residues allows cross-linking to stabilize the triple helical structure of tropocollagen, a subunit of procollagen, before excretion. Figure 1 1. The figure depicts ketoamide cross-linking of collagen triple helix molecules. Ketoamide cross-links are reducible cross-links capable of binding only two collagen molecules. Ketoamide cross-links are immature divalent cross-links.

Figure 12. The figure depicts non-reducible Hydroxylysyl pyridinoline (PYD) cross- linking of collagen molecules. PYD cross-links are mature trivalent cross-links that bind three collagen molecules.

Figure 13. The figure depicts fish muscle structure (top and middle) and collagen arrangement (bottom). Different collagen types are arranged in a complex structure and 27 distinct collagens are identified in mammals. Collagen in fish are located in the myoseptum (separates the myotomes), the perimysium (surrounds muscle bundles) and the endomysium (surrounds the individual cells). Collagen type I and V are the major and minor collagen in fish. Collagen is not equally distributed, but the majority of the collagen is located in the myoseptum.

Figure 14. (A) A flow diagram of collagen analysis as performed in Example 1 . (B) A flow diagram of the PYD cross-link assay performed in Example 1. The analyses were performed essentially as described previously by Li et al. 2005.

Example 1

Aims

A 12 week seawater trial was performed with 2.5 kg salmon to evaluate dietary impacts of free hydroxyproline (Hyp) added as a Sigma chemical (99.9%) or as a Hyp rich (1 1 .2%) bone gelatine product (Hydroxy Plus ™), relative to a control diet without added Hyp. The trial aimed to evaluate impacts of free Hyp and bone-bound Hyp on growth, feed intake, feed conversion and survival. Impacts of feed ingredients on muscle quality were further evaluated in a storage trial (4 ⁰ C) by measurements of:

• gaping score, • muscle texture/firmness,

• total soluble and insoluble collagen,

• PYD (mature cross-link formation),

• water content,

• proximal composition • amino acids and free amino acids in muscle

• free amino acids in blood.

The aim of the trial was to test the hypothesis that it is possible to modify collagen formation and the strength of connective tissue and/or muscle tissue by dietary supplements of hydroxyproline as hydroxyproline is essential to stabilize the tipple helical structure of collagen.

Materials and methods

Diets Three experimental extruded diets were produced either without added Hyp (Control, Diet 1 ) or with Hyp added in free form (Free Hyp, Diet 2) or bound in a bone gelatine product provided by Sonac BV (Bone Hyp, Diet 3), c.f. table 1 . The basal control diet was a standard commercial formulated high plant protein diet with slightly lower fish meal inclusion (18%) than usually provided in commercial diets (20%). Protein in the bone gelatine product was balanced with wheat gluten meal, otherwise the dietary composition was similar. Amino acids were balanced (Met, Lys, Thr) to satisfy the requirement for the predefined growth period of Atlantic salmon used for the trial. The Hyp administration level was similar to the one suggested to be an optimum for growth in Aksnes et al. (2008). In the feeding trial, this level of administration corresponded to 4.1 g / kg fish during the 12 week feeding trial. Table 1

DIET COMPOSITION

Composition of diet Control Hyp Hydroxy Pfus

Diet no 1 2 3

% % %

FM 221/07 17.9 17.9 18.0

SPC 197/08 16.00 16.00 16.0

Fullfθtt soya 147/05 4.0 4.0 4.00

Wheat gluten 224/07 4.00 3.72 1.30

NorSeaOil 01/07 31.70 31.70 31 .70

Wheat 134/08 10.50 10.50 10.50

Corn gluten 176/07 10.00 10.00 10.00

Vitamin - mix 2.0 2.0 2.0

Mineral mix uten P 0.4 0.4 0.4

Mono Calsium Fosfat MCP(22.7%) 2.200 2.200 2.200 Hyp

Bone gelatin 2.65 92.0 11.5 0.80

Carop. Pink (10%) 0.064 0.064 0.064

Methionine 99% 0.250 0.250 0.300

Lys (99 %, 19.41 % Cl) T26/07 0.75 0.75 0.70

OH-Pro 0.000 0.280 0.000 100.000 99.000 0.79

Threonine 98.5% 0.20 0.20 0.20

Sum 100.00 100.00 100.00

Tola) P {%) 1.00 1.09 1,11

Soluble P {%} 0,?4 0.74 0.74

Plant protein (% of protein in diet) 59.83 59.11 53.41 lys (g/100 protein) 6.76 6.74 6.77 meth (g/100 protein) 2.87 2.85 2.79 trp (g/100 protein) 0.97 0.97 0.91 thr (g/100 protein) 3.95 3.93 3.90

Arg (g/100 protein) 5.31 5.28 5.63

His (g/10Og prot) 1.52 1.51 1.46

HyptøiOøgprot) 0,46 1.25 1.25

Calculated chemical composition in the feed (% in diet)

Protein 34.9 35.0 35.1

Lipid 34.9 34.9 34.9

Carbohydrate 19.4 19.3 19.0

Ash 4.3 4.3 4.4

Water 6.2 6.2 6.3

Sum 99.7 99.7 99.7

Enerαi MJ/kα 25.5 25.5 25.5

Kg per diet 520.0 520.0 520.0 The content of Hydroxyproline in the three diets was as follows (Table 2):

Diet 1 : Basal control diet (0.33 g hyp / 100 g protein) Diet 2: Basal diet added free Hyp (1 .1 1 g hyp / 10O g protein )

Diet 3: Basal diet added bone-bound Hyp (1.1 O g Hyp / 10O g protein)

Ingredient composition and the chemical contents including total amino acids and free amino acids of the experimental diets are shown in Table 1 , 2 and 3. The diets were iso-caloric and the energy content ranged from 25.4 MJ kg ^"1 to 25.6 MJ kg ^"1 . All diets were extruded (10 mm pellet) at fixed and defined conditions (twin screw) and the physical quality appeared similar for all experimental diets.

Table 2. Chemical composition of experimental diets

Diet 1 Diet 2 Diet 3

Control Free Hyp Bone Hyp

Ingredient content

Protein, g kg ^"1 357 350 357

Lipid, g kg ^"1 358 367 357

Moisture, g kg ^"1 55 59 58

Ash, g kg ^"1 58 58 58

Gross energy ¹ , MJ kg ^{"1 1} 25.5 25.6 25.4

Total amino acids, g10Og prof ¹

Asp 8.8 8.8 9.4

Glut 19 19.4 18.2

Hyp 0.33 1.11 1.10

Ser 4.5 4.5 4.5

GIy 4.6 4.5 5.9

His 2.2 2.2 2.2

Arg 6.1 6.1 6.5

Thr 3.9 3.9 3.9

Ala 5.6 5.7 6.1

Pro 6.1 6.2 6.2

Tyr 3.1 3.0 2.9

VaI 4.6 4.7 4.6

Met 2.5 2.6 2.7 lso 4.2 4.2 4.1

Leu 8.6 8.9 8.6

Phe 4.5 4.6 4.5

Lys 6.8 6.8 7.0

Total sum aa 95.4 97.2 98.4 Table 3. Free amino acids of experimental diets

Diet 1 Diet 2 Diet 3

Control Free Hyp Bone Hyp

Free amino acid, gWOgprot

Kreatinin

Aspartic acid 0.04 0.04 0.04

Glutamic acid 0.09 0.08 0.09

Hydroxyprolin (free) 0.77

Ser 0.02 0.02 0.02

Asp 0.03 0.03 0.03

GIy 0.05 0.05 0.05

Glut

3-amino-propionic acid

Taurine 0.3 0.29 0.28

His 0.04

4-amino-butanic acid 0.02 0.02 0.02

Citrullin

Thr 0.52 0.42 0.40

Ala 0.08 0.08 0.08

Carnosine 0.05 0.18 0.03

Arg 0.1 0.09 0.12

Pro 0.02 0.02 0.02

Tyr 0.01 0.02 0.02

Anserin 0.02 0.02 0.02

VaI 0.04 0.04 0.04

Met 0.73 0.73 0.85

Cys lsoleu 0.02 0.02 0.02

Leu 0.06 0.06 0.06

Phe 0.03 0.03 0.04

Trp 0.05 0.03 0.04

Ornithine 0.02 0.02 0.01

Lys 1 .76 1 .63 1.54

Total sum free aa 4.10 4.69 3.82

Experimental fish and handling

Atlantic salmon, average weight at start 2359 ± 147 g, were randomly distributed to nine 5.0 x 5.0 m sea net pens, each holding 80 fish. The fish were fed one of the 3 experimental diets, in triplicate tanks, for a feeding period of 12 weeks. Fish were fed to satiation by automatic feeders, and the daily feed rations were adjusted according to assumed fish biomass and feed intake.

Individual weight and length of fish were measured at start and at the end of the trial. After a feeding period of 84 days, 5 fish from each tank were collected 24 hours post- feeding for evaluation of weight, length, gutted weight, liver weight, and for calculation of condition factor, hepatosomatic index (HSI) and dressing out percentage (DOP). The fish were anaesthetised, killed with a blow to the head and samples of whole body and tissues were sampled from each tank.

Another six individuals per sea net pen were collected to perform a storage trial in a controlled environment. The fish were anaesthesized (benzocaine) and killed with a sharp blow to the head, bled in ice-chilled seawater for 15 min, weighed, gutted and individually pit tagged and packed with ice for transport by plane. At Bodø University College, Faculty of Biosciences and Aquaculture, the fish were stored in a controlled environment in ice at 4 ^Q C in a cooling chamber. Fish were manually filleted after 5, 9 and 15 days of storage (6 fish per diet x 3 net pens; that is 18 individual fish per sampling per diet, 54 fish in total for each storage day).

Impacts of feed ingredients on product quality were further evaluated by measurements of pH, water content, gaping score, muscle texture (firmness), total alkaline-soluble (a- i) and insoluble (i-s) collagen, PYD (mature cross-link formation), proximal composition and free amino acids in blood and in muscle.

Chemical methods

All chemical analyses were carried out in duplicate by laboratories accredited by the Norwegian National Accreditation body. In feed ingredients, diets and fish tissue, crude protein (N x 6.25) was determined by the Kjeldahl method (ISO 5983-1997), moisture gravimetrically after drying for 4 h at 105 ^Q C (ISO 6496-1999) and ash after combustion for 16 h at 550 ^Q C (ISO 5984-2002). Lipid content in the diets was determined gravimetrically after petroleum ether extraction (Soxhlet technique), (AOCS Ba 3-38). Amino acids of feed and faeces were hydrolyzed in 6M HCI for 22 h at 1 1 O ⁰ C and analyzed by HPLC using a fluorescence technique for detection as described previously by Cohen and Michaud, 1993.

Collagen and PYD cross-links

The assay used for collagen and PYD crosslink analyses was as described by Li et al. (2005). 9 ml of cold deionised water (4 ^Q C) was added to each of the 50 ml tubes containing exactly 1 g of fast muscle. The sample was homogenised at full speed (22000 rpm) for one minute using a Polytron (mod. PT 1200CL, knife (mod. PT-DA

1207/2EC)). After homogenization 10 ml of cold 0.2 M NaOH was added to each tube, making it up to a total volume of 20 ml 0.1 M NaOH solution. The tubes were then placed on a rotator (Stuart rotator SB3, Bibby Sterilin LTD, Straffordshire, UK) in a cold o

room (2 ^Q C) for 4 h. Immediately after NaOH extraction tubes were centrifuged (Eppendorf mod. 5804R) at 10000 g for 30 minutes at 4 ^Q C. From the supernatant, containing the a-s collagen fraction, 0.5 ml was removed to a 4 ml glass vial with screw cap containing 0.5 ml of 12 M HCI. The remaining of the supernatant was gently poured off. The a-i collagen fraction (pellet) was re-suspended in 2 ml of 6 M HCI and transferred to an 8 ml hydrolyse vial with screw cap. The a-s and a-i collagen fraction was hydrolysed in an oven at 1 10 ^Q C for 2O h. 10 μl and 2 μl was then removed from the a-s and i collagen fraction respectively, and re-suspended in an 1.5 ml eppendorf tube containing 100 μl of deionised water. The rest of the a-i collagen fraction, was used for hydroxylysyl pyridinoline (PYD) cross-link analysis, and was dried down together with the re-suspended a-s and a-i collagen samples using a vacuum rotary evaporator (Jouan RC1022, Nantes, France). The dried PYD cross-link fraction was diluted in 3 ml consisting of deionised H2O, acetic acid and butanol (v/v/v, 1 :1 :4). For PYD extraction a cellulose column (Varian Ltd., Oxford, UK) was used (Black et al., 1988; Eyre et al., 1984). The cellulose columns were attached to a vacuum block

(vacuum (15 Hg) generated by a water suction) (Varian/Holger, Oslo, Norway), and the re-suspended PYD fraction was loaded on to the column, followed by a series of washings (3x3 ml of 1 :1 :4 buffer). The PYD fraction was eluted from the cellulose column by adding deionised water which was collected in a glass tube, dried down and resuspended in 300 ml injection sample buffer (5% CH3CN, 1% HFBA) containing 250 nM pyridoxine (internal standard). The re-suspended PYD samples were filtered through 4 mm syringe filters (0.2 μm PVDF membrane) into micro-inserts in HPLC vials before being loaded on to the HPLC. Before each HPLC run (PYD and HYP) a validation sample was added in front of the sample series to check for any instrumental inaccuracies.

Muscle firmness

The texture of the flesh was determined in duplicate using a TA-XT2 texture analyser with Texture Expert Exceed 2.52 software from Stable Micro Systems (Surrey, England) using a shear test as described previously by Johnston et al., 2004b. Muscle blocks were cut from the deeper layer of dorsal fast myotomal muscle using a standardized frame (2.5x2.5x1 cm). The fish were filleted in groups of six and kept on ice prior to texture measurements to ensure fillets were not exposed to temperature fluctuations. The probe used in the shear test was a 60 ^Q knife edge blade (not sharpened). A 25 kg load cell was used and test speed was set to 1 mm s-1 . The texture profile was analysed using the Texture Expert Exceed 2.52 software, to determine maximum shear force described as the highest peak value measured in Newtons (N) whilst the area under the curve during shearing until fracture corresponds to the work done in millijoules (mJ) as described previously by Veland and Torrissen, 1999.

Muscle pH pH was analysed day 5, 9 and 15 by an use of an electrode" that was gently forced into homogenized fillet (PHM92 pH-meter from Radiometer Analytical, Copenhagen, Denmark).

Fresh gaping assessment

Gaping score was measured on a scale from 0 to 4; (0 = no gaping; 1 = slight gaping; 2 = serious gapiing and 4= fillet falling apart). As there were little gaping, the score was divided further into 0.5 values, i.e. 0, 0.5, 1 , 1.5 etc. Gaping is estimated by visual inspection of the fish fillet according to the knowledge of a person of skill.

Biological calculations

Growth, feed intake and feed conversion ratio were determined according to the following formulas (BW ₂ = final body weight, BW ₁ = initial body weight):

- Specific growth rate, % SGR = (In BW ₂ - In BW ₁ ) ^* 100/ feeding days.

- Thermal growth coefficient, TGC = (BW ₂ ^(1/3) - BW/ ¹⁷³ ') ^* 1000 / Σ (temp.(°C) ^* feeding days) according to Cho (1992).

- Daily feed intake per fish = g feed intake / days / number of fish.

- Daily feed intake, % of weight gain = g feed intake / days /((BW ₂ + BWi)/2 ^* 100) / fish no.

- Feed conversion ratio = g live weight gain / g dry feed eaten

Hepasomatic index (HSI), gonadosomatic index (GSI), dress out percentage (DOP), spleen somatic index (SSI) and head index (HI) were calcuated as follows: - Tissue index = 100 x g tissue weight / g body weight

Condition factor was calculated as follows: Condition factor (CF) = (100 x g body weight) / (body length) ³

Statistical methods and calculations

Biological and analytical data were subjected to ANOVA one-way analysis of variance (ANOVA) using STATISTICS (Ver 7.1 , StatSoft, Tulsa, OK, USA) and differences between means were tested using Tukey HSD test (Sokal and Rohlf, 1981 ). Effects with a probability P < 0.05 were considered significant. Pearson correlation and Spearman's rank correlation analysis was further used to examine possible relationships between feed parameters and dietary responses.

Results

Dietary composition

Proximate composition of experimental diets showed that the intended levels of nutrients and calculated levels of energy were approximately similar (Table 2) with a mean protein, lipid, water, ash and energy content of 355 ± 8 gkg ^'1 , 361 ± 6 gkg ^'1 , 57 ± 2 gkg ^'1 , 58 ± 0 and 25.5 ± 0.1 MJKg ^'1 , respectively. Total amino acids showed that dietary level of Hyp was increased more than 3 times in fish fed Hyp in free form (Diet 2) and in bone-bound form (Diet 3), Table 2. Analyses of the free amino acids showed that Hyp was present in the free form only in Diet 2, whereas below detection limits in Diet 1 and Diet 3 (Table 3). The bone-gelatine diet (Diet 3) increased dietary level of Asp, GIy and Arg by 6.8, 28 and 6.6 %, otherwise dietary differences in amino acid composition were small.

Growth, feed intake and feed conversion

The growth rate was acceptable for slaughter size 3.5 kg salmon (Table 4), showing mean SGR values of 0.51 ± 0,05 % and TGC of 1.88 ± 0.18 for all dietary groups. Diet composition did not affect final body weight, weight gain or growth as measured by SGR and TGC (P > 0.05), Table 4. Further, no significant difference in total feed intake, feed consumption or feed conversion rate (FCR) appeared (P > 0.05). Mortality was low for all dietary groups, showing maximum 0 to 2 dead fish in individual tanks (< 1.5% per diet) throughout the experimental period, Table 4. Mean SGR and FCR are shown in Fig. 1. Slaughter quality of fish

Body weights, lengths, gutted weight, milt and roe of sampled fish (n=3, each of 5 fish per net pen) as well as the related calculated indices C-factor, hepatosomatic index (HSI), gonad somatix index (GSI) and dressing out percentage (DOP) did not show dietary differences (P > 0.05), selected parameters are shown in Table 4.

Results - Storage trial

Chemical composition and free fatty acids (FFA)

With one exception, body weights, lengths and gutted weights of fish sampled after 5, 9 and 15 days of storage (n=3, each of 6 fish per net pen), and the related indices C- factor and DOP, did not show dietary differences throughout the storage trial (P > 0.05), Table 5. DOP of fish fed Hyp in free and bound form was significantly lower after 15 days of storage, as compared to control fish (P < 0.05).

During 15 days of storage at stable temperature (4 ⁰ C), slight reductions in muscle protein, lipid and dry matter contents occurred in all dietary groups. The chemical composition and FFA of fish did not show significant changes (P > 0.05). FFA was fairly stable and low in fish from all diets throughout the complete storage period, indicating low level of lipid degradation.

Table 4 Growth, feed intake, feed conversion and morphometries of Atlantic salmon fed a high plant protein control diet, and Hyp added in _^ ^ Diet ϊ Diet 2 Diet 3 P ^τ <

?.9.πir.9i ^F χ ^e . ^e . Uyp ^B °. ^ne - yyp

Body weight at start, g 2344(17) 2367(31) 2366 (80) ns Body weight at end, g 3555(127) 3587 (88) 3522 (63) ns Weight gain, g/fish 1211 (110) 1220(82) 1157(63) ns SGR, % 0.51 (0.04) 0.51 (0.03) 0.49 (0.03) ns TGC 1.91 (0.14) 1.91 (0.11) 1.83(0.10) ns

Total feed intake, kg 105.1 (8.4) 102.3(5.8) 100.3(4.1) ns Feed consumption, g feed/fish/day 16.1 (1.24) 15.9(0.97) 15.7(0.5) ns Feed consumption, % of mean BW /day 0.55 (0.03) 0.53 (0.02) 0.53(0.01) ns Feed conversion rate, g feed eaten /g fish 1.08(0.02) 1.06(0.01) 1.10(0.03) ns

Mortality, no fish per group 0 C

Weight of sampled fish, g 3645(151) 3624(123 3714(200) ns

HSI 1.34(0.01) 1.39 (0.04) 1.39(0.04) ns

Condition-factor 1.41 (0.07) 1.40(0.05) 1.42(0.07) ns

DOP 16.0(0.4) 16.5(0.7) 16.8(0.3) ns

Gonad somatic index, % 0.22 (0.02) 0.16(0.04) 0.19(0.01) ns

¹ Statistical differences within rows are shown with different superscript letters, P < 0.05. Ns = not significant

² Morphometries calculated on sampled fish, n=3 per diet (5 individual fish sampled per tank).

Table 5 Storage trial, Atlantic salmon fed a high plant protein control diet, and Hyp added in free (Free Hyp) and bone-bound (bone Hyp) form, all values given as means and SEM (n=3).

Diet 1 Diet 2 Diet 3 ^" FT

PP Control Free Hydroxy Plus

5 days

Body weight of sampled fish, g 3725 (76) 3624(91) 3734 (30) ns

Condition-factor 1.36(0.02) 1.33(0.01) 1.35(0.02) ns

DOP 15.1 (0.3) 15.5(0.2) 15.0(0.6) ns

Protein 19.4(0.1) 19.2(0.3) 19.6(0.1) ns

Lipid 17.1 (0.5) 17.7(0.4) 17.0(0.6) ns

Ash 1.17(0.03) 1.10 (<0.01) 1.13(0.03) ns

Dry matter 36.1 (0.5) 35.9(0.1) 36.4 (0.4) ns

FFA 1.87(0.12) 1.73(0.09) 2.0 (0.06) ns

9 days

Body weight of sampled fish, g 3661 (132) 3726(135) 3560(167) ns

Condition-factor 1.35(0.02) 1.36(0.03) 1.35(0.03) ns C

DOP 15.6(0.05) 14.7(0.22) 15.4(0.3) ns O

Protein 18.9(0.1) 19.1 (0.12) 19.1 (0.1) ns

Lipid 18.0(0.7) 17.6(0.35) 17.1 (0.3) ns

Ash 1.13(0.03) 1.10 (<0.01) 1.13(0.03) ns

Dry matter 36.3 (0.7) 35.9 (0.35) 35.4 (0.4) ns

FFA 1.83(0.03) 1.80 (<0.01) 1.90(0.15) ns

15 days

Body weight of sampled fish, g 3550 (28) 3506(18.2) 3424 (85) ns

Condition-factor 1.35 (0.04) 1.28(0.03) 1.28(0.02) ns

DOP 16.0(0.3) ^a 14.4(0.1) ^b 14.4(0.3) ^b 0,01

Protein 19.1 (0.1) 18.8(0.4) 19.0(0.1) ns

Lipid 17.0(0.7) 17.2(0.6) 15.8(0.3) ns

Ash 1.10 (<0.01) 1.13(0.03) 1.17(0.03) ns

Dry matter 35.6 (0.5) 35.2 (0.5) 34.6(0.18) ns

FFA 1.93(0.03) 2.07(0.17) 2.07 (0.07) ns

¹ Statistical differences within rows are shown with different superscript letters (b indicates statistical significance), P < 0.05. Ns = not significant (P > 0.05)

Amino acids and free amino acids

Total sum of muscle amino acids were increased by approximately 4% in fish fed Hyp in free and bound form (Table 6), in particular due to an increase in some of the non- essential amino acids (NEAA). Although the dietary impacts were insignificant (P > 0.05), they were all consistent and seem to reflect the dietary Hyp stimulation. Results suggest stimulated uptake and/or synthesis of the non-essential amino acids Hyp, Asp and Glut in particular, but also of Ala that appeared in fish fed both free and bound Hyp in the diet. Of the essential amino acids (EAA), the weakly increased Lys level accounted for 80% of the increase in EAA in Diet 2 (Free Hyp). In fish fed diet 3 (bone Hyp), exactly the same essential and non-essential amino acids were weakly stimulated, while in addition, muscle Arg level was also slightly increased and reflected amino acid composition of Diet 3.

At the sampling day 5, small dietary difference was seen in muscle free amino acids (Table 7), except for soluble Hyp in muscle that was increased in diet 2 and 3, in response to the higher dietary Hyp supplementation levels.

Free amino acids in blood and urine

Blood samples were collected 24 hours after feeding the fish. The timing for blood sampling is an important factor for understanding metabolic changes. Free amino acids in blood plasma 24 hours post-feeding showed significant differences related to Hyp, GIy, Thre, Tyr and Lys (p < 0.05), Table 8, fig 7. Plasma levels of Hyp and Tyr was significantly increased and decreased, respectively in fish fed diets supplemented with both free and bound Hyp. Plasma level of GIy was increased, while significant only in fish fed the bone-bound Hyp. Plasma levels of Thr and Lys were significantly reduced in fish fed Hyp in the free form (p < 0.05). The ratio EAA/NEAA was reduced in fish fed Hyp in free and bound form, whereas significant only in fish fed free Hyp (p < 0.05), reflecting the clear tendency to reduced essential amino acids in the blood.

The results further show 20% less EAA secretion in urine of fish fed free Hyp. Secretion of the amino acids Thre, Tyr, Lys, His, Trp and Leu were reduced compared to control. Hence, Free Hyp supplementation results in decreased urinary secretion of at least some amino acids. Table 6 Amino acids in muscle of Atlantic salmon fed a high plant protein control diet, and Hyp added in free (Free Hyp) and bone-bound (bone Hyp) form), all values given as means and SEM (n=3).

Diet 1 Diet 2 Diet 3 P ¹ <

Control Free Hyp § _, 9 ^ne _, dYP

Total amino acid, gloόgprof ¹

Asp 8.9 (0.5) 10.2 (0.3) 9.8(0.5) 0.20

Glut 12.6(0.6) 14.0 (0.4) 13.7(0.6) 0.20

Hyp 0.17(0.02) 0.24(0.02) 0.23 (0.1) ns

Ser 3.8(0.12) 3.9 (0.1) 3.9 (0.1) ns

GIy 4.8(0.12 4.9 (0.1) 4.9 (0.2) ns

His 2.9 (0.03) 2.8(0.1) 2.8(<0.1) ns

Arg 6.0 (0.13) 6.0 (0.1) 6.3 (0.1) 0.34

Thr 4.4(0.1) 4.4(0.1) 4.5(0.1) ns

Ala 5.8(0.1) 6.1 (0.1) 6.1 (<0.1) 0.27

Pro 3.2 (0.1) 3.2 (0.1) 3.2 (0.1) ns

Tyr 3.3 (0.1) 3.2 (0.1) 3.3 (0.1) ns

VaI 5.4(0.1) 5.5(0.1) 5.5(<0.1) ns

Met 3.2 (0.1) 3.2 (0.1) 3.2 (<0.1) ns lso 4.9 (0.1) 4.9 (0.1) 5.0 (<0.1) ns

Leu 7.7(0.2) 7.8(0.1) 7.8(0.1) ns

Phe 4.1 (0.1) 4.0 (0.1) 4.1 (<0.1) ns

Lys 9.8(0.4) 10.3 (0.3) 10.1 (0.2) ns

Total sum aa 91.0 (1.9) 94.7(1.7) 94.3 (1.2) 0.29

EAA 48.4(1.0) 49.0 (1.0) 49.2 (0.3) ns

NEAA 42.6(1.4) 45.7(0.8) 45.1 (0.9) ns

EAA/NEAA 1.14(0.04) 1.07(0.01) 1.09 (0.02) ns_

¹ Statistical differences within rows are shown with different superscript letters, P < 0.05. Ns = not significant (P > 0.05)

² Morphometries calculated on sampled fish, n=3 per diet (6 individual fish sampled per tank).

Table 7. Free amino acids in muscle of Atlantic salmon, all values given as means (n=3).

Day 5 Day S I Day 15

Control Free Hyp Bone Hyp Control Free Hyp Bone Hyp Control Free Hyp Bone Hyp

Free amino acid, g/16g N

Creatinine ^* 0,01 0,01 0,01 0,02 0,02 0,01 0,02 0,02 0,03

Aspartic acid 0,02 0,02 0,04 0,01 0,01 0,01 0,02 0,01 0,01

Glutamic acid 0,38 0,42 0,37 0,50 0,53 0,50 0,37 0,33 0,41

Hyp (free) 0,04 0,14 0,08 0,05 0,13 0,15 0,04 0,10 0,11

Ser 0,02 0,02 0,05 0,02 0,06 0,02 0,02 0,03 0,03

Asp

GIy 0,19 0,18 0,17 0,19 0,19 0,22 0,18 0,18 0,23

Glut 0,02 0,02 0,01 0,02 0,02 0,01

S-amino-propionic acid 0,05 0,05 0,06 0,07 0,07 0,07 0,1 1 0,10 0,12

Taurine ^* 0,21 0,21 0,23 0,21 0,20 0,21 0,26 0,29 0,25

His 0,05 0,04 0,08 0,03 0,02 0,01 0,06 0,05 0,03

4-amιno-butanιc acid 0,01 0,01 0,01

Citrulline 0,01 0,01 0,01

Thr 0,06 0,06 0,08 0,04 0,04 0,04 0,07 0,06 0,04 C

C

Ala 0,20 0,19 0,24 0,17 0,15 0,16 0,20 0,22 0,20

Carnosine ^* 0,08 0,05 0,06 0,07 0,06 0,04 0,08 0,07 0,04

Arg 0,02 0,02 0,02 0,01 0,01 0,01 0,02 0,02 0,01

Pro 0,11 0,1 1 0,06 0,15 0,15 0,12 0,1 1 0,08 0,08

Anserine ^* 2,66 2,61 2,75 2,38 2,40 2,35 2,6 2,77 2,55

Tyr 0,047 0,04 0,05 0,04 0,04 0,05 0,04 0,04 0,04

VaI 0,04 0,04 0,06 0,04 0,03 0,03 0,04 0,04 0,04

Met 0,01 0,01 0,02 0,02 0,01 0,04 0,02 0,02 0,01

Cys - - - - lsoleu 0,017 0,013 0,02 0,01 0,01 0,01 0,01 0,02 0,02

Leu 0,06 0,05 0,07 ,05 0,03 0,05 0,05 0,05 0,05

Phe 0,04 0,04 0,05 0,04 0,04 0,03 0,03 0,03

Trp - - - 0,01 0,01

Ornithine 0,01 0,01 0,01 0,01 0,01

Lys - - - - - - - -

Total sum free aa 4,34 4,34 4,58 4,14 4,21 4,15 4,42 4,58 4,37

Essential free aa 0,30 0,27 0,40 0,24 0,21 0,24 0,31 0,28 0,23

Non-essential free aa 1 ,03 1 ,13 1 ,07 1 ,14 1 ,26 1 ,23 1 ,01 1 ,01 1 ,12

Other (Creat, Tau, Cam, Ans) ^* 2,96 2,89 3,05 2,68 2,67 2,61 2,96 3,15 2,87

Table 8. Free amino acids in blood plasma of Atlantic salmon fed a high plant protein control diet, and Hyp added in free (Free Hyp) and bone-bound (bone Hyp) form all values given as means and SEM (n=3).

Diet 1 Diet 2 Diet 3 P ¹

Control Free Hyp Bone Hyp Total amino acid. gWOgprof ¹

Glutamic acid 3.67(0.67) 3.33 (0.33) 3.33 (0.33) ns

Hyp (free) 2.00 (<0.0) ^a 4.00 (0.58) ^b 4.00 (0.0) ^b 0.01

Ser 2.67(0.33) 2.33 (0.33) 3.0 (0.0) ns

Asp <0.01 <0.01 <0.01 ns

GIy 4.00 (0.00) ^a 4.67 (0.33) ^ab 5.67 (0.33) ^b 0.01

Glutamine 14.00 (1.53) 13.33 (1.45) 12.0 (0.0) ns

Taurine 2.33 (0.67) 2.67(0.33) 2.5 (0.5) ² ns

His 1.67(0.33) 1.33 (0.33) 1.0 (0.0) ns

Citrulline 2.033 (0.67) 3.00 (0.58) 3.00 (0.58)

Thr 7.00 (0.00) ^a 5.67 (0.33) ^b 7.33 (0.33) ^a 0.01

Ala 16.67(1.20) 15.33 (0.33) 16.0 (0.58) ns

Arg 3.67(0.67) 3.0 (0.0) 3.67(0.33) ns

Pro 5.00 (0.58) 4.33 (0.33) 5.0 (1.0) ns

Tyr 4.67(0.33) ^a 3.00 (0.58) ^b 3.0 (0.0) ^b 0.01

VaI 9.00 (0.58) 8.00 (1.00) 7.33 (0.33) ns

Met 3.67(0.33) 3.00 (0.58) 3.33 (0.33) ns lsoleu 4.67(0.67) 4.33 (0.33) 3.67(0.33) ns

Leu 12.33 (1.45) 11.0 (1.53) 10.0 (0.58) ns

Phe 3.33 (0.33) 2.67(0.33) 3.0 (0.0) ns

Trp 1.00 (0.00) 1.00 (0.0) 1.0 (0.0) ns

Lys 6.33 (0.33) 4.67(0.33) 6.0 (0.58) 0.07

EAA 52.7(3.5) 44.67(3.84) 46.3 (0.33) 0.22

NEAA 52.7(3.2) 50.33 (2.03) 52.0 (1.53) ns

EAA/NEAA 1.00 (0.01) ^a 0.88 (0.04) ^D 0.89 (0.02) ^aD 0.05

¹ Statistical differences within rows are shown with different superscript letters (b indicates statistical significance). P < 0.05. Ns = not significant (P > 0.05)

o

Results - Muscle quality during storage

Weight of fish, water content, gaping score and pH

Fish weight of stored fish and the analysed water content and gaping score were unaffected by diet during the storage trial (5, 9 and 15 days), p > 0.05, Table 9. In general, the gaping score was very low (< 0.5 %) in all dietary groups. Water content of fish showed small changes during storage, and was increased by maximum 1 % at day

15. Muscle pH increased slightly by 2.6 -3 % for all dietary groups during storage (Fig

2) and a significant increase in muscle pH occurred at day 5 of storage in fish fed free

Hyp (p < 0.05), but not following 9 and 15 days of storage in fish fed Hyp in either free or bone-bound form.

Muscle firmness

Muscle firmness or texture (analysed as shear force and work done) was non- significantly improved day 5, 9 and 15 in fish fed dietary Hyp in free form by 5, 10 and 6.5 %, respectively relative to control fish when measured as shear work (Fig. 3), and by 5, 1 1 and 9 % when measured as shear force, Table 9 and Fig. 4. In all dietary groups, muscle firmness decreased as expected during the storage trial due to autolysis. The terms firmness and texture are used interchangeably in the application.

In fish fed Hyp in free form, the difference in muscle firmness relative to control fish was highest following 9 days of storage, as shown by the 10 % difference. This indicates that Hyp supplementation in the diet can improve muscle firmness and possibly also maintain a higher firmness of flesh in the period between slaughtering and until the fish reach the marked.

Acid soluble (a-s) and insoluble (a-i) Hyp and PYD cross-links

Acid insoluble (a-i) Hyp, as indicator of collagen in the muscle tissue, was significantly increased in fish fed Hyp in free form (Fig. 5) after 5 days of storage (p < 0.05), but not after 9 days of storage (p > 0.05). In fish fed dietary Hyp in bone-bound form, a tendency towards improved collagen formation relative to the control fish also appeared at day 5, although the result was not statistical significant (p > 0.05). Similar to Hyp in free form, no differences in a-i Hyp appeared following 9 days of storage.

The a-s Hyp in muscle was significantly increased in fish fed Hyp in both free and bone-bound form following 5 and 9 days of storage (Fig. 6), as also reflected when measured as free amino acids in muscle (Table 7, fig 8B) and in blood (Table 8, fig 7) after 5 days of storage. Results suggests that more Hyp in the free form is available for collagen synthesis in the tissue, possibly explaining the higher level of a-i Hyp as a measure of collagen formation after 5 days (Table 5 and Fig. 5).

PYD cross-links in the muscle tissue was increased by 9.7, 17.9 and 6 % at day 5, 9 and 15, respectively, in fish fed free Hyp as compared to control fish, Table 9 and Fig. 9. Dietary differences were significant after 9 days of storage (p < 0.05), but not after 5 and 15 days of storage. PYD cross-links was non-significantly decreased by 7, 4 and 4 % at day 5, 9 and 15, respectively, in fish fed bone-bound Hyp as compared to control fish (p > 0.05).

Table 9 Storage trial, Atlantic salmon fed a high plant protein control diet, and Hyp added in free (Free Hyp) and bone-bound (bone Hyp) form), all values given as means and SEM (n=3). Diet ^" ? DΪ ^' e ^' fέ ^' Diet ^" 3 F ^" <

Control Free Hyp §.9.. ^ne . UYP 5 days

Body weight of fish, g 3725 (87) 3624(75) 3734(60) ns

Water, % 70.9 (0.2) 71 (0.3) 70.6 (0.2) ns kb pH 6.14(0.02) ^a 6.21 (0.01 ) ^b 6.17(0.1) ^" <0.01

Gaping score, % 0.39 (0.10) 0.5 (0.09) 0.36 (0.17) ns

A-I Hyp, umol g ¹ muscle 1.18 (0.08) ^a 1.58 (0.12) ^b 1.41 (0.67) έ ^" ib <0.05

A-S Hyp, umol g ^"1 muscle 2.59 (0.19) ^a 5.84 (0.61 ) ^b 4.13(0.45) ^'ab2 <0.001

Texture, Shear force (N) 26.0 (1.0) 27.3 (1.3) 25.3 (1.10) ns

Texture, Area under curve 219.6(7.3) 231.2 (8.5) 219.2(9.1) ns

PYD cross-links, pmolg ^"1 360.2 (28.7) 395.1 (33.8) 334.6 (60.4) ns 9 days

Body weight of fish, g 3662 (121) 3726 (90) 3560 (86) ns

Water, % 71.2(0.3) 71.3 (0.3) 71.6 (0.25) ns pH 6.20 (0.02) 6.19(0.02) 6.22 (0.01) ns

Gaping score, % 0.47(0.12) 0.33(0.11) 0.36 (0.08) ns

A-I Hyp, umol g ^"1 muscle 1.40 (0.06) 1.36 (0.07) 1.34(0.05) ns

A-S Hyp, umol g ^"1 muscle 2.85 (0.23) ^a 4.65 (0.3) ^b 4.43 (0.19) ^b <0.001

Texture, Shear force (N) 22.4 (1.3) 24.9 (1.12) 22.8(1.1) ns

Texture, Area under curve 191.3(11.1) 210.6 (9.0) 196.2(7.9) ns

PYD cross-links, pmolg ^"1 418.3 (19.9) ^a 493.1 (23.6) ^b 399.7 (22.4) ^a <0.01 15 days

Body weight of fish, g 3550 (118) 3506 (68.5) 3424 (63.4) ns

Water, % 71.9(0.3) 71.4(0.3) 71.5(0.2) ns pH 6.30 (0.01) 6.37(0.03) 6.36 (0.03) ns

Gaping score, % 0.31 (0.07) 0.47 (0.09) 0.36(0.11) ns A-I Hyp A-S Hyp

Texture, Shear force (N) 21.2(1.5) 23.1 (1.7) 21.3(0.9) ns

Texture, Area under curve 197.9(13.3) 204.8 (12.6) 189.1 (6.76) ns

PYD cross-links, pmolg ^"1 497.8 (33.5) 530.2 (36.3) 479.6 (32.2) ns

¹ Statistical differences within rows are shown with different superscript letters (b indicates statistical significance. P < 0.05. Ns = not significant. ² Near significant, p = 0.051

Muscle firmness (shear work and shear force) correlated positively to a-i Hyp, PYD and pH at day 5, 9 and 15, p < 0.05 with one exception between pH and work done at day 15 (p > 0.05), Table 10. Hyp in the diet significantly increased pH of muscle day 5, and PYD cross-links day 9. According to the correlation values, pH at day 5 and PYD cross- links at Day 9 were the factors that further showed the highest correlations to muscle firmness, supporting the evidence for a specific impact of free Hyp in the diet. Muscle firmness also showed negative correlations to fish body weights at all samplings (P < 0.05), indicating that the largest fish had the lowest firmness. As individual tagging was not used in this study, it is not possible to distinguish between impacts of fast growing fish and fish with final high body weights, relative to muscle firmness.

At the sampling day 5, a significant correlation between pH and water, pH and a-i Hyp, as well as a-s Hyp and a-i Hyp were found, while similar was not shown later in the storage trial.

Table 10. Factors that correlate significantly to muscle texture/firmness following storage of salmon for 5, 9 and 15 days.

Muscle firmness Work done Shear force Water p_H a-s Hyp

Day 5 a-i Hyp 0.31 0.32 0.32 0.49

PYD cross¬

0.37 0.38 link pH 0.47 0.47 0.34

Body weights -0.38 -0.38

Day 9 a-i Hyp 0.31 0.33

PYD cross¬

0.49 0.49 link

PH 0.35 0.33

Body weights -0.44 -0.39

Water 0.30

Day 15 a-i Hyp - -

PYD cross¬

0.55 0.53 link

PH 0.28

Body weights -0.39 -0.39

Water -0.27 Preliminary conclusions

- Growth and feed conversion unaffected in a 12 weeks feeding trial

.- The changes in Hyp and other amino acids in plasma, urine and muscle indicate metabolic changes in response to dietary Hyp supplementation, possibly related to the observed changes in collagen synthesis or PYD cross-link formation.

- During storage of salmon in cooling chamber (4 ⁰ C), dietary Hyp inclusion in free form was found to: • Significantly increase pH (Day 5)

• Improved muscle texture (5 - 10%)

• Significantly increase collagen production (a-i Hyp), Day 5

• Significantly increase free Hyp in blood and muscle

o Substrate for collagen formation

• Significantly increase PYD cross-links in salmon muscle (Day 9)

- Dietary Hyp in bone-bound form tended to increase a-i Hyp (day 5) and increased a-s Hyp in muscle significantly (Day 5 and 9), but did not improve muscle firmness, a-i Hyp or PYD formation in the muscle.

- Muscle firmess was significantly influenced by a-i HYP (collagen), PYD cross-links and pH at all samplings during storage for 5, 9 and 15 days (4 ⁰ C), and a negative relation to fish body weight was found at all samplings (Day 5, 9 and 15).

- The results indicate that supplementation of free Hyp in the diet may improve the shelf-life of fish, such as salmon.

Administration of Hyp was found to improve muscle texture. The shown data indicate that the mechanism responsible for the improved muscle texture (increased muscle strength) is a hyp-induced increase in muscle collagen production and increased levels of mature cross-links in the muscle tissue. Hydroxyproline may be a semi essential amino acid and necessary for optimal nutrition, health and welfare of fish fed high plant protein diets. Hydroxyproline may also play a role on strength and functional properties of other connective tissue compartments in the body, such as in the skin, scale, intestine and bone.

Example 2

The example relates to the determination of the effect of Hydroxyproline on muscle tissue.

Method for measuring muscle mass

The effect of Hydroxyproline on muscle mass can be measured by any method known in the art including the method disclosed in United States Patent 5628328. This method for determining muscle mass in a human comprises administration of a bolus dose of a metabolic marker for 3-methylhistidine, the use of a three-compartment model to describe data from blood samples collected periodically thereafter, and calculation of muscle mass as a function of specific values generated by the model. The muscle mass before and after the intake of Hydroxyproline over period of time are compared.

Alternatively the effect of the Hydroxyproline composition can be monitored e.g. in a human being by measurement of muscle to fat ratios, such as by the Bioelectric Impedance Analysis (BIA) test. The BIA test is a simple, non invasive, painless procedure. By using a very small amount of electric current, this test can not only show the ratio of lean muscle to fat, but also other important indicators such as the amount of intra-cellular and extra cellular-water.

Method for measuring muscle strength

The effect of the Hydroxyproline on muscle strength can be measured by any method known in the art. The muscle strength of e.g. biceps muscle can be determined by lifting, pushing or pulling larger and larger weight loads until you reach the limit of your strength. The muscle strengths before and after the intake of Hydroxyproline over period of time are compared.

The maximum strength you measure is termed one repetition maximum (1 RM). This is a measurement of the greatest load (in kilograms) that can be fully moved (lifted, pushed or pulled) once without failure or injury. This value is difficult to measure directly because the weight must be increased until you fail to carry out the activity to completion.

It is safer to estimate 1 RM by counting the maximum number of exercise repetitions you can make using a load that is less than the maximum amount you can move. This number is called the repetitions to fatigue (RTF) - you stop counting the repetitions when you can no longer perform the exercise properly or when you slow down too much and cannot keep a steady pace.

1 RM can be used as an indication of your strength development. Repeat this measurement at regular intervals can determine if a person is gaining strength.

The values of the load you used and the number of repetitions you counted (the RTF) are entered into a prediction equation that calculates an estimate of your 1 RM.

One prediction equation for 1 RM that was published by Epley in 1985 has the formula: 1 RM = (0.033 x RTF x load) + load

So, if a person can lift a 50 kg weight for nine repetitions before tiring significantly, their estimated 1 RM is:

1 RM = (0.033 x 9 x 50) + 50 = 14.85 + 50 = approximately 65 kg

This means the person should just be able to lift 65 kg and no more. It also means they would need a rest of several minutes before they could lift the same weight again.

There are a number of equations that have been constructed by other sports science researchers over recent years to estimate 1 RM and a number of calculators that use various 1 RM prediction equations have been developed - search for them online using the keywords "1 RM calculator".

Furthermore, the muscle strength can be determined by the Apparatus for measuring muscle strength disclosed in United States Patent 2680967.

Grip strength can be measured using a dynamometer. A dynamometer is held in the hand and is designed to measure muscle strength in a simple way.

Determination of amino acid content in muscle The effect of the Hyproxyproline on the total amino acid content in muscle can be measured by any method known in the art. The total amino acid content in muscle before and after the intake of Hydroxyproline over period of time are compared.

One method for determination of the total amino acid content in muscle comprises one or more muscle biopsies and the analysis technique described herein below. Muscle biopsies of e.g. the quadriceps are carried out. For amino acid measurement, one muscle fragment is immediately weighed and homogenized with sulfosalicylic acid; plasma is precipitated with 3% sulfosalicylic acid. Amino acids in the acid extracts are measured using a Carlo Arba 3A 29 Automatic Aminoanalyzer. Amino acid concentrations are expressed as uM per Kg of muscle wet weight (AAm) or per Kg of plasma water (AAEcw).

Example 3

The example relates to the determination of the effect of Hydroxyproline on subcutaneous connective tissue.

The effect of the Hydroxyproline on skin elasticity can be measured by any method known in the art. The skin elasticity before and after the intake of Hydroxyproline over period of time are compared.

The elasticity measurements are performed with a Cutometer®. For this test, a suction cycle of 1 second at 500 mBar followed by a release cycle of one second was selected.

In elastometric measurement, the skin surface is aspirated from the depression induced by the machine into the aperture of the elastometer's measuring probe. The depth of the skin penetration inside the probe is measured by an optic sensor. Cutaneous elasticity reflects the skin's potential capacity (measured in mm) for retraction.

A graph is obtained that represents the deformation curve of skin undergoing aspiration, and includes two components.

• An elastic component (Ue), which corresponds to the part of the curve that rises rapidly, and is reversible to the deformation;

• A plastic component (Uv), which corresponds to the part of the curve that rises slowly and is not completely reversible to the deformation.

The total distension of the skin obtained at the end of an aspiration cycle is defined as skin extensibility (Uf). During the releasing phase, the quantity of deformation remaining in the skin can be observed (Ua=residual deformation). Cutaneous elasticity is defined as the ratio:

Elasticity = Uf-Ua/Uf

It represents the recovery degree of the maximum deformation reached, whose values range between 0 and 1 (maximum elasticity).

Example 4

The example related to the determination of the effect of Hydroxyproline on lung function. The effect of Hydroxyproline on lung function can be determined by any test in the art including the tests described herein below.

Spirometry

Spirometry is often the first lung function test done. It measures how much and how quickly you can move air out of your lungs. For this test, you breathe into a mouthpiece attached to a recording device (spirometer). The information collected by the spirometer may be printed out on a chart called a spirogram.

The more common lung function values measured with spirometry are:

• Forced vital capacity (FVC). This measures the amount of air you can exhale with force after you inhale as deeply as possible. • Forced expiratory volume (FEV). This measures the amount of air you can exhale with force in one breath. The amount of air you exhale may be measured at 1 second (FEV1 ), 2 seconds (FEV2), or 3 seconds (FEV3). FEV1 divided by FVC can also be determined.

• Forced expiratory flow 25% to 75%. This measures the air flow halfway through an exhale (FVC).

• Peak expiratory flow (PEF). This measures how quickly you can exhale. It is usually measured at the same time as your forced vital capacity (FVC).

• Maximum voluntary ventilation (MVV). This measures the greatest amount of air you can breathe in and out during one minute. • Slow vital capacity (SVC). This measures the amount of air you can slowly exhale after you inhale as deeply as possible. • Total lung capacity (TLC). This measures the amount of air in your lungs after you inhale as deeply as possible.

• Functional residual capacity (FRC). This measures the amount of air in your lungs at the end of a normal exhaled breath.

Expiratory reserve volume (ERV). This measures the difference between the amount of air in your lungs after a normal exhale (FRC) and the amount after you exhale with force (RV).

Gas diffusion tests Gas diffusion tests measure the amount of oxygen and other gases that cross the lungs' air sacs per minute. These tests evaluate how well gases are being absorbed into your blood from your lungs. Gas diffusion tests include:

• Arterial blood gases, which determine the amount of oxygen and carbon dioxide in your bloodstream.

Carbon monoxide diffusing capacity (also called transfer factor, or TF), which measures how well your lungs transfer a small amount of carbon monoxide (CO) into the blood. Two different methods are used for this test. If the single-breath or breath- holding method is used, you will take a breath of air containing a very small amount of carbon monoxide from a container while measurements are taken. In the steady-state method, you will breathe air containing a very small amount of carbon monoxide from a container. The amount of carbon monoxide in the breath you exhale is then measured. Diffusing capacity provides an estimate of how well a gas is able to move from your lungs into your blood.

Body plethysmography

Body plethysmography may be used to measure:

Total lung capacity (TLC), which is the total amount of air your lungs can hold. For this test, you sit inside an airtight booth called a plethysmograph and breathe through a mouthpiece while pressure and air flow measurements are collected.

• Residual volume (RV), which is the amount of air that remains in your lungs after you exhale as completely as possible. For this test, you sit inside the plethysmograph booth and breathe a known amount of a gas (either 100% oxygen or a certain amount of helium in air). The test measures how the concentration of the gases in the booth changes.

Inhalation challenge tests

Inhalation challenge tests are done to measure the response of your airways to substances (allergens) that may be causing asthma or wheezing. The tests also may determine the effect of chemicals such as histamine or methacholine on your airways. These tests are also called provocation studies.

During inhalation testing, increasing amounts of an allergen are inhaled through a nebulizer, a device that uses a face mask or mouthpiece to deliver the allergen in a fine mist (aerosol). Alternatively, increasing amounts of a substance (histamine or methacholine) may be inhaled through the nebulizer. Before and after inhaling the substance, spirometry readings are taken to evaluate lung function.

In rare cases, a bronchospasm can occur with inhalation challenge testing. You will be closely monitored during and after the test.

Exercise stress tests

Exercise stress tests evaluate the effect of exercise on lung function tests. Spirometry readings are done after exercise and then again at rest.

Lung function results are measured directly in some tests and are calculated in others. No single test can determine all of the lung function values, so more than one type of test may be done. Some of the tests may be repeated after you inhale medicine that enlarges your airways (bronchodilator).

Example 5

The example relates to the determination of the effect of Hydroxyproline on edema. The effect of Hydroxyproline on edema can be determined by any test in the art including the tests described herein below.

Technique for evaluation of peripheral edema:

Examiner impresses thumb into skin over bony surface such as over Tibia, Fibula and or Sacrum. Thumb is withdrawn and the depth of the pit is measured and record in millimetres. The pit depths before and after the intake of Hydroxyproline over a period of time are compared.

Example 6 The example relates to the determination of the effect of Hydroxyproline on joints, ligaments and tendons. The effect of Hydroxyproline on joints, ligaments and tendons can be determined by any test in the art describing flexibility testing including the tests described herein below.

Evaluation of the hvpermobilitv syndrome:

The hypermobility syndrome may be assessed using the Beighton score:

The total scores, before and after the intake of Hydroxyproline over a period of time, are compared.

Example 7

The example relates to the determination of the effect of Hydroxyproline on varicose veins. The effect of Hydroxyproline on varicose veins can be determined by any test in the art including the tests described herein below.

Varicose veins can be assessed using following methods:

• Physical exam

• X-rays

• Ultrasound

The severity of varicose veins, before and after the intake of Hydroxyproline over a period of time, is compared. Example 8

The examples relates to the determination of the effect of Hydroxyproline on incontinence. The effect of Hydroxyproline on incontinence can be determined by any test in the art including the tests described herein below.

Components of a Basic Continence Evaluation:

The basic evaluation should include a history, physical examination, estimation of post- void residual (PVR) urine volume, and urinalysis.

Urinary incontinence may be assessed by one or more of the following methods:

• Urination diary

• Stress test

• Urinalysis • Blood tests

• Ultrasound of kidneys and urinary system

• Cystoscopy

• Urodynamics