# Patent

## Title
Bear derived isolate and method

## Abstract

A bear derived isolate found in denning black bears or black bears from which food has been withheld for at least two weeks when injected into another mammal produces reduced heart rate, reduced respiratory rate, reduced temperature, and a wakeful tranquility not unlike the phenomena of the denning black bear. The isolate is taken from either the serum or urine of such denning bear. The method of the invention involves the steps of fractionating the serum or urine of a denning black bear to produce fractions of varying potency.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of Ser. No. 10/232,880, filed Aug. 30, 2002 now U.S. Pat. No. 6,855,337 by the same inventor's herein; which application is a continuation of Ser. No. 08/833,096, filed Apr. 4, 1997 now abandoned, which is a continuation-in-part of 08/470,750, filed Jun. 6, 1995 now abandoned; which is a continuation-in-part of Ser. No. 08/259,788, filed Jun. 14, 1994 now abandoned; which is a continuation-in-part of Ser. No. 08/079,089, filed Jun. 16, 1993 now abandoned.


FIELD OF INVENTION
The present invention relates to the discovery and isolation of a substance called bear derived isolate (BDI) which can be found in fasting and denning black bears which, in combination and with various carriers and various doses, based upon studies conducted with guinea pigs, bone cultures, and rats, will likely have beneficial results on humans in promoting bone growth in those persons having osteoporosis, in conserving nitrogen to a point where hemodialysis and kidney transplants need not be done in patients with chronic or end stage renal disease, in inhibiting protein breakdown in humans suffering burns and trauma, in permitting long-term flights into space by conserving bone integrity and preventing muscular atrophy, and in producing weight loss in obese subjects in the form of fat reduction while conserving lean body mass and promoting tranquility while in an alert state at normal body temperature. A related aspect of the invention is directed to a method of the isolation and purification of the bear derived isolate, whether from a fasting bear or a denning bear, to a form where predictable results in the above phenomena are readily achieved alone or in combination with other known metabolic substances. The further discovery that a fasting or otherwise normal summer bear, as distinguished from a denning bear, will produce the equivalent of a bear derived isolate (BDI) requires that this invention be considered in terms of a fasting bear, despite the fact that the bulk of the investigation has evolved around the isolate from a denning bear.
A better understanding of the field of invention, the invention itself, and the description of preferred embodiments will follow from an understanding of the definitions of various terms which are used, and which appear in the following “Glossary of Terms”.
GLOSSARY OF TERMS
Aliquot: A specified portion.
Alkaline Phosphatase Activity: Activity of this enzyme increases in bone as part of osteoblastic stimulation of bone growth.
Anorexia: Loss of appetite.
Aqueous Fraction: That portion containing water.
Bone Remodeling: A function of bone in which osteoblasts form bone and osteoclasts resorb bone. Positive bone remodeling occurs when the osteoblastic activity exceeds the osteoclastic activity; or when the osteoclastic activity is diminished; or where the osteoblastic activity is increased. In any of these events there is a positive addition to bone. Negative bone remodeling occurs when the osteoclastic activity outstrips the osteoblastic activity, or the osteoblastic activity is reduced from its normal balance with the osteoclastic activity; and any combination of the foregoing.
Bone Resorption: Occurs when bone is subjected to osteoclastic activity. Countercurrent Chromatography (CCC): A technique used to separate substances of different molecular characteristics by using solvents of aqueous and organic properties with centrifugation. Some substances are retained on the coil while others pass through.
Deproteination: Subject the sample to any of various procedures for removing all or part of the original protein in the sample.
Differentiation: To develop into specialized organs or cells.
Eluted: Drawn down, through or off (e.g. liquid through a filter).
Eluted Isocratically: Separate substances off of a column using one solvent system without changing concentration of that solvent system.
Fasting: A voluntary or involuntary state represented by states of non-ingesting, hypophagia, or anorexia. In the context of a fasting active summer bear, while food may be withheld, water is available on demand.
Fibroblast: A stellate or spindle-shaped cell with cytoplasmic processes present in connective tissue, capable of forming collagen fibers.
Gas Chromatography(GC): A method of chromatography in which the substance to be separated into its components is diffused along with a carrier gas through a liquid or solid adsorbent for differential adsorption.
High Performance Liquid Chromatography (HPLC): Method of partitioning chromatography that employs high pressures to propel the solvent through a thin column resulting in a high resolution of complex mixture.
Intraperitoneally: Inside the abdominal cavity.
Latin Square Design: An experimental design which gives statistical meaning to data when using small numbers of experimental units (e.g. numbers of animals, samples, etc.). The number of treatments tested is always equal to the number of experimental units being used and each experimental unit receives all treatments over time.
Lyophilization: The creation of a stable preparation of a biological substance or isolate (blood serum, plasma, etc.), by rapid freezing and dehydration of the frozen product under high vacuum.
Lyophilize: Freeze dry.
Mass Spectrometry (MS): A procedure used to determine the masses of atoms or molecules in which a beam of charged particles is passed through an electric field that separates particles of different masses.
Metabolites: Any of various inorganic or organic compounds produced by metabolic pathways in the body such as urea, creatinine, amino acids, hydroxy acids, fatty acids, glucose, ions, etc.
Monocyte: Cells with a single nucleus derived from marrow monoblasts. They have deeply indented and irregularly shaped nuclei and bundled and scattered single filaments in the cytoplasm. Marrow monocytes are responsible for forming osteoclasts.
Ninhydrin: Agent used to develop color on TLC plates.
Nuclear Magnetic Resonance (NMR): The absorption of electromagnetic radiation of a specific frequency by an atomic nucleus that is placed in a strong magnetic field, used especially in spectroscopic studies of molecular structure.
Osteoblast: A cell from which bone develops.
Osteoclast: A large multinuclear cell that resorbs bony tissue in the process of osteoclasis.
Osteoid: Relating to or resembling bone ossiform; newly formed organic bone matrix prior to calcification.
Osteoporosis: Demineralization of bone; decrease in bone mass or structure.
Ovariectomy: Surgical removal of the ovaries.
Pellet by Centrifugation: Spin sample to force protein residues to bottom of test tube.
Phosphomolybdic Acid Detection: Method used to develop color on TLC plates.
Renal Failure: Inability of kidney to function properly; one aspect is failure to excrete the amount of urea formed by the body daily. This leads to a gradual elevation of urea which may result in uremia, a toxic condition, that requires dialysis or kidney transplantation for treatment.
Resolution Factor (Rf): The distance that the midpoint of the compound travels on a given plate divided by the distance the solvent travels on the plate.
Resorb: To dissolve and assimilate.
Silica Gel/Column Chromatography: Sandlike material is placed in a long glass tube which is wet with solvents and is used to separate the materials by retaining some components on the silica while other components pass through depending on the solvents used.
Sham: A subject is subjected to surgical procedure without removal of organs (ovaries) in order to duplicate the physical and mental impact of the surgical procedure on test animals.
Silica Plate: Glass plate or microscope slide coated or painted with sand-like material. Used to separate and detect substances.
Stirring Rod: Metal or glass rod used to stir mixtures (e.g. spoon in coffee).
Supernatant: Liquid fraction of a liquid solid mixture where the solid has settled to the bottom of its container (e.g. in water and sand, water is the supernatant).
Thin Layer Chromatography (TLC): Method used to separate chemical constituents which can then be identified by color or other properties upon development.
Transamination: A process involved in the metabolism of amino acids in which amino groups (—NH2) are transferred from amino acids to certain keto acids yielding new keto and amino acids.
Triturate: Treat certain dry materials by dissolving part of them into solution leaving behind components that do not dissolve in said solution.
Ultrasonication: Using sound waves to remove particles from small places (e.g. used to clean jewelry).
BACKGROUND OF THE INVENTION
It is known that denning, fasting black bears, fasting polar bears, and pregnant female polar bears who den possess blood factors that can recycle harmful body waste products back into usable protein for building tissue, and that denning, fasting black bears can continue to build bone when the bear is immobile for months at a time. Upon isolating the substance which controls this phenomena in the bear, there is the possibility that the same can be used to prevent toxic buildups that endanger humans with kidney failure that now require the stressful, expensive treatments of dialysis and kidney transplant to sustain life. The isolate (BDI) also includes the possibility that it can prevent protein breakdown which leads to life threatening situations in humans suffering burns and trauma.
It is believed that such knowledge can lead to strategies to combat bone loss, which afflicts millions of middle aged and elderly people, especially post-menopausal women and astronauts in weightlessness of space. Loss of bone mass in space is one of the major problems that prevents long term space flights by humans.
Bears preparing to enter the denning phase go through a period of hyperphasia during which they eat enough food to store enough fat to last through the denning period. During denning, bears do not eat, do not drink, and do not urinate or defecate. Exiting the den after a four to five month period, the bears resume normal eating patterns. Knowledge and/or the isolate (BDI) may be useful in developing strategies and/or products for the treatment of eating disorders such as anorexia nervosa and bulimia.
Black bears in particular, during their three to five month denning, show a reduction in body temperature of at least 2° C., remain alert and expend energy normally; yet they do not eat, drink, urinate, or defecate and exhibit no problems with waste building to toxic levels. Other mammals, including humans, can recycle some waste, but under similar conditions must quickly rid themselves of the rest of their waste or die.
It has been determined that bears in a non-denning state during summer months are induced to produce the isolate (BDI) after 20 days of fasting, even though they are allowed to drink water. Under these circumstances, bears urinated and did not exhibit the tranquility associated with a denning bear.
Other mammals (including deep hibernators such as ground squirrels who continually awaken throughout hibernation and generate waste they must get rid of) break down protein mainly from muscle to supply energy and other essential nutrients for life. This process not only depletes body muscle, it also releases the toxic form of nitrogen as ammonia. Mammals, including humans, convert the ammonia to urea, which is much less toxic but must be eliminated in urine. During denning, black bears also produce urea, but close this loop and recycle the urea nitrogen back into protein. They produce no waste and maintain muscle mass while eliminating the need to urinate or defecate. The process is so efficient that normal urea concentration in blood decreases and body protein increases. The bear is the only animal known that fasts completely (no food or water) yet ends a 100 day or longer fast with a little more protein (lean tissue) than when it started. During the denning period, the bear steadily consumes body fat that had been stored during the pre-denning period.
This unique response extends to maintenance of bone mass. The bear shows no bone loss even when supine over more than 100 days. In contrast, deep hibernators lose bone and exhibit osteoporosis when hibernating. The bear does not develop osteoporosis and is able to maintain skeletal integrity despite the harsh conditions. Under similar stimuli, humans would suffer severe bone loss.
Taken in the context of the foregoing, it is a desirable forward goal in the treatment of human ailments to be able to isolate the bear derived isolate (BDI) which permits the foregoing phenomena in bears, and to translate it into meaningful metabolic and curative processes in the human.
These goals appear possible. For instance, a bile salt produced by the bear has been shown to improve liver function in humans with the fatal disease of primary biliary cirrhosis. In humans, this bile salt also reverses serious rejection reactions against bone marrow transplants. Further, this bile salt, ursodeoxycholic acid, is the most effective dissolver of human gall stones. Thus, a isolate produced by bears has direct positive application to human disorders.
Important to the present invention is the skill of the technician practicing the invention in identifying when the true state of denning exists in the bear and when the denning bear accomplishes the unique management of wastes such that none accumulate.
Experiments and observations directed to studies in denning bears have been under way for more than 23 years. During that time, it has been established that the recycling of body wastes causes the blood ratio of urea to creatinine (U/C) both expressed in mg/dl to decrease from 20 or more (sometimes ranging as high as 70 after eating a high protein diet) to 10 or less—something impossible for any other mammal that is not drinking fluid. A U/C ratio of 10 or less due to a significant decrease in urea and a significant increase in creatinine indicates that recycling of urea is in progress. The low U/C ratio found throughout denning sometimes occurs in wild bears in the fall just before denning. At this point, wild bears have stored enough fat for denning. They stop eating and drinking; complete waste recycling has begun before they enter the den.
The bear continues to degrade amino acids and form urea. In turn, the urea molecule is quickly degraded by transferring nitrogen from it to substances such as pyruvic acid or alpha-ketoglutaric acid to reform amino acids. This latter process is called transamination. The substances necessary for transamination (pyruvic acid and alpha-ketoglutaric acid) are generated from glycerol which has been released from fat. The newly formed amino acids are then reincorporated into protein.
The overall process of urea recycling consists of two processes: 1) formation of urea from amino acids, and 2) reformation of amino acids from urea which are then reincorporated into protein. Since (2) is faster than (1), there is net formation of new protein. Based on our knowledge, no other fasting animal can accomplish this feat.
Some amino acids formed in the bear are: alanine, serine, ornithine, arginine, glycine, leucine, threonine, phenylalanine, and tyrosine. These amino acids are found in such proteins as albumin and fibrinogen.
Humans can recycle only about 25% of the urea they form. The bear, on the other hand, recycles urea back into protein a little faster than it makes it. Thus, its blood urea concentration diminishes even though it does not drink water or urinate. The amino acids that serve as vehicles for urea recycling are ordinarily found in all mammals, but not in the concentrations shown by bears when fasting. Therefore, it is assumed that they may become vehicles to be used with the bear derived isolate when duplicating the bear's unique recycling.
During denning, the kidney of the bear continually forms urine. Upon reaching the urinary bladder, the urine (which contains BDI) is completely absorbed by the wall of the bladder. Thus, in a highly concentrated form, BDI moves across the bladder wall into blood, circulates, and stimulates all tissues of the bear. When compared to the blood of fasting humans, blood of the denning bear differs in concentrations of some amino acids, bear ketones are much lower, and there is a difference in some other essential substances. While concentrations of many of these substances decrease during human fasting; they do not decrease in the bear. Therefore, exact profiles of these known metabolites may have to be added to BDI in order to duplicate the bear's unique recycling in humans.
Recycling urea, the waste product of protein breakdown, back into protein leads to maintenance of lean body mass.
To prevent bone loss, bone remodeling occurs normally while in the supine state. In the human, a supine state inhibits normal bone remodeling and leads to severe loss of calcium and bone.
All of these stages of prior art were possible only by developing the state of the art that permits bears to den in captivity and to design the definitive studies to explain the processes.
SUMMARY OF THE INVENTION
The present invention results from the discovery of the method and results from isolation of a material in bears, particularly black bears, called Bear Derived Isolate or BDI, that enables denning so that BDI can be used alone or identified with one substance or combination of substances either novel and unique or previously identified to help human beings and other mammals. All predictable results are based upon in vivo studies with guinea pigs, in vivo studies with rats, in vitro organ studies of calvarial mouse bone, and in vitro studies of prevention of proliferation of cells that resorb bone and stimulation of proliferation of cells that form bone using cell cultures of monocytes, osteoclasts, osteoblasts and fibroblasts. BDI is present in the serum (blood) of denning bears. BDI is also present in urine of denning bears. However, because the bear is an omnivore, fasting in summer is extremely rare. What has been discovered however, is that when the normally active black bear is fasted in the summer time, but water not withheld, over a period of two to three weeks it will develop in the urine the same BDI referred to with regard to denning black bears. Post-fast data showed that urea recycling was induced. This was evidenced by a low serum urea/creatinine ratio, a slight increase in total proteins, and a marked increase in beta-hydroxybutyric acid. Accordingly where the term BDI is used, it includes fasting bears from which food has been withheld but which are not in the traditional denning season. The same can be extrapolated for active polar bears. Because the U/C ratio of polar bears is near 10 or less when fasting, urea recycling is indicated.
In order to obtain the research material (BDI) blood (serum) and urine are collected from black bears during their denning period. Quantities of 100 ml may be drawn monthly from each bear or on a more frequent schedule as required. The urine and/or serum is then subjected to the isolation method as described herein.
As illustrated in Table 1, isolation of BDI requires precipitation of protein from winter urine or serum using methanol, centrifuging the sample and removing precipitated protein as pellets, and drying the BDI into a visible extract. Further, by the use of thin layer chromatography (TLC), countercurrent chromatography (CCC), preparative thin layer chromatography, or column chromatography, at least two compounds, both in urine and blood, can be isolated in BDI. Thus, the method of isolating these compounds permits predictable separation of BDI into Fractions. These Fractions are suitable for biologic testing. One component is an as-yet-unidentified compound. It is called the Miers-Nelson Component (MNC) after the researchers. The other component is beta-hydroxybutyrate (BHB).
BDI can be divided into three Fractions which are sufficiently purified to test for their biological activity in guinea pigs, rats, and bone culture assays. These Fractions are:

    
    
        Fraction I=BDI−[BHB+MNC] (Early fractions),
        Fraction II=BHB (Middle fractions), and
        Fraction III=MNC (Late fractions).
    
    


OBJECTIVES OF THE INVENTION
It is a primary object of the present invention to isolate and evaluate BDI which is present in a denning bear or fasting bear.
A further object of the present invention is to permit the isolation of BDI in such quantities that BDI used alone, or in combination with other metabolites and carriers, may be administered orally or by injection to other animals or humans for various treatments.
Being on the cutting edge of a pioneer area of analysis, yet another object of the present invention is to produce BDI (which permits denning) in order to facilitate further research concerning various beneficial results that can be achieved regarding the kidney, liver, bone growth and remodeling, brain, and nitrogen cycles in the body.
Yet another object of the present invention, and an important one, is to produce BDI in a form which, upon further analysis, will permit synthesis of BDI in larger volumes and at significantly reduced expenditures.
Further objects and advantages of the present invention will become apparent as the following description proceeds, taken in conjunction with the accompanying data.
Following is a Table illustrating the process for the isolation of BDI and two compounds found in it.







TABLE 1







Chemical Process for Isolation of BDI


and Two Compounds Found In It


Research Procedure for Isolating BDI and Its Fractions










STEP
SAMPLE
PROCESS
YIELDS













One
Urine (50 ml)
1. MeOH Deproteinization
Dry Sample (BDI)




2. n-BuOH Trituration


Two
Dry BDI (3.5 g)
CCC (n-BuOH:AcOH:H2O)
Dry Sample




20:1:20


Three
Dry sample (2 mg)
CCC (n-BuOH:AcOH:H2O)
Fractions:













20:1:20
A.
Fraction I






BDI − [BHB + MNC]






Early CCC Fractions





B.
Fraction II






BHB






Middle CCC Fractions





C.
Fraction III






MNC






Late CCC Fractions











DESCRIPTION OF PREFERRED EMBODIMENT
The Denning Process of Bears
The denning process of bears has been defined in the statement of Background of the Invention above. In order to obtain the bear derived isolate successfully, denning bears must be available quickly and throughout the denning period as is the case at The Carle Foundation Bear Research Station, Champaign County, Illinois. At this facility, after food intake decreases in October or November, food is removed, inducing the bear to enter the denning state. At all times where reference is made to the bears which were used to produce BDI, such bears were the well known North American Black Bears (Ursus americanus).
Thereafter, blood and urine samples are taken from the bears. This continues until March when the bear leaves its den and has access to food and water. At first (for approximately two to three weeks), the bears slowly begin to eat after they emerge from their dens in the spring. Food intake reaches normal levels, and weight gain continues until early June in preparation for mating. By mid June the bears have normalized their body stores of fat that were diminished during denning and will continue to eat throughout the summer to maintain body weight. Slight increases in body weight throughout the summer can be attributed to continued growth. In late August, in preparation for the subsequent denning season, the bear increases its food intake from 5,000 to 8,000 Calories/day to 20,000 Calories/day. The bear eats almost to a calorie the quantity of food required to store enough fat to support energy requirements of denning, fetal support, and lactation. For a 400 pound bear, energy expenditure during denning is about 4,000 Calories/day.
Bears that have been fasted for a period of not less than 21 days during the summer or non-denning period, whose urine, when subjected to isolation methods, yielded a material (BDI) which produced bone remodeling effects and urea creatinine ratios comparable to that of the material (BDI) taken from a denning bear. The experiment related to 14 bears which were given free access to drinking water, but food was withheld for 21 days. The group was fasted during the month of July, a recognized non-denning period for bears. This was in an attempt to determine whether fasting is the controlling factor in the production of BDI.
Defecation stopped after approximately 2–3 days in the fasting bears, but occasionally bile stain material passed per rectum in some of the bears. With free access to water, the bears drank enough to stimulate urination. (Excess water was required because the only mechanism bears have to regulate body temperature is through evaporation via the respiratory tract. In summer, ambient temperature is much higher than experienced by denning bears, thus there is a need for increased evaporative water loss. This, in turn, stimulated drinking, which exceeded the bears' requirements for body temperature control and thus stimulated urination.) Even though the fasted bears drank water, thirteen of fourteen bears showed an increase in serum creatinine. Eleven of fourteen bears showed a reduction in serum urea, which resulted in a significant reduction in the U/C ratio. Five animals demonstrated values previously known to be associated only with denning bears (Table 2).







TABLE 2







SUMMER BEAR FASTING EXPERIMENT: Jul. 13, 1994 to Aug. 2, 1994









DATE



















Jul. 13,











1994 to



Jul. 13,
Aug. 2,
Aug. 2,
Jul. 13,
Aug. 2,
Jul. 13,
Aug. 2,
Jul. 13,
Aug. 2,



1994
1994
1994
1994
1994
1994
1994
1994
1994



PRE-FAST
POST-FAST
WEIGHT
PRE-FAST
POST-FAST
PRE-FAST
POST-FAST
PRE-FAST
POST-FAST



WEIGHT
WEIGHT
LOSS
UREA
UREA
CREATININE
CREATININE
U/C
U/C


BEAR
(lbs.)
(lbs.)
(lbs.)
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
RATIO
RATIO



















1-524
256
214
−42
22.39
21.89
1.4
2.1
15.99
10.42


2-523
186
150
−36
29.61
36.70
1.4
2.2
21.15
16.68


3-519
358
298
−60
31.70
27.47
1.7
2.6
18.65
10.56


4-521
226
186
−40
32.60
41.85
1.7
2.1
19.18
19.93


5-522
350
302
−48
30.90
18.24
1.8
2.1
17.17
8.69


6-520
298
248
−50
32.20
30.90
2.1
2.4
15.33
12.88


♀ 7-513  
210
178
−32
30.70
26.61
1.5
2.1
20.47
12.67


♀ 8-514  
216
190
−26
45.50
27.47
1.7
2.6
26.76
10.56


9-515
306
260
−46
37.98
30.26
2.2
2.3
17.26
13.16


♀ 10-516  
162
140
−22
33.00
31.55
1.6
2.2
20.63
14.34


11-518 
304
262
−42
19.74
36.48
1.6
2.6
12.34
14.30


12-517 
306
260
−46
44.40
24.46
2.3
2.0
19.30
12.23


U.P.
412
356
−56
49.35
24.46
2.4
2.7
20.56
9.06


Caruso
388
328
−60
42.30
31.76
1.9
2.4
22.26
13.23


MEANS
284 ± 77
241 ± 67*
−35 ± 15
34.46 ± 8.5
29.29 ± 6.3
1.8 ± 0.3
2.3 ± 0.2*
19.08 ± 3.47
12.75 ± 3.0*





*Indicates a significant difference between the Pre-fasting and Post-fasting values using a paired t test, p < 0.01.


SUMMARY


Active bears eating normally were fasted 21 days. After fasting:


1. 11 out of 14 bears showed a decrease in the concentration of serum urea.


2. 13 out of 14 bears showed an increase in serum creatinine.


3. 12 out of 14 bears showed a decrease in the U/C ratio with 5 bears showing values ≦ 10.






Data collected from fasted summer bears (after they had been eating normally during the non-denning period) were compared with data collected from fasted winter bears. Although bears usually den (and don't eat) during the winter, these bears had been eating prior to entering the Carle Bear Research Facility. The data collected from fasted summer bears were similar to data collected from the same bears after a three week winter fast (Table 3).







TABLE 3







WINTER BEAR FASTING EXPERIMENT: Feb. 14, 1994 to Mar. 7, 1994









DATE



















Feb. 14,











1994 to


Feb. 14,
Mar. 7,



Feb. 14,
Mar. 7,
Mar. 7,
Feb. 14,
Mar. 7,
1994
1994
Feb. 14,
Mar. 7,



1994
1994
1994
1994
1994
PRE-FAST
POST-FAST
1994
1994



PRE-FAST
POST-FAST
WEIGHT
PRE-FAST
POST-FAST
CREAT-
CREAT-
PRE-FAST
POST-FAST



WEIGHT
WEIGHT
LOSS
UREA
UREA
ININE
ININE
U/C
U/C


BEAR
(lbs.)
(lbs.)
(lbs.)
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
RATIO
RATIO



















1-524
280
230
−50
15.02
10.73
1.5
2.0
10.01
5.37


2-523
192
156
−36
17.17
19.31
1.6
2.2
10.73
8.78


3-519
384
332
−52
30.04
15.02
2.1
2.7
14.31
5.56


4-521
288
238
−50
32.18
12.88
1.7
2.1
18.90
6.13


5-522
380
324
−56
19.31
15.02
1.7
2.3
11.36
6.53


6-520
282
244
−38
23.61
10.73
2.2
2.5
10.73
4.30


♀ 7-513  
228
206
−22
27.90
10.73
1.8
2.1
15.50
5.11


♀ 8-514  
222
198
−24
36.48
21.46
2.2
2.4
16.58
8.94


9-515
328
282
−46
32.19
32.19
2.2
2.3
14.63
14.0


♀ 10-516  
184
152
−32
27.90
27.90
1.6
1.8
17.44
15.50


11-518 
318
286
−32
32.19
21.46
2.4
2.9
13.41
7.40


12-517 
354
316
−38
17.17
10.73
1.5
2.0
11.44
5.36


*U.P.
380
374
−06
10.73
10.73
3.3
3.4
3.25
3.16


*Caruso
436
426
−10
6.40
6.44
3.2
3.2
2.01
2.01


MEANS
286 ± 69
247 ± 62
−43 ± 15
25.88 ± 7.19
17.30 ± 7.21**
1.9 ± 0.3
2.3 ± 0.3**
13.72 ± 2.92
7.73 ± 3.57**





*Bear was already denning


**Indicates a significant difference between the Pre-fasting and Post-fasting values using a paired t test, p < 0.01.


SUMMARY


Of the bears who were not previously denning (ie. had access to food during the winter), after fasting:


1. 9 out of 12 bears showed a decrease in the concentration of serum urea.


2. 12 out of 12 bears showed an increase in serum creatinine.


3. 12 out of 12 bears showed a decrease in the U/C ratio with 10 bears showing values ≦ 10.






It was concluded that after both the summer fast and the winter fast, the bears were in the urea recycling mode previously only characterized during denning.
The prefasted BDI from summer urine tested in bone cultures was from catheterized specimens while the post BDI from urine was collected without anesthesia from the specially adapted metabolic cages. As described later, BDI from the latter sample significantly increased osteoblast activity.
CHEMISTRY OF THE INVENTION
Introduction
The presentation to follow is divided into two parts. The first deals with the chemical process of isolation and characterization of BDI and two compounds characteristic of the winter denning bears (BHB and MNC) found in BDI. The second part describes the biologic activity of BDI and three of its component Fractions. The chemical isolation of BDI using chromatography makes it possible to divide purified BDI. Countercurrent chromatography yields 50 fractions in successive order: 1–50. The first group of CCC fractions (1–17) does not contain either BHB or MNC. The second group of CCC fractions (18–22) contains BHB. The third group of CCC fractions (23–50) contains MNC, found mainly in fractions 25–29. The CCC machine is then washed out to collect anything left in it. The third division also includes the wash; nothing is discarded. CCC fractions are grouped for further studies and labeled Fraction I, Fraction II, and Fraction III.
The specific fractions related to CCC samples may vary slightly. For instance, BHB may elute in fractions 19–23, and MNC in fractions 24–29. However, all CCC samples at division points are tested by thin layer chromatography so that no BHB appears in either Fraction I or Fraction III and so that no MNC appears in Fraction II.
Therefore, through the use of CCC, two characteristic components can be isolated. They also serve as logical points for division of BDI into three Fractions in order to test biologic activity: Fraction I (BDI−[BHB+MNC]), Fraction II (contains BHB), and Fraction III (contains MNC). When separated by CCC, these Fractions are known to contain amino acids, ammonia, urea, creatinine, creatine, and other animal products.
Identification of Bear Derived Isolate (BDI) Derived from Urine
A 50 ml aliquot of bear urine is deproteinated by diluting with methanol (1:1 v/v) and allowing proteins to precipitate out overnight at −20° C. The proteins are then pelleted by centrifugation (20 minutes @ 2500 r.p.m., 10° C.) and the supernatant is extracted. To completely dry the supernatant extract, nitrogen gas is used to remove methanol. Samples are then frozen (−80° C.) and lyophilized. Once dry, samples are weighed using Mettler Analytical Balance AE163. Fifty milliliters of winter bear urine yields approximately 3.5 g of dry residue known as BDI. For observation of the effects of BDI, the dry deproteinated sample (BDI) is reconstituted with 2 or more ml of saline. This solution can then be used for guinea pig and bone culture studies.
Isolation and Characterization of the Miers-Nelson Component (MNC)
Step 1: Verification of MNC Presence In BDI
BDI containing MNC is prepared as before and dried to a residue using nitrogen gas or lyophilization. The BDI is then:

    
    
        Dissolved in 100–500 μl of methanol depending on sample weight.
    
    


To test for presence of MNC in number (1) above, approximately 4–6 μl is applied to normal phase TLC plates (EM Science, P.O. Box 70, 480 Democrat Road, Gibbstown, N.J. 08027-1296 Silica Gel 60 F254, 0.25 mm) in successive μl applications.
The silica plate is then developed in a 4:1:1 1-butanol:acetic acid:water solvent system contained in a TLC chamber. Once developed, the plate is removed, dried by heat gun, and finally detected by ninhydrin spray (0.3% w/v in 1-butanol).
Location of MNC is detected with vigorous heating by heat gun and/or hot plate until edges of the TLC plate are charred.
At this point in isolation, MNC is visualized as a pink spot at Rf=0.74–0.80.
Step II: Purification of MNC
Approximately 1.75 g of BDI containing MNC is then prepared for the next purification step involving countercurrent chromatography. This procedure utilizes a bi-phasic solvent system of 1-butanol:acetic acid:water (20:1:20) and a Countercurrent Chromatography System with #10 semi-preparative coil (P.C. Inc.).
Two liters of the bi-phasic solvent described above is prepared at least one day prior to using CCC.
This butanol-acetic acid-water solvent system is mixed by shaking and allowed to settle 2 to 4 hours before separation of the organic and aqueous bilayers.
Two liters of solvent yields approximately 1200 ml of the organic stationary phase (primarily composed of butanol) and approximately 800 ml of the aqueous mobile phase (primarily composed of water).
The dried sample of BDI that has been prepared prior to the aqueous/organic solvent system still contains MNC. This sample is reconstituted in 5 ml of the solvent system (2 ml stationary phase:3 ml mobile phase) and loaded on to a 10 ml injection loop interfaced to the CCC.
The CCC coil is first loaded with 385 ml of stationary (organic) phase.
Using the mobile (aqueous) phase, the triturate is injected onto the coil for separation.
The coil is rotated at approximately 800 r.p.m., flow rate=4 ml/min (LDC Analytical Mini Pump). Five minute samples are collected (Gilson Microfraction Collector #203).
Fifty (20 ml) samples are collected and the coil is washed with methanol:water (1:1 by volume).
All samples are then frozen (−80° C.) and lyophilized (freeze dried).
Once dry, the 50 samples are analyzed by TLC/ninhydrin to determine which samples contain MNC.
MNC elutes in samples 25–29 (approximately 520–580 ml post coil).
Next, those usable, isolated MNC samples are combined with each other for further purification. Sample weight at this stage of purification has been reduced from 1.75 g to 1–2 mg. At this point, samples containing concentrated MNC also contain biological salts and significantly reduced concentrations of other impurities as detected by TLC/ninhydrin, UV, iodine vapor, and phosphomolybdic acid.
Then, samples containing MNC, the remainder of the CCC samples, and the wash of the CCC (fractions 22 through 50 plus wash) are recombined and passed through CCC a second time under the exact conditions described above.
Step III: Harvesting MNC: Preparative Thin Layer Chromatography
Final purification of Fraction III (MNC) entails the use of preparative thin layer chromatography.
The dried combined samples of MNC from the second countercurrent chromatography run are the sources of samples to be applied across an 8×12 cm silica thin layer plate. MNC is first reconstituted in 100 μl of methanol and then applied in ten 1 microliter (μl) spots across the plate.
Application of MNC in solution (to the TLC plate) is then repeated 10 times.
In order to achieve the best resolution, between each application the μl spots are allowed to air dry. When finished, each spot on the plate will contain 10 microliters (μl) of MNC in solution forming a band across the TLC plate.
The plate is then resolved in 4:1:1 BuOH:AcOH:H2O. Once the solvent rises to 80% –90% of the TLC plate, the plate is removed from the solvent and dried by heat gun.
Without developing the plate, the MNC band is removed by scraping the silica from the plate at the Rf region of 0.74–0.80.
The silica is then wetted in approximately 1–2 ml of 1-butanol with vigorous vortex mixing.
The 1-butanol and silica mixture is then centrifuged for 20 minutes at 2500 r.p.m. This allows the silica to pellet to the bottom of the tube.
The MNC containing butanol supernatant is then removed and dried down under nitrogen gas.
At this step in purification, the 1–2 mg sample has been reduced to 100–200 μg of MNC and is separated from salts and other impurities as detected by TLC/UV, ninhydrin, and iodine vapor. A lipid contaminant is apparent under phosphomolybdic acid development at the solvent front of normal phase TLC plates at this point. However, MNC remains the only significantly concentrated material present as detected by TLC/ninhydrin, UV, iodine vapor, and phosphomolybdic acid detection.
Properties of MNC
The harvested MNC has the following properties:

    
    
        1. It is soluble in water, methanol, and 1-butanol.
        2. It is insoluble in less polar organic solvents such as chloroform, toluene, and hexane.
        3. It is stable when stored frozen at −20° C. to −85° C. for at least eight years.
        4. It is stable at room temperature (20° C.–22° C.) for at least four days.
        5. It is heat resistant to 65° C.
        6. It is slightly UV active by detection of TLC and UV spectroscopy at 280 and 320 nm wavelengths.
        7. It is ninhydrin positive only with extended heating as previously described.
        8. It can be identified as pink in color at Rf 0.77–0.80 when purified on normal phase silica TLC plates, sprayed with ninhydrin and heated.
        9. It can be detected using iodine vapor development of normal phase silica TLC plates.
        10. To date, no tested substances in blood and urine of mammals show characteristics similar to the ninhydrin reaction at Rf range of 0.77–0.80 on the thin layer chromatography used in isolation.
        11. Recommended storage of the harvested MNC is to freeze it in a light resistant container under nitrogen gas.

Isolation and Characterization of Beta-hydroxybutyric Acid (BHB) Preparative Thin Layer Chromatography

    
    


The verification, purification, and harvesting of BHB is similar to MNC, except that CCC samples 18–22 are used to elute BHB. Further, BHB is extracted using the same method of preparative thin layer chromatography except that the silica is scraped from the plate at the Rf region of 0.82 to 0.92.
Flash Column Chromatography
An alternative method of harvesting BHB called Flash Column Chromatography can be used. When this method is used, BHB samples obtained from CCC purification are combined and dried.
The combined samples are reconstituted in 250 μl of 1-butanol. Mixing and ultrasonication are used to induce the sample into a homogeneous solution.
Once the samples are completely solubilized in the 250 μl of butanol, 250 μl of acetone is added to the solution. The resultant 500 μl sample is ready for subsequent purification by silica gel flash column chromatography.
A 15×230 mm silica gel (0.040–0.063 mm particle, 230–400 mesh) column is packed and wetted with five column volumes of acetone: 1-butanol (99:1). This ratio significantly contributes to purity and yield.
The 500 μl samples, in 1-butanol:acetone (1:1), are applied to the column and are desirably eluted isocratically with acetone: 1-butanol (99:1) under nitrogen gas pressure (5 psi) at a rate of approximately 2 in/min. Fifty (1 ml) samples are collected in approximately 20–30 minutes.
Since acetone is the primary solvent, all collected samples are dried by nitrogen gas or allowed to air dry, and then visualized by TLC/ninhydrin. BHB elutes off the column in samples 19–21 with good reproductibility and resolution given the method employed.
SUMMARY OF PREPARATION OF PRE-FASTED AND FASTED URINE
The bears were fasted overnight before the day of the experiment. They were allowed unlimited access to water. On the day of the experiment bears were anesthetized with Telazol, i.m. 4–5 mg/kg body weight. Baseline blood and urine (catheterized) were taken as pre-fast controls. Catheterized urine was only collected from three of the bears, numbers 4/521; 9/515; and 12/517. The urine was pooled and treated with an equal amount of methanol (165 ml). After sitting overnight at 0° C., the urine was centrifuged at 1650 gravity×15 minutes. The supernatant was removed and the precipitate discarded.
Next, the supernatant was placed under a nitrogen stream until most of the methanol had been removed. The sample was then frozen at −80° C. After freezing, the sample was placed on the lyophilizer. YH 11-9-1 (BDI-U) was then used either for use in the bone culture or further purification by countercurrent chromatography (CCC).
Twenty-one days later, the bears were again anesthetized to collect serum and urine in the same fashion as the pre-fasted controls. Prior to this, beginning Jul. 28, 1994 until Aug. 2, 1994, urine was also collected from beneath the cages. All male urine was pooled and female urine was pooled. Catheterized urine was collected from bears and kept separately and treated with an equal volume of methanol after aliquots were removed for urea and creatinine analysis: 6/520 (4 ml, YH 11-13-2), 9/515 (119 ml, YH 11-13-3), and 11/518 (17 ml, YH 11-13-4). Also collected from two of the older bears was 125 ml from Caruso (YH 11-13-5), and 6.5 ml from UP (YH 11-13-6).
The samples were purified by countercurrent chromatography in the following manner. The dried, deproteinated serum (BDI, 0.5 to 1.0 g), was reconstituted in three to four ml of a lower phase 1-butanol:acetic acid:water (20:1:20) mixture. Ten fractions were collected in one run according to the standardized protocol (as attached). The samples were then lyophilized, reconstituted in methanol for transfer to pre-weighed vials, and then dried down under nitrogen for weight determination. At this point, samples were then evaluated for further bone cultures, lc/ms or further purification by HPLC. The cultures which were run with urine produced enhanced bone remodeling both of the osteoblastic enhancement and the osteoclastic diminution.
Formation of the Organic Bone Matrix—Osteoid
Both osteoblasts and fibroblasts are involved with formation of osteoid, the matrix of bone. BDI directly stimulates proliferation of osteoblasts, increasing their numbers by 129%. In a similar fashion, BDI directly stimulates proliferation of fibroblasts by 205%. BDI was tested in fibroblast cultures of NIH-3T3 cells. The concentration of BDI that achieved maximum results was 10 mg/ml, the same concentration that achieved maximum results in the osteoblast cultures of MC-3T3 cells. Thus, BDI coordinates the final stage of bone remodeling by furnishing a place to put new bone. BDI induces a similar significant proliferation of fibroblasts (the cells that form matrix or osteoid), the supporting structure of bone, as BDI induced in osteoblasts. Furthermore, the proliferation response of fibroblasts to BDI is similar to proliferation and the bone production response of osteoblasts to BDI.
Thus, BDI orchestrates bone remodeling in a remarkable fashion. In order to form bone while under the combined stresses of not eating or drinking, remaining non-weight bearing, and in the absence of sex steroid production, the bear makes enough bone to avoid osteoporosis. To do this, the bear must shut down bone resorption, stimulate bone formation, and prepare a place to put the newly formed bone. The bear accomplishes this by inhibiting bone resorption while simultaneously stimulating bone formation.
Vitamin D and Bone Integrity in the Denning Bear
During denning, unopposed action by the active form of vitamin D, 1,25-dihydroxyvitamin D3 would produce bone loss, high blood calcium, and death. Ordinarily, 1,25-dihydroxyvitamin D3 stimulates the gut to absorb calcium to replace calcium lost in urine. If insufficient calcium is in food, 1,25-dihydroxyvitamin D3 stimulates bone to release calcium (bone resorption) to keep blood levels of calcium constant.
Since the denning bear is fasting and not urinating, unopposed action of 1,25-dihydroxyvitamin D3 on bone would constantly stimulate bone to release calcium, causing blood calcium to rise to high enough levels to cause cardiac standstill and death. To prevent this occurrence, the bear reduces production of 1,25-dihydroxyvitamin D3 while increasing production of another form of vitamin D—24,25-dihydroxyvitamin D3. Considered by most a metabolite of vitamin D that has no metabolic action and normally excreted from the body, the 24,25 form actually stimulates bone deposition. The effect of increasing production of 24,25-dihydroxyvitamin D3 while decreasing production of 1,25-dihydroxyvitamin D3 has a favorable effect. The ratio of 24,25 to 1,25 changes from 186 to 300 in captive denning bears (who have ample vitamin D in their summertime food rations) and from 16 to 89 in wild, denning bears.
The large increase in the ratio of 24,25 to 1,25 (61% in captive and 456% in wild bears) serves two purposes:

    
    
        1. The ability of 1,25-dihydroxyvitamin D3 to release calcium from bone is reduced, and
        2. The increase in 24,25-dihydroxyvitamin D3 is enough to recycle calcium that continues to be lost from bone back into bone. The ideal regulation of vitamin D metabolites to prevent high blood calcium only works if the bear can prevent bone loss. We have found that although the bears exists in a state similar to a post-menopausal woman, the bear makes bone normally, protects its skeleton from osteoporosis, and prevents high blood calcium and death.
    
    


Female rats grow normally when receiving daily injections of BDI at a concentration similar to that which enters the blood stream each day from the urinary bladder of a denning bear. No untoward, observable signs or symptoms indicative of adverse reactions to BDI were observed in these rats.
Fasting Summer Bear Conclusions
The fasting summer bear exhibits substantially the same decrease in urea to creatinine ratio as the denning winter bear. Moreover, it exhibits essentially the same bone remodeling enhancement as the denning winter bear. Accordingly, the beneficial aspects of the bear isolate as it relates to renal disorders and osteoporosis appear to be equally as potent with the summer fasting bear as with the winter denning bear.
BIOLOGY OF THE INVENTION
EVALUATION OF BDI AND ITS FRACTIONS
IN VIVO STUDIES: INDUCING DENNING BEAR BEHAVIOR IN GUINEA PIGS AND IN VITRO STUDIES: STIMULATION OF BONE REMODELING
In Vivo Studies
Introduction
The first study was exploratory. It evaluated BDI that had been isolated from winter urine. The second study determined the effects on vital signs of the guinea pig of a lyophilized sample of winter urine and of the precipitate isolated from the urine during deproteination. The third study used a Latin Square Design. It was an in-depth investigation of BDI and three of its isolated Fractions. The fourth study compared fifth study compared BDI derived from winter, denning bears with serum from active, eating bears. As described under “Chemistry of the Invention”, serum from winter, denning bears (BDI) and serum from active, eating bears were deproteinized with methanol, the proteins were pelleted by centrifugation, and the supernatants were removed and lyophilized. The dry samples were then reconstituted in 2 ml of saline.
Study One: Exploratory Study Comparing Effects of Summer and Winter Urine on Body Temperature, Heart Rates, and Tranquility in Guinea Pigs
Methods
Urine from denning and non-denning bears was processed in similar fashion. Guinea pigs received BDI in the same relative concentration as it appears in the denning bear. Thus, the predicted concentration in the blood of the guinea pig was about equal to the predicated concentration of BDI in the blood of the denning bear. Blood volume was estimated as five percent of body weight. 50 ml of urine was deproteinated, lyophilized, and reconstituted in 2 ml of sterile saline as described above. A 2 ml sample was delivered by intraperitoneal injection into each animal.
Results
Five minutes post injection, the animals receiving BDI presented signs of tranquility, reduced heart rate [from approximately 256 to 96 beats per minute (BPM)], and reduced body temperature (from approximately 38° C. to 35° C. or 100.4° F. to 95° F.). The tranquil effects lasted approximately 50 minutes. The tranquil effects were evidenced by the fact that animals could be held on their backs without signs of struggle and that the guinea pigs were alert to their surroundings, but were simultaneously very calm and indifferent to external stimuli such as sudden loud noises. Body temperatures did not return to normal for up to 15 to 20 hours post injection.
Guinea pigs receiving urine from non-denning bears that had been processed in a manner similar to the processing of BDI showed no decreases in body temperature or heart rate. They did not develop a tranquil state.
Conclusion
These data indicate that BDI induces responses of the denning bear in the guinea pig.
Study Two: Comparing Effects of Whole Urine and Precipitate on Heart Rates and Body Temperature in Guinea Pigs
Methods
Four guinea pigs were injected with varying doses of lyophilized samples of winter bear urine or the precipitate resulting from deproteination of winter bear urine. Rectal body temperature was measured and an electrocardiogram (ECG) was taken every 15 minutes after time of injection. The material to be injected was prepared in the following manner.
Whole bear winter urine was aliquoted out into 20 ml, 40 ml, and two 50 ml samples.
The 20, 40, and one of the 50 ml samples were lyophilized and placed in the freezer until the day of the experiment.
The second 50 ml sample was treated with an equal volume of methanol, vortexed, and allowed to set in the freezer overnight.
The next day, the methanol treated urine was centrifuged and the supernatant removed.
The remaining precipitate was dried under a nitrogen stream and then frozen until the day of the experiment.
On the day of the experiment, each of the four samples were reconstituted into 2 ml of bacteriostatic 0.9% saline for injection. After a control ECG and rectal body temperature (° F.) were taken, each guinea pig was injected intraperitoneally. ECG recordings and rectal temperatures were then taken every 15 minutes for up to 90 minutes.
Results (Table 4 and Table 5)
The guinea pig receiving the protein precipitate (0.0148 g) had an average increase in heart rate of 18 bpm during the 90 minute observation period. The maximum change in heart rate was +28 bpm and occurred 15 minutes after injection. Rectal temperature changes ranged from −1.2° F. to +0.7° F.
The guinea pig that received the lyophilizate from 20 ml of urine (0.5384 g) exhibited an average decrease in heart rate of 49 bpm with the lowest heart rate measured at 15 minutes after injection. Rectal temperature decreased an average of 2.1° F. over the 90 minutes.
In the animal that received the lyophilizate from 40 ml of urine (1.2164 g), heart rate decreased by an average of 60 bpm within 15 minutes after injection. However, heart rate returned to normal more rapidly in this particular animal than in the guinea pig that received only 20 ml of the lyophilized urine. Therefore, the average change in heart rate for this animal was only −4 bpm. In contrast, rectal temperature decreased by 5.5° F. and remained lowered even at 90 minutes.
The guinea pig that received the highest dose of the lyophilizate from 50 ml of urine exhibited a maximum decrease in heart rate (−154 bpm) at 15 minutes. Rectal temperature decreased by 7.3° F. and was still 6° lower than control 90 minutes after injection.
All animals survived.







TABLE 4







GUINEA PIG STUDY: WHOLE URINE AND PRECIPITATE


MEAN CHANGES IN HEART RATES (BPM)


(Treated Rates - Control Rates) 














Protein






Post Injection Time
Precipitate
20 ml
40 ml
50 ml

















15 minutes
+28
−83
−60
−154



30 minutes
+18
−34
+19
−129



50 minutes
+17
−50
+15
−103



75 minutes
+20
−43
+6
−135



90 minutes
+9
−37
0
−120



Mean of Means
+18.4
−49.4
−4.0
−128.2

















TABLE 5







GUINEA PIG STUDY: WHOLE URINE AND PRECIPITATE


CHANGES IN BODY TEMPERATURE (° F.)


(Treatment Temperature - Control Temperature)














Protein






Post Injection Time
Precipitate
20 ml
40 ml
50 ml

















15 minutes
—
−0.5
−0.3
−4.3



30 minutes
0.0
−2.6
−2.8
−5.0



45 minutes
+0.7
−4.4
−5.5
−7.3



60 minutes
−1.2
−3.2
−5.3
−6.8



90 minutes
−0.7
−0.0
−5.1
−6.8



Mean
−0.3
−2.14
−3.8
−6.0










Summary

Fifty ml of winter bear urine that had been lyophilized and reconstituted in 2 ml of normal saline caused a 45% decrease in heart rate within 15 minutes of injection.
Fifty ml of winter bear urine that had been lyophilized and reconstituted in 2 ml of normal saline caused a decrease in rectal temperature that was maximal at 45 minutes post injection.
Both effects were sustained throughout the 90 minute observation period.
In the guinea pigs that received the lower doses of the lyophilizate from bear urine, heart rate and rectal temperature still decreased with maximal effects measured at 15 minutes for heart rate and 45 minutes for temperature.
The magnitude of the effects produced by 20 ml and 40 ml of urine were smaller when compared to 50 ml of urine.
The animal that received the precipitate intraperitoneally exhibited an increase in heart rate rather than a decrease with little or no change in rectal body temperature.
Conclusions
The lyophilized winter bear urine injected intraperitoneally into conscious guinea pigs produced a decrease in heart rate and rectal body temperature similar to changes previously noted with BDI. The precipitate from the same volume of urine did not produce the same effects; it did not decrease heart rate and had little or no effect on rectal body temperature.
Study Three: Latin Square Designed Studies—The Effect of BDI in a Non-Hibernating Animal, The Guinea Pig
Introduction
This study was designed to test the effects of BDI and its Fractions in guinea pigs. To ensure unbiased observations, the study was blinded so that the researchers did not know which animal was injected with BDI, with Fraction I, with Fraction II, with Fraction III, or with saline. The Latin Square Design permitted use of animals as their own controls. Thus, in each animal, changes in heart rate and temperature after experimental injections were compared to the guinea pig's own recorded normal heart rate and temperature prior to each injection. In addition, all animals received a control injection of sterile saline during the five week experimental period in an effort to measure the physiologic response in each animal to the pain of the injection itself. Food and water intake, urine output, and urea and creatinine excretion in urine were measured daily for four days after each injection. Therefore, each animal is used as its own control, and each sample injection can be compared to a saline control injection in all animals.
Methods
Heart rates were intermittently monitored by electrocardiograms. Rectal temperatures were intermittently monitored via inserted thermistors calibrated to National Bureau of Standard requirements. Recordings were made every 15 to 30 minutes throughout the two to three hour study. A video camera was used to record behavioral activity in each animal throughout the study. Research observers were asked to comment on each animals' tranquility by observing animal handling and animal reaction when exposed to a loud snapping noise. Thereafter, the animals were housed in a metabolic cage throughout the five-week experiment in order to measure food and water intake and urine output. Urine urea and creatinine concentrations were measured. Effects of the following fractions were compared with BDI, with the saline control, and with each other: Fraction I, representing BDI−[BHB+MNC]; Fraction II, representing BHB; and Fraction III, containing MNC.
Design
Fractions were obtained by combining appropriate samples from the second CCC run. They were lyophilized as those for BDI. Thereafter, they were reconstituted in a saline solution.
After collecting Fraction I, Fraction II, and Fraction III, the study was blinded so that the researchers did not know which animal was injected with Fraction I, with Fraction II, with Fraction III, with saline, or with BDI. Animals were used as their own controls in a Latin Square Design. Heart rates were intermittently monitored by electrocardiograms. Rectal temperatures were intermittently monitored via inserted thermistors. Results were recorded every 15 to 30 minutes throughout the two to three hour study. A video camera was used to record behavioral activity in each animal throughout the study.
To measure effects on body temperature (° C.), heart rates (BPM), and tranquility from each injection on the five guinea pigs, the data were grouped into the following time categories: Zero minutes (pre-injection control), 15–25 minutes, 30–40 minutes, 41–59 minutes, 60–74 minutes, and 75–95 minutes (post injection). Each animal was used as its own control. Treatment means were reported as the difference of each injection effect from the zero minutes (control) result. Therefore, positive or negative treatment mean values indicate an increase or decrease in the effect measured. A similar approach was used for daily determinations of food and water intake and urine excretion of urea and creatinine.
Results
Body Temperature (Table 6)
Beginning at 30 minutes and extending through to the end of the study, BDI produced a significant reduction in body temperature. The overall mean of temperature reduction was seven fold greater than that experienced by the animal when it received saline as a control measure.
Effects of Fraction I, Fraction II, and Fraction III were not different from control observations throughout the study.







TABLE 6







GUINEA PIG STUDY: 5 × 5 LATIN SQUARE


MEAN CHANGES IN BODY TEMPERATURE (° C.)


(Treatment Temperature - Control Temperature)













Post








Injection


Time
I
II
III
BDI
C
p < 0.05
















15 to 25 minutes
0.33
0.41
0.35
0.34
0.01
N.S.


30 to 40 minutes
0.10
0.34
0.19
−0.31
−0.31
N.S.


41 to 59 minutes
0.03
0.22
0.17
−0.84
−0.24
N.S.


60 to 74 minutes
−0.15
0.21
0.10
−1.14
0.01
*


75 to 95 minutes
−0.42
0.12
0.38
−1.54
−0.15
*


Mean of Means
−0.02
0.26
0.24
−0.70
−0.10
—





I = BDI − (BHB + MNC)


II = BHB


III = MNC through Wash


C = Saline Control


* Treatments are significantly different at p < 0.05






BDI produced a significant reduction in heart rate. Animals receiving Fraction I showed a significant heart rate reduction of approximately 50% of that shown by BDI. Animals receiving Fraction III showed a moderate but not a statistically significant reduction in heart rate (approximately 20% of that shown by BDI). Compared to BDI, those receiving Fraction II showed only a 10% reduction in heart rate. Saline injection failed to reduce heart rate (Table 7).







TABLE 7







GUINEA PIG STUDY: 5 × 5 LATIN SQUARE


MEAN CHANGES IN HEART RATES (Beats per Minute)


(Treatment Rates - Control Rates)













Post








Injection


Time
I
II
III
BDI
C
p < 0.05
















15 to 25 minutes
−34.4
−7.2
−15.2
−54.0
9.2
*


30 to 40 minutes
−29.4
−4.4
−9.2
−53.0
6.8
**


41 to 59 minutes
−25.0
−7.6
−11.4
−62.8
6.8
*


60 to 74 minutes
−19.8
2.2
−13.4
−53.8
4.4
N.S.


75 to 95 minutes
−23.4
−7.6
−10.2
−51.6
0.2
N.S.


Mean of Means
−26.4
−4.9
−11.9
−55.0
5.5
—





I = BDI − (BHB + MNC)


II = BHB


III = MNC through Wash


C = Saline Control


* Treatments are significantly different at p < 0.05


** Treatments are significantly different at p < 0.01






Food and Water Intake

Guinea pigs that received BDI showed a decreased intake of food that was significant by the third and fourth day post injection.
Water intake by guinea pigs that received BDI was not changed.
Urine urea to creatinine ratios were profoundly reduced in guinea pigs receiving BDI.
Tranquility (Table 8)
Only animals receiving BDI were rated more tranquil than those receiving saline.







TABLE 8







GUINEA PIG STUDY; 5 × 5 LATIN SQUARE


TRANQUILITY














Number of




Substance
Fraction
Animals
Tranquility*
















BDI
—
5
3.6



BDI − (BHB + MNC)
I
5
2.0



BHB
II
5
2.8



MNC
III
5
2.8



Saline (Control)
C
5
2.6







*Animals rated 1 to 4 (anxious to tranquil) when exposed to a brief snapping sound and turned over on their backs






Deaths

Two animals died within 24 hours. One received Fraction I; the other received BDI.
Summary
BDI demonstrated significant and profound reductions in body temperature when compared to its Fractions—I, II, or III.
The reductions in body temperature stimulated by BDI increased over time with temperatures remaining low for up to 24 hours.
Individual components of BDI (Fraction I, Fraction II, and Fraction III) had no effect on body temperature.
BDI demonstrated significant and profound reductions in heart rate when compared to its Fractions—I, II, or III.
Heart rates were reduced significantly within 30 to 60 minutes after the injection of BDI and tended to return to normal within two to three hours post injection.
In order of responses, Fraction I, Fraction III, and Fraction II reduced, but to a much lesser degree, heart rates independently.
The decrease in urea to creatinine ratios were profoundly reduced in guinea pigs receiving BDI.
Only BDI induced tranquility over that shown by animals receiving the saline control.
Conclusion
BDI contains components that target specific physiologic changes independently, but BDI exhibits the greatest overall effects when all the components of BDI are present. The performance of BDI exceeds the results of any of the above fractional components.
Study Four: Effects of Combination of Fraction I, Fraction II, and Fraction III Isolated From Urine In A Non-Hibernating Animal, the Guinea Pig
Introduction
Samples were defined as follows:

    
    
        1. Combination A: Fraction I plus Fraction III representing BDI−BHB; contains MNC.
        2. Combination B: Fraction I plus Fraction II representing BDI−MNC; contains BHB.
        3. Combination C: Fraction II plus Fraction III representing BHB+MNC.
    
    


The above Combinations were obtained by combining appropriate samples from the second CCC run. They were dried as those for BDI. Thereafter, they were reconstituted in a saline solution.
Methods
BDI obtained from urine taken from early, mid, and late denning bears was used for isolation of Fraction I, Fraction II, and Fraction III. The combinations were injected intraperitoneally.
Body temperature (° C.), heart rates (BPM), and tranquility were measured for each treatment on three guinea pigs.
Data were grouped into time categories: 0 minutes (pre-injection control), 30 minutes, 60 minutes, 75 minutes, and 260 minutes (post injection).
Each animal was used as its own control. Treatment means are reported as the difference of each treatment effect from the 0 minutes (control) result. Therefore, as in the Latin Square Study, positive or negative treatment mean values indicate an increase or decrease in the effect measured. A mean of the Combination means was then calculated from each Combination over all animals and all time categories. All research observers (blinded study) were asked to comment on each animals' tranquility by observing the animal handling and animal reaction when exposed to a loud snapping noise.
In these studies, comparison between guinea pigs, sample potency was expressed as the ratio of averaged treatment means to g dry weight of each sample injected.
Results
Temperatures (Table 9) were reduced in all three guinea pigs receiving Combination A, Combination B, and Combination C with the largest decreases in temperatures occurring in animals receiving Combination A or Combination B.
When temperature responses were related to weight of the injected sample (Table 9—Potency), Combination A, Combination B, and Combination C were potent in reducing body temperatures. Combination C had the greatest potency (Table 9).







TABLE 9







GUINEA PIG STUDY: COMBINED FRACTIONS


CHANGES IN BODY TEMPERATURE (° C.) AND POTENCY


(Treatment Temperature - Control Temperature)










Post Injection





Time
Combination A
Combination B
Combination C





 30 minutes
−0.21
−0.67
−0.17


 60 minutes
−1.21
−1.68
−0.17


 75 minutes
−1.60
−2.01
−0.34


260 minutes
−4.49
−3.63
−1.50


Mean
−1.88
−2.00
−0.55


Sample Weight
  3.3833 g
  1.9917 g
  0.1699 g


Potency*
−0.56
−1.00
−3.24





Combination A = Fraction I + Fraction III = BDI − BHB (Contains MNC)


Combination B = Fraction I + Fraction II = BDI − MNC (Contains BHB)


Combination C = Fraction II + Fraction III = MNC + BHB (Through Wash)






Heart rates were reduced in all three guinea pigs. The largest reductions occurred in animals receiving combinations A and B (Table 10).
Combination C was most potent in reducing heart rate (Table 10).







TABLE 10







GUINEA PIG STUDY: COMBINED FRACTIONS


MEAN CHANGES IN HEART RATES (Beats


per Minute) AND POTENCY


(Treatment Rates - Control Rates)










Post Injection





Time
Combination A
Combination B
Combination C













30 minutes
−88.0
−54.0
−14.0


60 minutes
−70.0
−67.0
−50.0


75 minutes
−79.0
−60.0
−68.0


Mean of Means
−70.0
−60.3
−44.0


Sample Weight
   3.3833 g
   1.9917 g
   0.1699 g


Potency*
−23.4
−30.3
−258.8





Combination A = Fraction I + Fraction III = BDI − BHB (Contains MNC)


Combination B = Fraction I + Fraction II = BDI − MNC (Contains BHB)


Combination C = Fraction II + Fraction III = MNC + BHB (Through Wash)






Combination A, Combination B, and Combination C produced tranquility in the animals (Table 11).







TABLE 11







GUINEA PIG STUDY: EFFECT OF COMBINED


FRACTIONS, TRANQUILITY












Number of



Substance
Combination
Animals
Tranquility*













BDI − BHB
Combination A
1
4.0


(Contains MNC)
(Fraction I + Fraction III)


BDI − MNC
Combination B
1
4.0


(Contains BHB)
(Fraction I + Fraction II)


MNC + BHB
Combination C
1
3.0



(Fraction II + Fraction III)





*Animals rated (anxious to tranquil) when exposed to a brief snapping sound and turned over on their backs






Animals receiving Combination A or Combination B died within 24 to 48 hours post injection.
Summary
Combination A, Combination B, and Combination C greatly reduced body temperature and heart rate.
Reductions in body temperature increased over time with temperatures remaining low for up to 24 to 48 hours.
Heart rates were reduced within 30 to 60 minutes after the injections and remained low throughout the 75 minutes that the animals were monitored.
Combination C gave the largest potency effect in temperature and heart rate reduction. The animal survived. This suggests that the components of Combination C may be the predominantly active ingredients in BDI containing no toxic side effects.
Conclusions
BDI from urine and its combined components demonstrate dramatic decreases in body temperature and heart rate in non-denning guinea pigs.
BDI from urine and its combined components also produce alert tranquility in this non-denning animal model.
Study Five: Comparison of BDI Derived From Denning Serum and Serum from Active Bears in a Non-Hibernating Animal, the Guinea Pig
Methods
As previously described, equal volumes of BDI and summer active serum were processed by deproteinization, centrifugation, supernatant removed, lyophilization, and residue reconstitution into 2 ml of saline. The reconstituted samples were each intraperitoneally injected into guinea pigs. Body temperatures, heart rates, and tranquility ratings were recorded as described in Study One, Study Two, and Study Three.
Results
The mean decrease in body temperature associated with BDI was −0.19° C. This is approximately two-fold greater than the −0.10° C. shown by serum from active bears and by saline controls in the Latin Square Design.
No significant change in heart rates occurred after injection. BDI was associated with an overall mean decrease of 8 beats/minute; active bear serum showed a mean decrease of 7 beats/minute.
Neither animal showed signs of tranquility.
Conclusions
BDI from serum showed only a mild response in lowering body temperature.
Active bear serum showed no response in lowering body temperature.
Neither BDI from serum nor active bear serum affected the heart rate or induced tranquility.
The lack of response may be attributable to an extremely low concentration of BDI in the samples.
Overall Conclusions of Guinea Pig Testing
When given intraperitoneally to the guinea pig, BDI induces the responses of the bear: tranquility, decreased heart rate, and decreased body temperature.
No differences in guinea pig results were noted when BDI was isolated from early, mid, or late denning bears.
BDI was most effective when used in full strength.
Isolated Fractions of BDI by themselves were inactive.
Combination of BDI into Combination A (Fraction I plus Fraction II), Combination B (Fraction I plus Fraction III), and Combination C (Fraction II plus Fraction III) also elicited positive results. Combination A and Combination B were associated with side effects which were, most likely, due to Fraction I. Three of seven animals died. They either received Fraction I or Combinations A and B that contained Fraction I.
A definite, safe, and highly active response with no observable side effects was noticed in the animal receiving purified Combination C (Fraction II plus Fraction III).
Treatment of Osteoporosis in Ovariectomized Rats
Our next step was to treat a living animal model similar to the post menopausal woman with BDI.
We used a pharmaceutical industry accepted animal model. Growing rats, less than six months old, were randomized into three groups of six rats each. One group was control (sham operated), one was ovariectomized, and one was ovariectomized and received BDI via subcutaneous injection. Similar volumes of saline were injected into the other two groups. BDI was given in amounts similar to its daily production in bears but proportionally scaled to body weight of the rat.
At the end of eight weeks, the ovariectomized group had become osteoporotic. When compared with this group, the ovariectomized group treated with BDI showed a 3% increase in bone mineral density (BMD) of the femur and a 4% increase in the lumbar vertebrae.
When compared with two month results of treating post menopausal women with estrogen, progesterone, and calcium, BDI results in rats showed a 16-fold greater increase in the BMD in lumbar vertebrae and a 3-fold greater increase in BMD of the femur. Another group of women on similar hormone replacement therapy showed only a 1.7% increase in BMD of the lumbar spine even though they were treated for 1.6 years.
In vitro Studies: Evaluation of BDI and its Fractions in Stimulating Bone Remodeling
Introduction
These studies focused on serum and urine obtained from denning bears. The bone mass of denning bears remains constant even though they exist in a non-weight bearing state, a condition that induces loss of bone. Unlike other mammals, the bear maintains bone mass, structure, and strength. In the bear, the cells that produce bone (osteoblasts) are as active as the cells that resorb bone (osteoclasts). Under similar conditions, other mammals (including humans) lose bone by reducing bone formation, by maintaining or increasing bone resorption, or by a combination of these changes.
Test One: Inhibition of the Resorption Activity of Chicken Osteoclasts
Introduction
Unprocessed serum from active eating bears and unprocessed serum from denning bears both showed an inhibition of osteoclast resorption activity. The studies focused on the denning bear because it continues to make bone despite the fact that its non-weight bearing state lasts for months.
Methods
BDI Serum Studies (Table 12)
BDI, BHB, and BDI−BHB (containing MNC) were prepared from serum of bears as described under “Chemistry of the Invention” in this application.
Results
BDI from three bears in concentrations of 1 mg/ml of sample reduced osteoclast resorption activity to values of 24, 46, and 55 percent of control. More dilute samples were not effective (0.1, 0.01, 0.001 mg/ml).
The sample BDI−BHB that contains MNC also proved effective in two bears at concentrations of 1 mg/ml, reducing osteoclast resorption activity to 10 and 75 percent of control.
BHB by itself had no effect on osteoclast resorption.







TABLE 12







BEAR SERUM: INHIBITION OF FORMATION OF CHICKEN OSTEOCLASTS


FROM CHICKEN MONOCYTES OBTAINED FROM BONE MARROW













Percent Reduction from Control



Bear
Weight
Concentration of Test Sample (mg/ml)














Substance
Name
(g)
CCC Samples
0.001
0.01
0.1
1.0

















BDI
Amanzo
0.017
not run
125
115
108
55



Caruso
0.012
not run
80
106

46



UP
0.020
not run
152
93
90
24


BDI − BHB
Amanzo
0.026
Fraction I and III
119
103
108
75


(Contains MNC)



UP
0.078
Fraction I and III
84
90
60
10


BHB
Amanzo
0.0006
Fraction II

130
130
135



Caruso
0.0023
Fraction II

95
95



UP
0.002
Fraction II
80
105
110









Conclusion

Direct action of BDI isolated from serum with or without BHB produced an environment conducive for bone formation by inhibiting resorption activity of osteoclasts, the cells that dissolve bone.
BDI Urine Studies (Table 13)
Methods
BDI was prepared from urine from three bears as described previously under “Chemistry of the Invention” of this application.
Results
BDI in concentrations of 10 mg/ml of sample inhibited resorption activity of osteoclasts to values of 25, 35, and 38 percent of control. More dilute samples were not effective (Table 13).







TABLE 13







BEAR URINE: INHIBITION OF FORMATION


OF CHICKEN OSTEOCLASTS FROM CHICKEN


MONOCYTES OBTAINED FROM BONE MARROW













Percent Reduction from




Sample
Control Concentration of



Bear
Weight
Test Sample (mg/ml)














Substance
Name
(g)
0.01
0.1
1
3
10

















BDI
Amanzo
0.268
147
110
130
95
25



Caruso
0.255
125
85


35



UP
0.270
123
107


38









Conclusions

BDI isolated from urine induces bone formation by inhibiting bone resorption by osteoclasts.
BDI isolated from serum is approximately 10 times more effective than BDI isolated from urine in reducing bone resorption by osteoclasts.
Test Two: Simultaneous Evaluation of Osteoblast and Osteoclast Activity
Methods and Materials
Experiments utilized an in vitro bone culture system. Calvaria (skull) of 4 to 6 day old neonatal mice were dissected out and cultured in individual capped test tubes in 2 ml of culture media (DMEM+glutamine, heparin, inactivated horse serum, and antibiotics). Each calvaria was gassed and incubated in a rotating roller drum at 37° C. Osteoblast activation (increased bone formation) was evaluated as a function of alkaline phosphatase activity (ALP). Osteoclast activity (bone resorption) was evaluated as a function of beta-glucuronidase activity. For testing purposes, two samples of serum from bears were used: 1) unprocessed bear serum, and 2) processed bear serum (BDI). Horse serum was used as a serum control to ensure that stimulation was not due to serum growth factors.
Results
Unprocessed bear serum from active, eating, weight-bearing bears increased ALP activity from 600 to 1200 nmole ALP/bone/30 minutes.
Unprocessed bear serum from denning, non-eating, non-active, non-weight bearing bears also significantly increased ALP activity from 600 to 1200 nmole ALP/bone/30 minutes.
Horse serum showed no change in ALP activity.
Unprocessed bear serum from denning bears showed a dose response result. The saline control value of 250 ALP/bone/30 minutes significantly increased to 600, to 800, and to 1000 ALP/bone/30 minutes in response to 50, 100, and 200 μl of serum respectively.
BDI increased ALP activity from 310 to 520 ALP/bone/30 minutes, about 55% of the response elicited by unprocessed bear serum that, in the same test, increased ALP to 700 ALP/bone/30 minutes.
The ability of BDI to increase ALP activity proved significantly greater than effects of calcitonin.
Inactivating serum proteins in unprocessed bear serum by heat produced results similar to BDI; ALP activity increased.
BDI failed to activate beta-glucuronidase. Combining these findings with the above indicated that BDI primarily stimulated bone formation by osteoblasts.
Unprocessed serum from active and denning bears showed both mild stimulation and failure to stimulate beta glucuronidase activity. However, when osteoclasts were stimulated, the response was less than one-half of the osteoblast stimulatory response. Therefore, bone formation activity continued to exceed bone resorbing activity.
Conclusions
Unprocessed serum from active and denning bears stimulates osteoblasts.
Unprocessed serum from active and denning bears varied in its ability to stimulate osteoclasts. At times no changes were observed; at other times mild stimulation was observed.
BDI stimulates osteoblasts to about 55% of that shown by unprocessed serum.
BDI does not stimulate osteoclasts.
The overall effect on bone remodeling is creation of an environment conducive to bone formation—stimulation of the limb that forms bone (osteoblasts) while not stimulating bone resorption (osteoclasts).
Test Three: The Effect of Summer Fasted BDI on Osteoblast and Osteoclast Activities
Introduction
As previously described, fasted bears (who had access to water) during the summertime revealed changes in levels of serum urea, creatinine, and a U/C ratio similar to changes noted when bears were denning. Thus, it was concluded that the summer fasting bears were in the mode of urea recycling (See Tables 1 and 2). Test Three was done to determine if bone remodeling was also stimulated when the bears were fasting. The effect of the 21 day summer fast on bone remodeling was determined by evaluating the activity of BDI obtained from these bears in an in vitro bone culture system.
Materials and Methods
As described in the discussion Test Two, calvaria of 4 to 6 day old neonatal mice were used for the in vitro bone culture system. Alkaline phosphatase activity (ALP) was used as a means of evaluating osteoblast activity (increased bone formation).
Because previous tests using beta glucuronidase activity to evaluate osteoclast activity (increased bone resorption) were inconclusive, a more sensitive test was employed. The production of tartrate resistant acid phosphatase (TRAP) was used as a measure of osteoclast activity (Lau et al., 1987; Delamis 1988). For testing purposes, BDI was prepared from urine of bears before and at the end of the 21 day fast. Denning bear plasma served as a positive control. Pre-fasted BDI was compared with fasted BDI. Both were compared with denning bear plasma and all three samples were compared with the phosphate buffered saline control.
Results
Osteoblast Results (Table 14)
Pre-fasted BDI results were similar to results of denning bear plasma. Both showed a moderate, significant increase in osteoblast activity (55% and 50% above control respectively). However, BDI from the final day of fasting significantly stimulated osteoblasts some 300% above control, about a six-fold increase over results from denning bear plasma or pre-fasted BDI.







TABLE 14







Changes in Medium Alkaline Phosphatase Activity In Calvaria


Incubated with Normal Denning Bear Plasma and BDI Processed


from Urine Before and At the End of a 21-Day Fast










Treatment Group
ALP Activity1















PBS (Phosphate
444.8a ±
108.5



Buffered Saline)



BP (Denning
666.4a,b ±
127.2



Blood Plasma)



Fasted (BDI from Urine
1337.7c ±
346.3



of Fasted Bears)



Pre-Fasted (BDI from Urine
690.9b ±
120.9



of Non-Fasting Bears)







1nmol of p-nitrophenol/30 min/bone



Different letters indicate a significant difference, p < 0.05, n = 6






Osteoclast Results (Table 15)

When using TRAP as an indicator of osteoclast activity, results clearly demonstrate BDI's ability to inhibit osteoclast function. Both the fasted and pre-fasted results showed similar, significant inhibitory effects on osteoclast function, reaching levels 40% to 46% of normal. These results confirmed results using the chicken osteoclast tissue culture assay (Tables 12 and 13) as an indicator of osteoclast activity. Denning bear plasma showed no effects on osteoclast function.







TABLE 15







Changes in Medium Tartate Resistant Acid Phosphatase Activity


In Calvaria Incubated With Normal Denning Bear Serum


and BDI Processed from Urine


Before and at the End of a 21-Day Fast










Treatment Group
TRAP Activity1















PBS (Phosphate
142.5a ±
53.5



Buffered Saline)



BP (Blood Plasma)
182.8a ±
58.2



Fasted (BDI from Urine
77.4b ±
4.1



of Fasted Bears)



Pre-Fasted (BDI from Urine
84.0b ±
4.9



of Non-Fasting Bears)







1nmol of p-nitrophenol/60 min/bone



Different letters indicate a significant difference from the phosphate buffered saline control, p < 0.05, n = 6






Conclusions

Summer fasting in black bears induces a significant increase in potency of BDI in stimulating bone formation through activation of osteoblasts. Simultaneously, BDI significantly inhibits osteoclast activity. Thus, fasting in summer potentiates BDI's ability to stimulate bone formation.
Overall Conclusions of Bone Remodeling Studies
Results of the two separate studies independently performed at two institutions in two different states show complementary findings that support the conclusion that BDI stimulates bone formation and inhibits bone resorption since: BDI stimulates osteoblasts to form bone, BDI does not stimulate osteoclasts already present in bone, BDI inhibits resorption of bone by osteoclasts, and the net effect of these changes is to form bone. Summer fasting induces similar results in bone remodeling.
BDI is extremely potent since it stimulates the bone forming process while simultaneously inhibiting the bone resorption process of bone remodeling. Summer fasting in bears duplicates these positive findings found in denning bears.
Occurrence of Fraction II (BHB) and Fraction III (MNC) in Fasting, Adult Humans
Methods and Materials
Initially, BHB was identified by TLC/ninhydrin in very low concentrations in serum samples obtained from two humans that fasted for 20 hours. The serum samples were also deproteinated using the same method established for BDI. A follow-up study was done in fifty adult humans who had fasted for twenty hours to determine if components contained in BDI, namely BHB and MNC, could be found.
Results
MNC was not detected in the serum of fasting humans.
BHB appeared in serum samples obtained from subjects after a food restricted 20 hour fast.
BHB was not detected in serum samples obtained from subjects in the fed state.
Little to no BHB was detected in the urine of subjects collected before and after the 20 hour fast.
Conclusions
MNC, found in BDI, was not found in fasting human serum or urine.
Serum and urine from fasting humans contains BHB.
Dosage Formulations
After BDI (containing both BHB and MNC) alone or in combination with existing identified metabolites of denning bears which are also found in humans, has been isolated as set forth above, it is combined with desirable solvents such as saline or 5% dextrose in water.
After the solvents have been applied, a carrier may also be involved. Such carriers include: peanut oil, propylene glycol, a 5% alcohol based elixir, or pills and capsules containing lactose and/or calcium carbonate fillers. Transdermals are available as an alternative means of delivering the necessary doses of BDI. For subcutaneous, intramuscular, intravenous, or other specialized routes such as into the cerebral spinal fluid, appropriate carriers such as saline, Ringer's lactate, or dextrose solutions may be used. BDI is stable, water soluble, and will not suffer dissolution after stirring or settling overnight.
Once the syringe has been loaded, or the pill compounded, the maximum dosages (which must first be assessed for safety) are calculated for the animal to be tested. The present means to predict maximum dosage was based only on the lyophilized BDI contained in aliquots of 50 ml of denning bear urine that also contained 200 micrograms (μg) of MNC. Next, the blood volume of the recipient is equated with 50 ml urine volumes from the bear. The concentration of MNC in 50 ml of urine is used for calculations.
Mammals have blood volumes of approximately 5% of total body weight. Therefore, a 1000 gram guinea pig has 0.05×1000 g=50 ml blood.
Fifty milliliters of denning bear urine containing between 2.0 and 3.6 grams of BDI also contains 200 micrograms (μg) MNC or 4 μg/ml.
Therefore, the dosage and formulation for a 1000 gram guinea pig was BDI containing 200 μg MNC, which equaled a dose of 0.2 μg MNC/g body weight.
Reaffirmation of Findings: Urea Recycling is Produced when BDI Injected into Guinea Pigs but not Necessarily its Basic Components.
A urea creatinine ratio indicative of urea recycling (10 or less) was produced when BDI was injected into guinea pigs. This effect of efficient recycling lasted for three days after the injection. BDI was then separated into its three basic components. These were done previously as set forth in connection with the Table 1. The three basic components were BDI minus (BHB+MNC); BHB; and MNC. When each of these three basic components was injected separately into guinea pigs, the urine of guinea pigs did not exhibit a urea to creatinine ratio indicative of urea recycling (see Table 16).







TABLE 16







Urine Urea to Creatine Ratio in Guinea


Pigs For Three Days Post-Injection










Treatment
Day 1
Day 2
Day 3













Control: Average U/C Ratio
34.28
34.28
34.28


Group A: BDI − (BHB + MNC)
26.33
22.13
26.09


(Contains 0.185 g urea)


Group B: BHB
31.86
29.45
23.69


Group C: MNC
26.23
33.20
34.55


Through Wash


Group D: BDI
8.33
12.25
7.66


(Contains 1.1 g urea)


Group E: Saline
17.39
13.01
14.93


Control









Thus, the combination of some substances contained in Fractions 1–17 of Table 1 (BDI minus [BHB+MNC]) and some substances from the fractions associated with BHB and/or MNC stimulate urea recycling.
Some of the individual components of these fractions are now known. The combination of the active substances in each fraction will stimulate urea recycling in the guinea pig, as distinguished from the lack of significant recycling when the three separate components are injected separately.
Further Refinement of Separation Techniques for BDI Isolated from Denning Bear Urine to: 1) Search for the Fractions in BDI Responsible for Stimulation of Osteoblasts, 2) Identify Known Chemicals in the Ten Fractions of BDI, and 3) Further Purify the Fractions of BDI by HPLC in order to Identify Structural Components of MNC by Nuclear Magnetic Resonance and Mass Spectrometry.
Chemical methods of obtaining BDI fractions and isolating the same were performed as previously set forth in Table 1. To support further analysis, ten newly defined fractions from the countercurrent coil were collected. For example, the new Fraction I was obtained by pooling the first five elutions acquired from the countercurrent centrifuge. Total volume per collection tube was 20 ml; therefore, Fraction I contains 100 ml.
The precise countercurrent apparatus and centrifuge is manufactured by P.C., Inc. of Potomac, Md., referred to as a Multi-Layer Coil CCC. The #10 coil having a volume of 385 ml was used in processing all of the elutions and rinse which resulted in new Fractions I–X (Table 17).







TABLE 17







Separation of BDI Into Ten Fractions After CCC










New Fractions
CCC Fractions







Fraction I
1–5



Fraction II
 6–10



Fraction III
11–15



Fraction IV
16–20



Fraction V
21–25



Fraction VI
26–30



Fraction VII
31–35



Fraction VIII
36–40



Fraction IX
41–45



Fraction X
Methanol Wash










The mobile phase (lower phase of 1-butanol:water:acetic acid, 20:20:1 mixture) of the first six of ten fractions were pumped through the CCC at 4 ml/minute. Collections were taken every twenty-five minutes. After collection of Fraction VI, the coil was stopped. Mobile phase continued pumping at an increased rate of 10 ml/minute. Collections were made at ten minute intervals. The mobile phase was discontinued while a 1:1 mixture of methanol and water was begun before beginning collection of Fraction IX. The methanol/water mixture was switched to 100% methanol at the beginning of Fraction X. After ten minutes, the pump was stopped and the coil was emptied by forcing compressed air through it. Everything collected from the coil at this point was added to Fraction X. All fractions were stored at −70° C. until lyophilization.
Search for Site of Osteoblast Stimulation in BDI
A sample of urine collected from a single denning bear was deproteinated and lyophilized. Up to one gram of BDI was then loaded on the CCC and separated into ten fractions through the procedure diagrammed in Table 17. Weights were obtained for each fraction. Fractions obtained from four separate runs of the CCC were combined before use in osteoblast cultures.
Each combined fraction was tested in a mouse calvaria bioassay to determine its effectiveness in stimulating osteoblasts. An increase in alkaline phosphatase production was interpreted as osteoblast stimulation.
The ability of each combined fraction to stimulate alkaline phosphatase in the mouse calvaria bioassay was measured and expressed as a percent of control. This was compared to the ability of BDI and of pooled blood serum from denning bears to stimulate alkaline phosphatase in the mouse calvaria bioassay (Table 18).







TABLE 18







Percent Stimulation of Osteoblast Activity By Blood


Serum, Bear Derived Isolate, and Its Fractions











Percent Above



Sample
Control/mg Specimen














Fraction III
23



Fraction II
78



BDI (Bear Derived Isolate)
75



BS (Blood Serum)
322



Fraction X
292



Fraction IV
401



Fraction IX
571



Fraction V
3,740



Fraction VI
4,281



Fraction VII
37,432










    
    
        Fraction II,
        BDI,
        Pooled blood serum from denning bears,
        Fraction X,
        Fraction IV,
        Fraction IX,
        Fraction V,
        Fraction VI, and
        Fraction VII

demonstrated stimulation of osteoblast activity. Fraction III inhibited osteoblast activity. Thus, Fraction III has the potential to arrest Paget's disease and other forms of neoplasms such as cancer resulting from overactivity of osteoblastic-induced bone growth. For a list of substances identified for Fraction III see Tables 19 and 20.

    
    









TABLE 19







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION III, BEAR URINE JZ4061:5










mM/M




CREATININE
Nrml Range













Organic Acids




LACTIC ACID
0
 0–75


PYRUVIC ACID
0
 0–20


GLYCOLIC ACID
6
 0–50


ALPHA-OH-BUTYRIC
0.0
0–1


OXALIC
0.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
0.2
 0–11


5-HYDROXYCAPROIC
4.4
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
0.0
0–8


SUCCINIC ACID
0
 0–20


GLUTARIC ACID
0.4
0–2


2-OXO-GLUTARATE
0
 0–210


FUMARIC
0.0
0–5


MALEIC
0.0
0


MALIC ACID
28.1
0–2


ADIPIC ACID
0.0
0–7


SUBERIC ACID
1.0
 0–11


SEBACIC ACID
0.0
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
0
0–3


METHYLSUCCINIC
0.0
0


METHYLMALONIC
0
0–5


ETHYLMALONIC
0.0
0–4


HOMOGENTISIC ACID
0.0
0–1


PHENYLPYRUVIC ACID
0.1
0–1


SUCCINYLACETONE
0.0
0–1


3-OH-ISOVALERIC
0.0
 0–21


PHOSPHATE
90
  0–3000


CITRIC ACID
24
 0–450


HIPPURIC ACID
11
  0–2000


URIC ACID
0
 0–360


Nutritionals


KYNURENIC ACID
0.6


FORMIMINOGLUTAMIC
0.15
0–3


4-PYRIDOXIC ACID
0.2
0–9


PANTOTHENIC ACID
14
 0–30


XANTHURENIC ACID
0.0
0–1


KYNURENINE
O.1
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
0.00
0–3


D-AM LEVULINIC
4.0
 0–18


3-METHYL HISTIDINE
0
 0–75


NIACINAMIDE
0.0
0–1


PSEUDOURIDINE
58
 10–220


2-DEOXYTETRONIC
0
 0–75


P-HO-PHEN-ACETIC
0
 0–12


XANTHINE
0
 0–18


UROCANIC ACID
0
0–3


ABSCORBIC ACID
1
 0–160


GLYCEROL
0
0–9


Carbohydrates


THREITOL
0
 0–40


ERYTHRITOL
0
 0–55


ARABINOSE
0
 0–30


FUCOSE
0.7
 0–12


RIBOSE
3.2
 0–12


XYLOSE
0
 0–70


FRUCTOSE
0
 0–115


GLUCOSE
3
 0–110


GALACTOSE
20
 0–200


MANNOSE
10
 0–70


N-AC-GLUCOSAMINE
1.0
0–3


LACTOSE
2
 0–60


MALTOSE
1
 0–40


XYLITOL
0.1
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
1.4
 0–10


GLUCURONIC ACID
113.6
 0–50


GALACTONIC ACID
12
 0–60


GLUCONIC ACID
5.2
 0–35


GLUCARIC
2.2
0–5


MANNITOL
11.5
 0–15


DULCITOL
2.2
 0–10


SORBITOL
3.2
 0–10


INOSITOL
3.4
 0–12


SUCROSE
0
 0–75


Neurotransmitters


GABA
0.0
0–1


HOMOVANILLIC ACID
0.0
 0–10


NORMETANEPHRINE
0.0
0–1


VANILLYLMANDELIC
0.0
0–6


METANEPHRINE
0.1
0–2


5-HIAA
0.0
0–6


MHPG
0.0
0–1


ETHANOLAMINE
0
10–90


Amino Acids and Glycine Conjugates


PROPIONYL GLY
0.3
0–1


BUTYRYL GLYCINE
0.1
0–1


HEXANOYL GLYCINE
0.1
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.0
0–1


ISOVALERYL GLY
0.0
0–1


TIGLY GLY
0.0
0–1


BETA MET CROT GLY
0.0
0–1


GLYCINE
1
 0–500


ALANINE
2
 0–130


SARCOSINE
0.0
0–8


BETA-ALANINE
0.1
0–2


B-AMINOISOBUTYRIC
0
 0–50


SERINE
0
 0–85


PROLINE
0.0
0–8


HYDROXY PROLINE
0
 0–75


HYDROXY LYSINE
0.1
0–1


ASPARTIC ACID
0.0
0–2


ASPARAGINE
0.0
0–2


N-AC ASPARTIC
0.0
 0–20


ORNITHINE
0.1
0–5


GLUTAMIC ACID
0.1
0–6


GLUTAMINE
1
 0–210


PIPECOLIC ACID
0.1
0–1


LEUCINE
0.0
0–9


KETO LEUCINE
0.0
0–1


VALINE
0.0
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
0.0
0–5


KETO-ISOLEUCINE
1.0
0–1


LYSINE
1
 0–35


HISTIDINE
1
 0–225


THREONINE
0
 0–45


HOMOSERINE
0.3
0–1


METHIONINE
0.0
0–3


CYSTEINE
0
 0–160


HOMOCYSTEINE
0.0
0–1


CYSTATHIONINE
0.1
0–1


HOMOCYSTINE
0.0
0–1


CYSTINE
0.1
0–5


PHENYLALANINE
16
 0–20


TYROSINE
1
 0–22


TRYPTOPHAN
0
 0–25
















TABLE 20







METABOLIC SCREENING LABORATORY


URINE ORGANIC CONSTITUENTS


FRACTION III, BEAR URINE JZ4061


CONCENTRATION: THIS SAMPLE CONTAINED


20.72 mM CREATININE/mL












PEAK

LIB
FIT
AREA
AREA


#
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















18
24, NU3131
2125
767
1.18
72.24


25
25
0
0
2.75
167.69


32
32
0
0
0.07
4.42


57
57
0
0
0.14
8.41


68
1,3 PROPANEDIOL DI-TMS
1675
854
0.35
21.28


78
78
0
0
0.30
18.24


83
PROPENE GLYCOL DI-TMS
50
868
0.86
52.40


94
GLYCOLIC ACID DI-TMS
55
925
1.83
111.85


97
GLYCOLIC ACID DI-TMS
55
947
1.46
88.88


101
92, NA3011
2070
711
0.09
5.63


112
104, NJ3031
2131
834
1.87
114.25


181
107, KA1051
2050
712
0.08
4.73


243
4-HYDROXY BUTYRIC ACID DI-TMS
97
799
0.12
7.40


257
MALONIC ACID DI-TMS
100
760
0.09
5.38


323
PHOSPHATE TRI-TMS
1413
929
0.16
9.94


351
PHOSPHATE TRI-TMS
1413
834
0.13
7.80


357
PHOSPHATE TRI-TMS
1413
852
0.60
36.50


362
PHOSPHATE TRI-TMS
1413
925
0.41
25.17


382
PHOSPHATE TRI-TMS
1413
933
0.08
4.58


387
PHOSPHATE TRI-TMS
1413
804
0.70
42.71


409
409
0
0
0.23
14.03


423
409, JZ4061
2327
959
0.73
44.75


430
409, JZ4061
2327
928
0.58
35.39


462
283, NF3091
2093
733
0.12
7.05


486
GLYCERIC ACID TRI-TMS
324
626
0.75
45.99


513
283, NF3091
2093
747
0.11
6.47


527
283, NF3091
2093
745
0.18
11.14


600
2,4 D1HYDROXYBUTYRIC ACID TRI-TMS
1889
922
0.23
13.89


628
628
0
0
0.09
5.22


638
3,4 DIHYDROXY BUTYRIC ACID TRI-TMS
361
887
0.88
53.73


658
CITRAMALIC ACID TRI-TMS, 675
2103
703
0.13
8.17


664
645, M27041
1836
863
0.13
7.74


694
CITRAMALIC ACID TRI-TMS, 675
2103
940
0.17
10.30


738
2-DEOXY PENTONIC ACID GAMMA LACTONE DI-TMS
176
795
0.15
8.91


764
1-AMINO CYCLOPENTANE CARBOXYLIC ACID DI-TMS
158
614
4.40
268.70


773
TETROSE TRI-TMS
362
938
3.31
202.06


787
TETROSE TRI-TMS
362
941
9.36
571.10


800
3-METHYL-2-TENTENEDIOIC ACID DI-TMS
2004
726
0.07
4.32


813
CREATININE ENOL TRI-TMS
1467
865
1.68
102.41


819
TETROSE TRI-TMS
362
683
1.09
66.57


825
4 DE-O TETRONIC TMS3, THREO
1649
671
0.65
39.52


836
4 DE-O TETRONIC TMS3, THREO
1649
902
5.55
338.69


859
4 DE-O TETRONIC TMS3; THREO
1649
886
1.97
120.42


886
ALANINE DI-TMS
78
546
0.08
5.08


903
PARA HYDROXY BENZOIC DI-TMS
202
635
0.07
4.53


910
D-ERYTHRO-PENTITOL, 2-DEOXY-1,3,4,5-TETRAKIS-
633
742
0.31
18.65


927
2,2 DIMETHYL 3-HYDROXY BUTRIC ACID DI–TMS
180
546
0.58
35.27


943
LACTULOSE METABOLITE?
1751
847
0.76
46.27


951
ARABINOFURANOSE TETRA-TMS
675
855
0.26
16.12


963
GLYCOLIC ACID DI-TMS
55
319
0.97
59.40


972
981, M21021
1829
752
0.46
27.86


985
RIBULOSE PER-TMS
1848
749
0.88
53.83


996
996
0
0
1.31
79.71


1005
965, JJ4011
2191
708
0.27
16.69


1011
ARABITOL
1841
752
0.31
19.21


1019
ARABITOL
1841
664
0.15
9.44


1024
1024
0
0
0.30
18.25


1034
D-ERYTHRO-HEX-2-ENOUIC ACID, DI-O-METHYLBIS-O
404
581
0.07
4.18


1041
6-DEOXY MANNOSE TETRA-TMS
719
873
0.28
16.91


1054
ARABITOL
1841
959
2.43
148.36


1060
ARABINONIC ACID, 2,3,5-TRIS-O-TMS-, .GAMMA. -L
464
731
0.17
10.45


1072
ARABITOL
1841
951
4.16
254.05


1077
1073, RT1051
2040
732
2.02
123.07


1099
CYSTEINE TRI-TMS
363
295
1.13
68.83


1107
D-XYLOPYRANOSE TETRA-TMS
679
783
0.93
56.63


1119
1357, M22011
1834
739
2.21
134.78


1126
6-DEOXY GLUCIOL PENTA-TMS
858
913
1.24
75.79


1131
1107, NU3081
2122
683
1.62
99.10


1138
4 DE-O TETRONIC TMS3, THREO
1649
691
0.97
59.50


1142
1142
0
0
0.11
6.54


1160
PROPANOIC ACID, 3- BIS TMS-OXY PHOSPHINYL OX
756
696
0.14
8.75


1167
CREATININE TETRA-TMS
1438
603
0.87
52.90


1176
ISO CITRIC ACID TETRA-TMS
775
891
3.14
191.49


1185
D-ARABINO-HEXITOL, 2-DEOXY-1,3,4,5,6-PENTAKIS
856
584
0.45
27.57


1195
1195
0
0
0.13
7.81


1203
1357, M22011
1834
683
1.48
90.53


1226
1224, YE1011
1884
638
0.99
60.32


1234
1234
0
0
0.08
5.12


1246
1246
0
0
0.99
60.46


1254
GALACTOSE PENTA-TMS
878
707
0.57
34.80


1258
NEO-INOSITOL HEXA-TMS
972
835
1.15
70.49


1269
BENZOIC ACID, 5-METHOXY-2-TMS-OXY - TRIMETH
293
336
0.33
20.21


1276
GLUCONIC ACID, 2,3,5,6-TETRAKIS-O-TMS- LACTO
737
816
0.73
44.42


1288
3,4,5 TRIHYDROXY FURAN 2-ACETALDEHYDE TETRA-T
743
680
0.31
18.72


1301
GLUCITOL TRI-TMS
979
899
1.51
92.20


1308
GLUCITOL TRI-TMS
979
895
1.60
97.44


1312
DULCITOL
1840
926
0.78
47.33


1318
1315, YE1011
1885
837
0.55
33.52


1325
2-DEOXY ERYTHROPENTONIC ACID TETRA-TMS
687
446
0.59
36.15


1334
GALACTONIC ACID HEXA-TMS
988
888
3.31
201.84


1354
TALOSE PENTA-TMS
896
883
0.45
27.31


1369
GALACTONIC ACID HEXA-TMS
988
789
0.58
35.69


1377
GALACTARIC ACID HEXA-TMS
993
772
0.46
27.82


1384
GALACTONIC ACID HEXA-TMS
988
811
0.83
50.75


1391
2-DEOXY ERYTHROPENTONIC ACID TETRA-TMS
687
529
0.20
12.26


1395
SCYLLO-INOSITOL HEXA-TMS
969
799
1.35
82.37


1403
.BETA.PHENYLPYRUVIC ACID DI-TMS
280
205
0.59
36.22


1424
ARABITOL
1841
584
1.31
79.85


1438
ARABITOL
1841
548
0.78
47.66


1443
MUCO-INOSITOL HEXA-TMS
974
802
0.98
59.86


1451
XYLULOSE TETRA-TMS
1771
658
0.17
10.36


1460
1460
0
0
0.08
4.63


1473
1473
0
0
0.06
3.85


1484
1484
0
0
0.07
4.16


1504
1504
0
0
0.07
4.18


1553
.BETA. -D-GALACTOFURANOSE, 1,2,3,5,6-PENTAKIS-
880
625
0.09
5.69


1561
1561
0
0
0.29
17.73


1591
1591
0
0
0.06
3.84


1596
PSEUDO URIDINE PENTA-TMS
1779
792
1.91
116.63


1615
D-RIBOFURANOSE TETRA-TMS
685
762
0.65
39.75


1658
1658
0
0
0.27
16.45


1704
D-XYLOPYRANOSE TETRA-TMS
679
650
0.08
4.71


1726
ARABINONIC ACID, 2,3,4-TRIS-O-TMS-, LACTONE,
461
629
0.08
5.11


1801
6-DEOXY MANNOSE TETRA-TMS
719
855
0.20
12.13





*The named compound matches the sample peak with a reliability given by “FIT”/1000






When results of this bioassay were expressed per mg of sample to represent potency of the sample, Fraction V, Fraction VI, and Fraction VII demonstrated the highest potency (Table 18). Fraction V exhibited a fifty-fold increase in potency when compared with BDI and a twelve-fold increase over the pooled denning bear serum. Similarly, Fraction VI exhibited a fifty-seven fold increase in potency when compared with BDI and a thirteen-fold increase over the pooled denning bear serum; Fraction VII exhibited a five hundred fold increase in potency when compared with BDI and a one hundred seventeen fold increase over pooled denning bear serum.
Identification of Known Substances in the Ten Fractions of BDI
The ten fractions of BDI collected from the CCC (including Fraction III above) were submitted to Dr. James Shoemaker, Director of the Metabolic Screening Laboratory and Assistant Professor of Biochemistry and Medicine in the College of Medicine, St. Louis University, St. Louis, Mo., for analysis by gas chromatography and mass spectrometry (GC/MS). The mass spectra of trimethylsilyl derivatives of the compounds in the CCC fractions were compared to a database of more than forty thousand chemicals.
Tables 21 and 22 depict data generated from Fraction V. Tables 23 and 24 depict data generated from Fraction VI; Tables 25 and 26 depict data generated from Fraction VII. Data on retention times are available for the substances depicted in Tables 19 through 38.







TABLE 21







QUANTIFIED TARGET PANEL


URINE ORGANIC COMPOUNDS


FRACTION V, BEAR URINE


JZ4081:7










um/L*
Nrml Range













Organic Acids




LACTIC ACID
55124


PYRUVIC ACID
10460


GLYCOLIC ACID
1123


ALPHA-OH-BUTYRIC
1274.5


OXALIC
0.0


4-OH-BUTYRIC
0.0


HEXANOIC ACID
0.0


5-HYDROXYCAPROIC
0.0


OCTANOIC
0.0


BETA-LACTATE
0.0


SUCCINIC ACID
23256


GLUTARIC ACID
0.0


2-OXO-GLUTARATE
*****


FUMARIC
0.0


MALEIC
0.0


MALIC ACID
0.0


ADIPIC ACID
0.0


SUBERIC ACID
0.0


SEBACIC ACID
0.0


GLYCERIC ACID
0.0


BETA-OH-BUTYRIC
2026.0


METHYLSUCCINIC
0.0


METHYLMALONIC
0.0


ETHYLMALONIC
0.0


HOMOGENTISIC ACID
0.0


PHENYLPYRUVIC ACID
0.0


SUCCINYLACETONE
0.0


3-OH-ISOVALERIC
231.5


PHOSPHATE
2.19
mg/dL


CITRIC ACID
2865


HIPPURIC ACID
486


URIC ACID
0.59
mg/dL


Nutritionals


FORMIMINOGLUTAMIC
0.00


4-PYRIDOXIC ACID
0.0


PANTOTHENIC ACID
0


XANTHURENIC ACID
0.0


KYNURENINE
0.0


QUINOLINIC
1871.0


OROTIC ACID
0.0


D-AM LEVULINIC
*****


3-METHYL HISTIDINE
*****


NIACINAMIDE
1121.0


PSEUDOURIDINE
11063


2-DEOXYTETRONIC
0


P-HO-PHEN-ACETIC
30


XANTHINE
0


UROCANIC ACID
0


ABSCORBIC ACID
0


GLYCEROL
7963.0


Carbohydrates


THREITOL
0


ERYTHRITOL
0


ARABINOSE
0


FUCOSE
0.0


RIBOSE
0.0


XYLOSE
0


FRUCTOSE
0


GLUCOSE
23
mg/dL


GALACTOSE
0


MANNOSE
84


N-AC-GLUCOSAMINE
0.0


LACTOSE
2869


MALTOSE
3113


XYLITOL
0.0


ARABINITOL
0.0


RIBITOL
0.0


ALLOSE
105.0


GLUCURONIC ACID
2467.5


GALACTONIC ACID
0


GLUCONIC ACID
0.0


GLUCARIC
0.0


MANNITOL
69.5


DULCITOL
0.0


SORBITOL
0.0


INOSITOL
0.0


SUCROSE
6311


Neurotransmitters


GABA
562.0


HOMOVANILLIC ACID
0.0


NORMETANEPHRINE
0.0


VANILLYLMANDELIC
*****


METANEPHRINE
20.0


5-HIAA
0.0


MHPG
500.0


ETHANOLAMINE
8655


Amino Acids and Glycine Conjugates


PROPIONYL GLY
863.0


BUTYRYL GLYCINE
*****


HEXANOYL GLYCINE
856.5


PHENYL PROP GLY
0.0


SUBERYL GLYCINE
49.0


ISOVALERYL GLY
0.0


TIGLY GLY
*****


BETA MET CROT GLY
0.0


GLYCINE
15925


ALANINE
192


SARCOSINE
86.0


BETA-ALANINE
0.0


B-AMINOISOBUTYRIC
798


SERINE
12428


PROLINE
1351.0


HYDROXY PROLINE
15079


HYDROXY LYSINE
0.0


ASPARTIC ACID
3027.5


ASPARAGINE
0.0


N-AC ASPARTIC
0.0


ORNITHINE
393.5


GLUTAMIC ACID
952.5


GLUTAMINE
577


PIPECOLIC ACID
0.0


LEUCINE
1799.0


KETO LEUCINE
*****


VALINE
3449.0


KETO-VALINE
0.0


ISOLEUCINE
1277.5


KETO-ISOLEUCINE
0.0


LYSINE
43


HISTIDINE
0


THREONINE
1750


HOMOSERINE
0.0


METHIONINE
599.0


CYSTEINE
*****


HOMOCYSTEINE
0.0


CYSTATHIONINE
0.0


HOMOCYSTINE
0.0


CYSTINE
0.0


PHENYLALANINE
860.5


TYROSINE
1398


TRYPTOPHAN
183.5





THIS SAMPLE CONTAINED 130.58 mg Creatinine/dL


*The numbers above are best used to make the qualitative judgement of normal versus abnormal and not for direct quantitative comparisons.













TABLE 22







METABOLIC SCREENING LABORATORY


URINE ORGANIC CONSTITUTENTS


FRACTION V, BEAR URINE


JZ4081


CONCENTRATION: THIS SAMPLE CONTAINED 0.01 mM CREATININE/mL












PEAK
CONSTITUENT'S BEST
LIB
FIT
AREA
CREAT


#
MATCH FROM LIBRARY*
ENTRY
vs 1000
%
NOT FOUND















7
10, STN031
1893
783
4.08



19
16, 011031
1989
806
6.95


34
31, NF3031
2090
757
0.78


57
49, AK2011
2047
836
0.69


66
SILANE, TRIMETHYLPHENOXY-
1122
887
2.82


70
ETHYL AMINE DI-TMS
22
589
12.54


77
PROPENE GLYCOL DI-TMS
50
867
0.84


107
107, JZ4011
2301
787
0.79


117
104, NJ3031
2131
860
12.78


121
119, J04011
2243
922
1.09


185
BETA-LACTATE DI-TMS
1654
773
2.17


292
283, NF3091
2093
747
5.88


361
TRIMETHYLSILYL ETHER OF GLYCEROL
273
917
0.77


600
2-METHYL PROPANOATE GLYCINE CONJUGATE DI-TMS
226
904
0.88


707
BUTYRIC ACID GLYCINE CONJUGATE DI-TMS
225
904
2.12


805
METHYL D3 CREATININE TRI-TMS
1466
745
8.61


825
BUTANEDIOIC ACID, OXO-TMS-, BIS-TMS- ESTER
401
698
0.68


878
878
0
0
1.72


940
940
0
0
0.80


1076
CIS-ACONITIC ACID TRI-TMS
540
874
2.34


1111
SALICYLIC ACID DI-TMS ORTHO-HYDROXY-BENZOIC
1720
286
3.95


1135
1135, JZ4011
2306
865
1.88


1223
VANILLYL MANDELIC ACID TRI-TMS
610
898
1.73


1284
1284
0
0
1.01


1364
1364, JZ4011
2312
888
1.05


1594
1594
0
0
17.08


1604
FROM GUAIFENESIN, 1813, NH3041
2169
688
6.27


1788
1527, 0G1021
1987
631
1.79





*The named compound matches the sample peak with a reliability given by “FIT”/1000













TABLE 23







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION VI, BEAR URINE JZ4011:1










mM/M




CREATININE
Nrml Range













Organic Acids




LACTIC ACID
2531
 0–75


PYRUVIC ACID
516
 0–20


GLYCOLIC ACID
53
 0–50


ALPHA-OH-BUTYRIC
6.9
 0–51


OXALIC
70.3
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
14.9
 0–11


5-HYDROXYCAPROIC
0.0
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
29.4
0–8


SUCCINIC ACID
49
 0–20


GLUTARIC ACID
272.8
0–2


2-OXO-GLUTARATE
26936
 0–210


FUMARIC
24.1
0–5


MALEIC
0.0
0


MALIC ACID
1.5
0–2


ADIPIC ACID
3.7
0–7


SUBERIC ACID
5.7
 0–11


SEBACIC ACID
0.0
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
55
0–3


METHYLSUCCINIC
8443.4
0


METHYLMALONIC
0
0–5


ETHYLMALONIC
0.0
0–4


HOMOGENTISIC ACID
25.6
0–1


PHENYLPYRUVIC ACID
7.7
0–1


SUCCINYLACETONE
2.6
0–1


3-OH-ISOVALERIC
0.6
 0–21


PHOSPHATE
8
  0–3000


CITRIC ACID
507
 0–450


HIPPURIC ACID
472
  0–2000


URIC ACID
218
 0–360


Nutritionals


KYNURENIC ACID
44.8


FORMIMINOGLUTAMIC
0.00
0–3


4-PYRIDOXIC ACID
0.0
0–9


PANTOTHENIC ACID
0
 0–30


XANTHURENIC ACID
0.0
0–1


KYNURENINE
0.0
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
0.00
0–3


D-AM LEVALINIC
1657.1
 0–18


3-METHYL HISTIDINE 2

 0–75


NIACINAMIDE
16.3
0–1


PSEUDOURIDINE
12665
 10–220


2-DEOXYTETRONIC
0
 0–75


P-HO-PHEN-ACETIC
5
 0–12


XANTHINE
38
 0–18


UROCANIC ACID
47
0–3


ASCORBIC ACID
0
 0–160


GLYCEROL
705
0–9


Carbohydrates


THREITOL
0
 0–40


ERYTHRITOL
12
 0–55


ARABINOSE
0
 0–30


FUCOSE
0.4
 0–12


RIBOSE
0.7
 0–12


XYLOSE
0
 0–70


FRUCTOSE
135
 0–115


GLUCOSE
99
 0–110


GALACTOSE
12
 0–200


MANNOSE
54
 0–70


N-AC-GLUCOSAMINE
2.7
0–3


LACTOSE
259
 0–60


MALTOSE
127
 0–40


XYLITOL
0.0
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
0.3
 0–10


GLUCURONIC ACID
10.2
 0–50


GALACTONIC ACID
15
 0–60


GLUCONIC ACID
1.0
 0–35


GLUCARIC
0.2
0–5


MANNITOL
10.2
 0–15


DULCITOL
0.4
 0–10


SORBITOL
9.7
 0–10


INOSITOL
8.5
 0–12


SUCROSE
1349
 0–75


Neurotransmitters


GABA
1.0
0–1


HOMOVANILLIC ACID
5.6
 0–10


NORMETANEPHRINE
41.3
0–1


VANILLYLMANDELIC
90.3
0–6


METANEPHRINE
1.1
0–2


5-HIAA
1.2
0–6


MHPG
0.0
0–1


ETHANOLAMINE
409
10–90


Amino Acids and Glycine Conjugates


PROPIONYL GLY
0.0
0–1


BUTYRYL GLYCINE
1196.9
0–1


HEXANOYL GLYCINE
0.0
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.0
0–1


ISOVALERYL GLY
0.0
0–1


TIGLY GLY
0.0
0–1


BETA MET CROT GLY
0.0
0–1


GLYCINE
1053
 0–500


ALANINE
12
 0–130


SARCOSINE
12.6
0–8


BETA-ALANINE
0.0
0–2


B-AMINOISOBUTYRIC
7
 0–50


SERINE
1106
 0–85


PROLINE
115.7
0–8


HYDROXY PROLINE
956
 0–75


HYDROXY LYSINE
0.0
0–1


ASPARTIC ACID
232.4
0–2


ASPARAGINE
5.0
0–2


N-AC ASPARTIC
191.8
 0–20


ORNITHINE
86.9
0–5


GLUTAMIC ACID
79.7
0–6


GLUTAMINE
4
 0–210


PIPECOLIC ACID
0.0
0–1


LEUCINE
141.2
0–9


KETO LEUCINE
611.7
0–1


VALINE
272.9
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
107.1
0–5


KETO-ISOLEUCINE
0.0
0–1


LYSINE
644
 0–35


HISTIDINE
140
 0–225


THREONINE
215
 0–45


HOMOSERINE
0.0
0–1


METHIONINE
2.7
0–3


CYSTEINE
1122
 0–160


HOMOCYSTEINE
0.0
0–1


CYSTATHIONINE
0.0
0–1


HOMOCYSTINE
0.0
0–1


CYSTINE
8.7
0–5


PHENYLALANINE
85
 0–20


TYROSINE
68
 0–22


TRYPTOPHAN
54
 0–25





This sample contained 0.02 uMoles Creatinine/1.00 ml.













TABLE 24







METABOLIC SCREENING LABORATORY


URINE ORGANIC CONSTITUENTS


FRACTION VI, BEAR URINE JZ4011


CONCENTRATION: THIS SAMPLE CONTAINED


0.02 uM CREATININE/ml












PEAK
CONSTITUENT'S BEST
LIB
FIT
AREA
AREA %


#
MATCH FROM LIBRARY*
ENTRY
VS 1000
%
OF CREAT















5
6, J14081
2189
780
1.67
422.70


8
10, STN031
1893
857
2.71
684.47


20
16, OI1031
1989
820
5.76
1454.73


35
35
0
0
0.75
190.42


58
49 AK2011
2047
835
0.52
132.24


67
SILANE, TRIMETHYLPHENOXY-
1122
932
2.18
551.58


73
1,3 PROPANEDIOL DI-TMS
1675
934
5.38
1358.88


78
LACTIC ACID DI-TMS
1510
927
0.74
187.43


107
107
0
0
0.59
148.59


118
104, NJ3031
2131
884
8.05
2032.64


122
119, J14011
2243
925
0.82
206.86


134
BLYCINE DI-TMS
51
822
0.25
64.34


186
BETA-LACTATE DI-TMS
1654
755
1.55
391.09


251
251
0
0
0.38
95.36


294
UREA DI-TMS
37
800
3.00
757.29


362
TRIMETHYLSILYL ETHER
273
904
1.33
336.55



OF GLYCEROL


383
OCTANOIC ACID, 2-OSO-,
72
707
0.27
69.11



TRIMETHYLSILYL ESTER


427
METHYLSUCCINIC ACID DI-TMS
173
948
3.17
799.71


502
SER1NE TRI-TMS
322
958
0.51
128.24


697
3-METHYL-2-PENTENEDIOIC
2004
619
0.31
77.45



ACID DI-TMS


706
BUTYRIC ACID GLYCINE
225
874
0.43
107.51



CONJUGATE DI-TMS


748
HYDROXY PROLINE DI-TMS
156
938
0.39
99.20


808
METHYL D3 CREATININE TRI-TMS
1466
705
12.91
3258.96


825
BUTANEDIOIC ACID, OXO-TMS-,
401
704
0.26
66.23



BIS-TMS-ESTER


828
828
0
0
0.42
105.07


894
PENTANEDIOIC ACID, 3-OXO-,
448
923
0.46
116.34



TRIS-TMS ESTER


901
PARA HYDROXY
202
912
0.38
95.59



BENZOIC DI = TMS


964
964
0
0
1.16
293.82


1013
1013
0
0
0.39
97.24


1078
CIS-ACONITIC ACID TRI-TMS
540
839
6.15
1152.41


1111
P-HO PHENYL GLYCOLIC TRI-TMS
532
927
2.98
753.39


1135
1135
0
0
0.70
175.75


1141
1141
0
0
1.39
351.33


1167
CITRIC ACID TETRA-TMS
774
870
0.67
169.16


1192
1192
0
0
1.20
302.08


1215
1215
0
0
0.40
101.36


1223
1223
0
0
0.28
69.72


1252
1252
0
0
0.78
197.12


1364
1364
0
0
0.30
76.77


1370
PALMITIC ACID TMS
335
821
0.24
60.76


1389
289, ND3031
2073
678
1.49
377.32


1417
PENTANEDIOIC ACID, 3,3-DIMETHYL-,
260
418
0.50
125.53



BIS-TMS-EST


1427
1427
0
0
0.55
138.13


1443
URIC ACID TETRA-TMS
1505
780
0.25
61.93


1462
1462
0
0
1.15
291.01


1492
PARA-HYDROXYPHENYLACETIC
2299
991
7.19
1816.50



GLYCINE CONJ TR


1500
1481, NU3091
2124
782
8.74
2207.43


1596
PSEUDO URIDINE PENTA-TMS
1779
768
8.67
2189.48


1628
1472, VST031
2031
737
0.25
63.50


1746
SUCROSE OCTA-TMS
1080
924
1.05
265.38





*The named compound matches the sample peak with a reliability given by “FIT”/1000













TABLE 25







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION VII, BEAR URINE JZ4021:2










mM/M
Nrml



CREATININE
Range













Organic Acids




LACTIC ACID
2166
 0–75


PYRUVIC ACID
211
 0–20


GLYCOLIC ACID
24
 0–50


ALPHA-OH-BUTYRIC
3.7
0–1


OXALIC
0.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
7.4
 0–11


5-HYDROXYCAPROIC
0.0
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
10.3
0–8


SUCCINIC ACID
7
 0–20


GLUTARIC ACID
0.0
0–2


2-OXO-GLUTARATE
0
 0–210


FUMARIC
6.4
0–5


MALEIC
0.0
0


MALIC ACID
0.0
0–2


ADIPIC ACID
55.2
0–7


SUBERIC ACID
0.0
 0–11


SEBACIC ACID
0.0
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
15
0–3


METHYLSUCCINIC
2082.5
0


METHYLMALONIC
0
0–5


ETHYLMALONIC
1711.8
0–4


HOMOGENTISIC ACID
14.6
0–1


PHENYLPYRUVIC ACID
3.4
0–1


SUCCINYLACETONE
10.4
0–1


3-OH-ISOVALERIC
0.6
 0–21


PHOSPHATE
208
  0–3000


CITRIC ACID
58
 0–450


HIPPURIC ACID
48
  0–2000


URIC ACID
3
 0–360


Nutritionals


KYNURENIC ACID
0.0


FORMIMINOGLUTAMIC
0.00
0–3


4-PYRIDOXIC ACID
0.0
0–9


PANTOTHENIC ACID
0
 0–30


XANTHURENIC ACID
0.0
0–1


KYNURENINE
4.8
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
0.00
0–3


D-AM LEVULINIC
274.3
 0–18


3-METHYL HISTIDINE
0
 0–75


NIACINAMIDE
0.0
0–1


PSEUDOURIDINE
8927
 10–220


2-DEOXYTETRONIC
0
 0–75


P-HO-PHEN-ACETIC
9
 0–12


XANTHINE
0
 0–18


UROCANIC ACID
11
0–3


ASCORBIC ACID
0
 0–160


GLYCEROL
470
0–9


Neurotransmitters


GABA
0.0
0–1


HOMOVANILLIC ACID
91.0
 0–10


NORMETANEPHRINE
0.7
0–1


VANILLYLMANDELIC
0.4
0–6


METANEPHRINE
0.4
0–2


5-HIAA
3.2
0–6


MHPG
0.0
0–1


ETHANOLAMINE
218
10-90


Carbohydrates


THREITOL
0
 0–40


ERYTHRITOL
4
 0–55


ARABINOSE
0
 0–30


FRUCTOSE
0.0
 0–12


FUCOSE
0.0
 0–12


RIBOSE
0
 0–70


XYLOSE
71
 0–115


GLUCOSE
101
 0–110


GALACTOSE
1
 0–200


MANNOSE
36
 0–70


N-AC-GLUCOSAMINE
0.9
0–3


LACTOSE
107
 0–60


MALTOSE
61
 0–40


XYLITOL
0.0
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
0.0
 0–10


GLUCURONIC ACID
35.8
 0–50


GALACTONIC ACID
10
 0–60


GLUCONIC ACID
4.5
 0–35


GLUCARIC
0.0
0–5


MANNITOL
12.7
 0–15


DULCITOL
1.0
 0–10


SORBITOL
12.7
 0–10


INOSITOL
2.0
 0–12


SUCROSE
577
 0–75


Amino Acids and Glycine Conjugates


PROPIONYL GLY
0.0
0–1


BUTYRYL GLYCINE
0.0
0–1


HEXANOL GLYCINE
0.0
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.0
0–1


ISOVALERYL GLY
279.7
0–1


TIGLY GLY
53.2
0–1


BETA MET CROT GLY
0.0
0–1


GLYCINE
584
 0–500


ALANINE
437
 0–130


SARCOSINE
5.2
0–8


BETA-ALANINE
0.0
0–2


B-AMINOISOBUTYRIC
2
 0–50


SERINE
675
 0–85


PROLINE
55.3
0–8


HYDROXY PROLINE
386
 0–75


HYDROXY LYSINE
0.0
0–1


ASPARTIC ACID
96.5
0–2


ASPARAGINE
0.0
0–2


N-AC ASPARTIC
10.3
 0–20


ORNITHINE
55.4
0–5


GLUTAMIC ACID
20.1
0–6


GLUTAMINE
0
 0–210


PIPECOLIC ACID
0.0
0–1


LEUCINE
54.5
0–9


KETO LEUCINE
64.7
0–1


VALINE
112.8
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
41.7
0–5


KETO-ISOLEUCINE
0.0
0–1


LYSINE
14
 0–35


HISTIDINE
5
 0–225


THREONINE
96
 0–45


HOMOSERINE
0.0
0–1


METHIONINE
32.3
0–3


CYSTEINE
713
 0–160


HOMECYSTEINE
0.0
0–1


CYSTATHIONINE
0.0
0–1


HOMOCYSTINE
0.0
0–1


CYSTINE
0.0
0–5


PHENYLALANINE
19
 0–20


TYROSINE
23
 0–22


TRYPTOPHAN
8
 0–25





This sample contained 0.02 uMoles Creatinine/1.00 ml.













TABLE 26







METABOLIC SCREENING LABORATORY URINE ORGANIC


CONSTITUENTS FRACTION VII, BEAR URINE JZ4021


CONCENTRATION: THIS SAMPLE CONTAINED 0.02


mM CREATININE/mL














LIB
FIT
AREA
AREA


PEAK
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















8
10, STNO31
1893
854
4.82
564.34


20
16, 011031
1989
819
6.98
817.58


35
35, JZ4011
2300
945
0.97
113.26


58
49, AK2011
2047
821
0.68
79.19


67
SILANE, TRIMETHYLPHENOXY-
1122
935
2.89
338.68


73
1,3 PROPANEDIOL DI-TMS
1675
931
6.05
708.72


78
LACTIC ACID DI-TMS
1510
931
1.23
144.38


108
107, JZ4011
2301
889
0.78
91.61


118
104, NJ3031
2131
880
11.50
1346.76


122
119, JQ4011
2243
920
1.13
131.83


186
BETA-LACTATE DI-TMS
1654
769
2.12
248.66


190
2-METHYL 2-HYDROXY BUTYRIC ACID DI-TMS
140
887
0.43
50.10


292
UREA DI-TMS
37
813
2.61
305.69


362
TRIMETHYLSILYL ETHER OF GLYCEROL
273
913
1.73
202.95


427
METHYLSUCCINIC ACID DI-TMS
173
943
1.52
178.04


501
501
0
0
1.45
170.19


697
697
0
0
1.05
123.17


750
697, JZ4021
2316
603
0.65
76.67


809
METHYL D3 CREATININE TRI-TMS
1466
683
26.41
3094.26


848
848
0
0
0.52
60.54


985
985
0
0
0.72
84.59


1239
P-HYDROXYPHENYL LACTIC ACID TRI-TMS
578
957
5.50
644.36


1496
1481, NU3091
2124
753
0.48
56.26


1596
PSEUDO URIDINE PENTA-TMS
1779
783
9.00
1054.48


1642
1631, M15041
1802
789
9.19
1076.96


1689
1689
0
0
0.58
67.59


1741
TREHALOSE PER-TMS
1850
773
2.86
335.16


1746
SUCROSE OCTA-TMS
1080
923
0.97
113.28





*The named compound matches the sample peak with a reliability given by “FIT”/1000.






Isolated compounds obtained from GC/MS were then compared to a database of chemical mass spectra for identification. Tables 21, 23, and 25 list the identified organic acids, nutritionals, carbohydrates, neurotransmitters, amino acids, and glycine conjugates of Fractions V, VI, and VII respectively.
Tables 22, 24, and 26 list peaks found in Fractions V, VI, and VII. The peaks are identified by retention time and correlated with the “best match” identified from the database library. Values of 700 or higher (1000 represents a perfect match) are considered indicative of substance identification. Peaks identified solely by a special number (peak #7 in Table 22 of Fraction V) indicate that this particular substance has been previously identified but that its chemical structure is unknown. When the peak number and the “best match from the library” are the same (as for peaks 878, 940, 1284, and 1594 in Table 22), it is an indication that these substances have not been identified by previous users of the database library. Similar data for Fractions I, II, IV, VIII, IX and X are in the following Tables 27 through 38.
BHB is found mainly in Fraction IV; MNC is found in Fractions V and VI. The most potent stimulators of osteoblast activity are found in Fractions V, VI, and VII.
Summary


    
    
        1. Separation techniques of BDI have been refined. BDI has been separated into ten small fractions. Fractions V, VI, and VII of BDI contain substances that produce the most potent stimulation of osteoblasts. The substances that most strongly inhibit osteoblast function are found in Fraction III of BDI.
        2. MNC is found in two fractions of BDI that produce the most potent stimulation of osteoblasts—Fractions V and Fraction VI. Preliminary data suggest that one or more components of MNC are found in Fraction VII.
        3. The presence of known and unknown substances contained in all ten fractions has been recorded by GC/MS.
    
    









TABLE 27







QUANTIFIED TARGET PANEL METABOLIC SCREENING


LABORATORY FRACTION I, BEAR URINE JZ4041:3











Nrml



uM/L*
Range















Organic Acids





LACTIC ACID
283233



PYRUVIC ACID
8387



GLYCOLIC ACID
1032



ALPHA-OH-BUTYRIC
19.5



OXALIC
0.0



4-OH-BUTYRIC
0.0



HEXANOIC ACID
227.5



5-HYDROXYCAPROIC
0.0



OCTANOIC
0.0



BETA-LACTATE
674.0



SUCCINIC ACID
0



GLUTARIC ACID
0.0



2-OXO-GLUTARATE
0.0



FUMARIC
35.0



MALEIC
0.0



MALIC ACID
0.0



ADIPIC ACID
49.5



SUBERIC ACID
47.5



SEBACIC ACID
0.0



GLYCERIC ACID
0.0



BETA-OH-BUTYRIC
2075.5



METHYLSUCCINIC
0.0



METHYLMALONIC
0.0



ETHYLMALONI
0.0



HOMOGENTISIC ACID
0.0



PHENYLPYRUVIC ACID
0.0



SUCCINYLACETONE
0.0



3-OH-ISOVALERIC
0.0



PHOSPHATE
3.71
mg/dL



CITRIC ACID
61



HIPPURIC ACID
0



URIC ACID
1.20
mg/dL



Nutritionals



FORMIMINOGLUTAMIC
0.00



4-PYRIDOXIC ACID
0.0



PANTOTHENIC ACID
0.0



XANTHURENIC ACID
0.0



KYNURENINE
0.0



QUINOLINIC
0.0



7OROTIC ACID
0.0



D-AM LEVULINIC
********



3-METHYL HISTIDINE
0.00



NIACINAMIDE
0.0



PSEUDOURIDINE
221791



2-DEOXYTETRONIC
0



P-HO-PHEN-ACETIC
10



XANTHINE
0



UROCANIC ACID
96



ASCORBIC ACID
0



GLYCEROL
5903.5



Carbohydrates



THREITOL
0



ERYTHRITOL
27



ARABINOSE
0



FUCOSE
0.0



RIBOSE
0.0



XYLOSE
13



FRUCTOSE
1067



GLUCOSE
35



mg/dL GALACTOSE
104



MANNOSE
988



N-AC-GLUCOSAMINE
0.0



LACTOSE
2921



MALTOSE
2684



XYLITOL
0.0



ARABINITOL
0.0



RIBITOL
0.0



ALLOSE
0.0



GLUCURONIC ACID
0.0



GALACTONIC ACID
440



GLUCONIC ACID
0.0



CLUCARIC
0.0



MANNITOL
681.5



DULCITOL
91.0



SORBITOL
681.0



INOSITOL
107.0



SUCROSE
12380



Neurotransmitters



GABA
89.5



HOMOVANILLIC ACID
0.0



NORMETANEPHRINE
0.0



VANILLYLMANDELIC
0.0



METANEPHRINE
0.0



5-HIAA
0.0



MHPG
0.0



ETHANOLAMINE
4416



Amino Acids and Glycine Conjugates



PROPIONYL GLY
0.0



BUTYRYL GLYCINE
0.0



HEXANOL GLYCINE
0.0



PHENYL PROP GLY
0.0



SUBERYL GLYCINE
0.0



ISOVALERYL GLY
0.0



TIGLY GLY
0.0



BETA MET CROT GLY
0.0



GLYCINE
10411



ALANINE
93



SARCOSINE
108.0



BETA-ALANINE
0.0



B-AMINOISOBUTYRIC
0



SERINE
10329



PROLINE
1125.5



HYDROXY PROLINE
10671



HYDROXY LYSINE
0.0



ASPARTIC ACID
1012.0



ASPARAGINE
27.0



N-AC ASPARTIC
116.0



ORNITHINE
390.0



GLUTAMIC ACID
343.5



GLUTAMINE
0



PIPECOLIC ACID
0.0



LEUCINE
1342.0



KETO LEUCINE
2776.0



VALINE
2256.0



KETO-VALINE
0.0



ISOLEUCINE
985.0



KETO-ISOLEUCINE
0.0



LYSINE
63



HISTIDINE
0



THREONINE
771



HOMOSERINE
0.0



METHIONINE
0.0



CYSTEINE
3314.5



HOMECYSTEINE
0.0



CYSTATHIONINE
0.0



HOMOCYSTINE
0.0



CYSTINE
0.0



PHENYLALANINE
308.5



TYROSINE
370



TRYPTOPHAN
28.0







This sample contained 7.61 mg Creatinine/dL.













TABLE 28







METABOLIC SCREENING LABORATORY URINE ORGANIC


CONSTITUENTS FRACTION I, BEAR URINE JZ4041


CONCENTRATION: THIS SAMPLE CONTAINED 0.00


uM CREATININE/mL














LIB
FIT
AREA
AREA %


PEAK
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















9
10, STN031
1893
849
12.44
50748.26


20
10, M13011
1782
755
12.97
52898.66


35
35, JZ4011
2300
942
1.24
5069.15


58
49, AK2011
2047
804
1.01
4129.25


67
SILANE, TRIMETHYLPHENOXY-
1122
934
3.83
15642.15


72
ETHYL AMINE DI-TMS
22
546
12.80
52202.81


79
LACTIC ACID DI-TMS
1510
959
7.49
30555.24


108
107, JZ4011
2301
939
0.99
4047.10


118
104, NJ3031
2131
882
16.86
68779.39


122
119, JQ4011
2243
930
1.60
6511.24


186
BETA-LACTATE DI-TMS
1654
770
2.91
11857.41


288
UREA DI-TMS
37
816
0.90
3654.45


361
TRIMETHYLSILYL ETHER OF GLYCEROL
273
911
1.17
4787.66


539
539
0
0
0.65
2647.54


807
METHYL D3 CREATININE TRI-TMS
1466
706
18.22
74308.42


1370
PALMITIC ACID TMS
335
857
0.92
3734.21


1519
STEARIC ACID TMS
434
870
0.70
2849.90


1595
PSEUDO URIDINE PENTA-TMS
1779
750
13.13
53567.98


1672
1669, P17031
1984
908
1.15
4703.70


1745
SUCROSE OCTA-TMS
1080
912
1.46
5942.59





*The named compound matches the sample peak with a reliability given by “FIT”/1000.













TABLE 29







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION II, BEAR URINE JZ4051:4










mM/M
Nrml



CREATININE
Range













Organic Acids




LACTIC ACID
94
 0–75


PYRUVIC ACID
6
 0–20


GLYCOLIC ACID
2
 0–50


ALPHA-OH-BUTYRIC
0.1
0–1


OXALIC
0.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
0.0
 0–11


5-HYDROXYCAPROIC
0.0
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
0.0
0–8


SUCCINIC ACID
3
 0–20


GLUTARIC ACID
0.0
0–2


2-OXO-GLUTARATE
0
 0–210


FUMARIC
0.0
0–5


MALEIC
0.0
0


MALIC ACID
0.0
0–2


ADIPIC ACID
0.0
0–7


SUBERIC ACID
0.0
 0–11


SEBACIC ACID
0.0
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
1
0–3


METHYLSUCCINIC
0.0
0


METHYLMALONIC
0
0–5


ETHYLMALONI
0.0
0–4


HOMOGENTISIC ACID
0.0
0–1


PHENYLPYRUVIC ACID
0.7
0–1


SUCCINYLACETONE
0.0
0–1


3-OH-ISOVALERIC
0.0
 0–21


PHOSPHATE
137
  0–3000


CITRIC ACID
0
 0–450


HIPPURIC ACID
13
  0–2000


URIC ACID
0
 0–360


Nutritionals


KYNURENIC ACID
0.0


FORMIMINOGLUTAMIC
0.00
0–3


4-PYRIDOXIC ACID
0.0
0–9


PANTOTHENIC ACID
0
 0–30


XANTHURENIC ACID
0.0
0–1


KYNURENINE
0.0
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
0.00
0–3


D-AM LEVULINIC
1.0
 0–18


3-METHYL HISTIDINE
7
 0–75


NIACINAMIDE
0.0
0–1


PSEUDOURIDINE
170
 10–220


2-DEOXYTETRONIC
0
 0–75


P-HO-PHEN-ACETIC
5
 0–12


XANTHINE
0
 0–18


UROCANIC ACID
0
0–3


ASCORBIC ACID
0
 0–160


GLYCEROL
3
0–9


Carbohydrates


THREITOL
1
 0–40


ERYTHRITOL
5
 0–55


ARABINOSE
0
 0–30


FUCOSE
0.0
 0–12


RIBOSE
0.0
 0–12


XYLOSE
0
 0–70


FRUCTOSE
0
 0–115


GLUCOSE
2
 0–110


GALACTOSE
0
 0–200


MANNOSE
0
 0–70


N-AC-GLUCOSAMINE
0.0
0–3


LACTOSE
1
 0–60


MALTOSE
1
 0–40


XYLITOL
0.9
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
0.4
 0–10


GLUCURONIC ACID
0.0
 0–50


GALACTONIC ACID
0
 0–60


GLUCONIC ACID
0.0
 0–35


CLUCARIC
0.0
0–5


MANNITOL
0.1
 0–15


DULCITOL
0.1
 0–10


SORBITOL
0.9
 0–10


INOSITOL
0.1
 0–12


SUCROSE
4
 0–75


Neurotransmitters


GABA
0.0
0–1


HOMOVANILLIC ACID
1.1
 0–10


NORMETANEPHRINE
0.0
0–1


VANILLYLMANDELIC
0.0
0–6


METANEPHRINE
0.2
0–2


5-HIAA
1.9
0–6


MHPG
0.0
0–1


ETHANOLAMINE
6
10–90


Amino Acids and Glycine Conjugates


PROPIONYL GLY
0.0
0–1


BUTYRYL GLYCINE
0.0
0–1


HEXANOL GLYCINE
0.0
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.0
0–1


ISOVALERYL GLY
0.0
0–1


TIGLY GLY
0.0
0–1


BETA MET CROT GLY
0.0
0–1


GLYCINE
10
 0–500


ALANINE
0
 0–130


SARCOSINE
0.2
0–8


BETA-ALANINE
0.0
0–2


B-AMINOISOBUTYRIC
0
 0–50


SERINE
9
 0–85


PROLINE
0.7
0–8


HYDROXY PROLINE
13
 0–75


HYDROXY LYSINE
0.0
0–1


ASPARTIC ACID
0.6
0–2


ASPARAGINE
0.0
0–2


N-AC ASPARTIC
0.0
 0–20


ORNITHINE
0.1
0–5


GLUTAMIC ACID
0.5
0–6


GLUTAMINE
0
 0–210


PIPECOLIC ACID
0.0
0–1


LEUCINE
0.9
0–9


KETO LEUCINE
13.4
0–1


VALINE
1.6
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
0.5
0–5


KETO-ISOLEUCINE
0.0
0–1


LYSIME
4
 0–35


HISTIDINE
0
 0–225


THREONINE
0
 0–45


HOMOSERINE
0.0
0–1


METHIONINE
0.0
0–3


CYSTEINE
9
 0–160


HOMOCYSTEINE
0.0
0–1


CYSTATHIONINE
0.0
0–1


HOMOCYSTINE
0.0
0–1


CYSTINE
0.0
0–5


PHENYLALANINE
0
 0–20


TYROSINE
0
 0–22


TRYPTOPHAN
0
 0–25





This sample contained 0.42 uMoles Creatinine/1.00 ml.













TABLE 30







METABOLIC SCREENING LABORATORY URINE ORGANIC


CONSTITUENTS FRACTION II, BEAR URINE JZ4051


CONCENTRATION: THIS SAMPLE CONTAINED


0.42 uM CREATININE/mL














LIB
FIT
AREA
AREA %


PEAK
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















6
10, STN031
1893
823
2.11
13.22


13
13
0
0
0.53
3.32


18
16, O11031
1989
785
6.94
43.44


33
35, JZ4011
2300
882
0.59
3.70


56
49, AK2011
2047
831
0.51
3.19


65
SILANE, TRIMETHYLPHENOXY-
1122
935
1.87
11.73


69
ETHYL AMINE DI-TMS
22
581
5.56
34.84


76
LACTIC ACID DI-TMS
1510
946
1.02
6.42


106
107, JZ4011
2301
785
0.58
3.62


116
104, NJ3031
2131
866
9.15
57.29


120
119, JQ4011
2243
913
0.75
4.71


184
BETA-LACTATE DI-TMS
1654
764
1.45
9.07


250
251, JZ4011
2302
923
0.47
2.97


282
UREA DI-TMS
37
721
0.83
5.23


308
283 NF3091
2093
745
18.17
113.79


354
PHOSPHATE TRI-TMS
1413
905
3.37
21.13


537
539 JZ4041
2320
956
0.56
3.53


810
CREATININE TRI-TMS
1784
946
35.05
219.48


846
3-PHENYL LACTIC TMS 2
1562
677
0.43
2.70


916
PARA-HYDROXYPHENYLACETIC ACID DI-TMS
1485
938
0.64
3.99


1189
1189
0
0
0.59
3.70


1204
1189, NU3061
2118
711
1.81
11.34


1230
MOUSE HORMONE?
1508
712
0.39
2.44


1234
1234, JD2031
2002
789
0.85
5.32


1261
STEROID M
1509
788
0.73
4.60


1369
PALMITIC ACID TMS
335
862
1.00
6.25


1519
STEARIC ACID TMS
434
918
0.38
2.38


1594
PSEUDO URIDINE PENTA-TMS
1779
816
5.75
36.03





*The named compound matches the sample peak with a reliability given by “FIT”/1000.













TABLE 31







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION IV, BEAR URINE JZ4071:6










mM/M
Nrml



CREATININE
Range













Organic Acids




LACTIC ACID
2393
 0–75


PYRUVIC ACID
15
 0–20


GLYCOLIC ACID
4
 0–50


ALPHA-OH-BUTYRIC
0.7
0–1


OXALIC
0.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
28.1
 0–11


5-HYDROXYCAPROIC
0.0
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
19.9
0–8


SUCCINIC ACID
1916
 0–20


GLUTARIC ACID
0.0
0–2


2-OXO-GLUTARATE
210
 0–210


FUMARIC
1.7
0–5


MALEIC
25.6
0


MALIC ACID
39.4
0–2


ADIPIC ACID
0.9
0–7


SUBERIC ACID
0.2
 0–11


SEBACIC ACID
1.6
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
5822
0–3


METHYLSUCCINIC
0.0
0


METHYLMALONIC
0
0–5


ETHYLMALONIC
0.0
0–4


HOMOGENTISIC ACID
0.0
0–1


PHENYLPYRUVIC ACID
1163.4
0–1


SUCCINYLACETONE
1.0
0–1


3-OH-ISOVALERIC
2.1
 0–21


PHOSPHATE
135
  0–3000


CITRIC ACID
8
 0–450


HIPPURIC ACID
25
  0–2000


URIC ACID
2
 0–360


Nutritionals


KYNURENIC ACID
13.8


FORMIMINOGLUTAMIC
16.80
0–3


4-PYRIDOXIC ACID
60.5
0–9


PANTOTHENIC ACID
20
 0–30


XANTHURENIC ACID
0.0
0–1


KYNURENINE
3.2
0–1


QUINOLINIC
37.4
0–6


OROTIC ACID
0.00
0–3


D-AM LEVULINIC
30.8
 0–18


3-METHYL HISTIDINE
9
 0–75


NIACINAMIDE
12.7
0–1


PSEUDOURIDINE
19
 10–220


2-DEOXYTETRONIC
2
 0–75


P-HO-PHEN-ACETIC
2
 0–12


XANTHINE
0
 0–18


UROCANIC ACID
1
0–3


ASCORBIC ACID
3
 0–160


GLYCEROL
36
0–9


Carbohydrates


THREITOL
0
 0–40


ERYTHRITOL
2
 0–55


ARABINOSE
0
 0–30


FUCOSE
1.4
 0–12


RIBOSE
1.0
 0–12


XYLOSE
2
 0–70


FRUCTOSE
0
 0–115


GLUCOSE
55
 0–110


GALACTOSE
7
 0–200


MANNOSE
1
 0–70


N-AC-GLUCOSAMINE
0.3
0–3


LACTOSE
11
 0–60


MALTOSE
11
 0–40


XYLITOL
0.0
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
0.8
 0–10


GLUCURONIC ACID
11.8
 0–50


GALACTONIC ACID
166
 0–60


GLUCONIC ACID
0.0
 0–35


CLUCARIC
0.0
0–5


MANNITOL
1.2
 0–15


DULCITOL
0.0
 0–10


SORBITOL
1.2
 0–10


INOSITOL
0.0
 0–12


SUCROSE
14
 0–75


Neurotransmitters


GABA
4.2
0–1


HOMOVANILLIC ACID
2.0
 0–10


NORMETANEPHRINE
20.2
0–1


VANILLYLMANDELIC
2.0
0–6


METANEPHRINE
0.5
0–2


5-HIAA
5.0
0–6


MHPG
2.7
0–1


ETHANOLAMINE
17
10–90


Amino Acids and Glycine Conjugates


PROPIONYL GLY
322.6
0–1


BUTYRYL GLYCINE
0.4
0–1


HEXANOYL GLYCINE
0.0
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.0
0–1


ISOVALERYL GLY
35.7
0–1


TIGLY GLY
18.7
0–1


BETA MET CROT GLY
150.5
0–1


GLYCINE
82
 0–500


ALANINE
50
 0–130


SARCOSINE
0.3
0–8


BETA-ALANINE
0.0
0–2


B-AMINOISOBUTYRIC
39
 0–50


SERINE
54
 0–85


PROLINE
4.8
0–8


HYDROXY PROLINE
92
 0–75


HYDROXY LYSINE
0.0
0–1


ASPARTIC ACID
14.0
0–2


ASPARAGINE
0.3
0–2


N-AC ASPARTIC
5.0
 0–20


ORNITHINE
12.0
0–5


GLUTAMIC ACID
2.4
0–6


GLUTAMINE
46
 0–210


PIPECOLIC ACID
0.0
0–1


LEUCINE
47.4
0–9


KETO LEUCINE
45.3
0–1


VALINE
9.1
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
6.3
0–5


KETO-ISOLEUCINE
0.0
0–1


LYSINE
45
 0–35


HISTIDINE
9
 0–225


THREONINE
6
 0–45


HOMOSERINE
2.2
0–1


METHIONINE
0.0
0–3


CYSTEINE
179
 0–160


HOMECYSTEINE
0.0
0–1


CYSTATHIONINE
1.2
0–1


HOMOCYSTINE
0.0
0–1


CYSTINE
0.3
0–5


PHENYLALANINE
3
 0–20


TYROSINE
5
 0–22


TRYPTOPHAN
238
 0–25





This sample contained 0.42 uMoles Creatine/1.00 ml.













TABLE 32







METABOLIC SCREENING LABORATORY URINE


ORGANIC CONSTITUENTS FRACTION IV,


BEAR URINE JZ4071


CONCENTRATION: THIS SAMPLE CONTAINED


0.23 uM CREATININE/mL














LIB
FIT
AREA
AREA %


PEAK
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















20
10, M13011
1782
716
1.28
48.98


28
10, M13011
1782
821
1.18
45.14


34
35, JZ4011
2300
836
0.25
9.56


57
49, AK2011
2047
814
0.20
7.79


66
SILANE, TRIMETHYLPHENOXY-
1122
879
0.80
30.66


71
ETHYL AMINE DI-TMS
22
529
2.92
111.91


78
LACTIC ACID DI-TMS
1510
927
4.23
162.24


107
107, JZ4011
2301
865
0.25
9.47


117
104, NJ3031
2131
872
4.13
158.52


122
119, JQ4011
2243
902
0.34
13.19


187
BETA HYDROXYBUTYRIC ACID DI-TMS
1622
930
14.85
569.62


251
251, JZ4011
2302
928
0.29
10.98


283
4-HYDROXY BUTYRIC ACID DI-TMS
97
724
0.16
6.05


293
283, NF3091
2093
745
0.25
9.61


305
283, NF3091
2093
744
1.83
70.32


355
PHOSPHATE TRI-TMS
1413
898
0.43
16.33


361
TRIMETHYLSILYL ETHER OF GLYCEROL
273
882
0.63
24.21


407
SUCCINIC ACID DI-TMS
1635
892
5.26
201.56


599
PROPIONATE GLYCINE CONJUGATE DI-TMS
165
961
1.11
42.71


611
564, JJ4021
2200
742
0.28
10.77


689
CITRAMALIC ACID TRI-TMS, 675
2103
944
0.40
15.18


111
NORLEUCINE DI-TMS
1540
656
2.48
95.07


749
749
0
0
1.11
42.72


797
259, 192TMS
1470
367
0.27
10.23


808
CREATININE TRI-TMS
1784
913
8.32
319.11


845
845
0
0
0.19
7.28


862
862
0
0
0.18
6.77


940
GLYCOLIC ACID DI-TMS
55
405
0.35
13.32


978
251, JZ4011
2302
390
0.16
6.22


985
985
0
0
2.58
98.95


997
996, GI1021
1958
790
0.24
9.35


1000
1000
0
0
0.25
9.60


1011
.BETA. PHENYLPYRUVIC ACID DI-TMS
280
887
3.95
151.29


1027
1027
0
0
0.93
35.63


1037
1037
0
0
0.41
15.72


1047
1047
0
0
0.19
7.19


1064
2-HYDROXY BENZAMIDE DI-TMS
198
421
0.51
19.63


1071
1071
0
0
0.22
8.29


1079
CIS-ACONITIC ACID TRI-TMS
540
792
6.66
255.42


1093
L-GLUTAMIC ACID, N-ACETYL-N-TMS, BIS-TMS EST
587
665
0.25
9.43


1098
862, JZ4071
2344
665
0.43
16.53


1103
1103
0
0
0.52
19.81


1114
1114
0
0
0.31
12.01


1120
1071, JZ4071
2350
685
0.64
24.48


1135
1135, JZ4011
2306
868
0.57
22.01


1178
1178
0
0
0.16
6.31


1183
6-AMINO HEXANOIC ACID DI-TMS
166
537
0.41
15.79


1196
QUINOLINIC TMS 2
1564
481
1.31
50.20


1202
1202
0
0
0.55
21.09


1228
1228
0
0
4.38
167.97


1237
1,6 DIHYDRO 1-METHYL 6-OXO 3-PYRIDINECARBOXAM
63
558
4.31
165.39


1253
MANNOSE PENTA-TMS
879
901
0.28
10.68


1277
4-PYRIDOXIC ACID TRI-TMS
580
697
0.37
14.00


1294
NORVALINE DI-TMS
128
402
0.75
28.82


1300
1300
0
0
0.39
14.89


1310
NORVALINE DI-TMS
128
432
0.25
9.50


1346
P-HO PHENYL GLYCOLIC TRI-TMS
532
735
0.17
6.61


1354
MANNOSE PENTA-TMS
879
913
0.38
14.67


1382
1382
0
0
0.64
24.60


1386
GLYCINE DI-TMS
51
477
0.18
6.93


1397
1217, NC1031
1992
543
0.16
6.32


1435
1435
0
0
0.20
7.49


1443
URIC ACID TETRA-TMS
1505
674
0.33
12.63


1510
TRYPTOPHAN TRI-TMS
1965
825
2.01
77.00


1515
1515
0
0
0.99
37.86


1545
1545
0
0
0.17
6.59


1589
1-PHENYL 2-AMINO PROPANE DI-TMS
190
712
0.16
5.96


1595
PSEUSO URIDINE PENTA-TMS
1779
945
2.48
95.21


1604
1631, M15041
1802
692
1.73
66.36


1616
1616
0
0
0.47
17.85


1631
2-PROPENOIC ACID, 2-TMS-OXY -3- 1-TMS-1H-IND
618
766
1.21
46.30


1641
1624, NU3061
2120
696
2.78
106.59


1659
1659
0
0
0.60
23.09


1665
1665
0
0
0.26
10.03


1731
TREHALOSE PER-TMS
1850
685
0.25
9.50


1745
TREHALOSE PER-TMS
1850
788
0.17
6.63





*The named compound matches the sample peak with a reliability given by “FIT”/1000.













TABLE 33







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION VIII, BEAR URINE JZ4091:8











mM/M
Nrml




CREATININE
Range














Organic Acids





LACTIC ACID
38661
 0–75


PYRUVIC ACID
0
 0–20


GLYCOLIC ACID
0
 0–50


ALPHA-OH-BUTYRIC
0.0
0–1


OXALIC
0.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
0.0
 0–11


5-HYDROXYCAPROIC
0.0
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
0.0
0–8


SUCCINIC ACID
0
 0–20


GLUTARIC ACID
0.0
0–2


2-OXO-GLUTARATE
0
 0–210


FUMARIC
0.0
0–5


MALEIC
0.0
0


MALIC ACID
0.0
0–2


ADIPIC ACID
3878.3
0–7


SUBERIC ACID
0.0
 0–11


SEBACIC ACID
244.7
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
89
0–3


METHYLSUCCINIC
0.0
0


METHYLMALONIC
0
0–5


ETHYLMALONIC
********
0–4


HOMOGENTISIC ACID
0.0
0–1


PHENYLPYRUVIC ACID
0.0
0–1


SUCCINYLACETONE
0.0
0–1


3-OH-ISOVALERIC
0.0
 0–21


PHOSPHATE
317
  0–3000


CITRIC ACID
37
 0–450


HIPPURIC ACID
84990
  0–2000


URIC ACID
125
 0–360


Nutritionals


KYNURENIC ACID
7544.8


FORMIMINOGLUTAMIC
0.00
0–3


4-PYRIDOXIC ACID
0.0
0–9


PANTOTHENIC ACID
0
 0–30


XANTHURENIC ACID
0.0
0–1


KYNURENINE
0.0
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
0.00
0–3


D-AM LEVULINIC
0.0
 0–18


3-METHYL HISTIDINE
0
 0–75


NIACINAMIDE
0.0
0–1


PSEUDOURIDINE
7176
 10–220


2-DEOXYTETRONIC
0
 0–75


P-HO-PHEN-ACETIC
1019
 0–12


XANTHINE
0
 0–18


UROCANIC ACID
907
0–3


ASCORBIC ACID
0
 0–160


GLYCEROL
8524
0–9


Neurotransmitters


GABA
0.0
0–1


HOMOVANILLIC ACID
4038.8
 0–10


NORMETANEPHRINE
0.0
0–1


VANILLYLMANDELIC
0.0
0–6


METANEPHRINE
374.2
0–2


5-HIAA
6190.5
0–6


MHPG
0.0
0–1


ETHANOLAMINE
3152
10–90


Carbohydrates


THREITOL
0
 0–40


ERYTHRITOL
0
 0–55


ARABINOSE
0
 0–30


FUCOSE
0.0
 0–12


RIBOSE
0.0
 0–12


XYLOSE
0
 0–70









FRUCTOSE
3266
 0–115


GLUCOSE
4435
 0–110


GALACTOSE
5127
 0–200


MANNOSE
2585
 0–70


N-AC-GLUCOSAMINE
11.8
0–3


LACTOSE
4679
 0–60


MALTOSE
4470
 0–40


XYLITOL
0.0
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
384.7
 0–10


GLUCURONIC ACID
0.0
 0–50


GALACTONIC ACID
13137
 0–60


GLUCONIC ACID
0.0
 0–35


GLUCARIC
42.7
0–5


MANNITOL
604.1
 0–15


DULCITOL
0.0
 0–10


SORBITOL
603.4
 0–10


INOSITOL
0.0
 0–12


SUCROSE
18255
 0–75


Amino Acids and Glycine Conjugates


PROPIONYL GLY
0.0
0–1


BUTYRYL GLYCINE
2523.4
0–1


HEXANOL GLYCINE
0.0
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.0
0–1


ISOVALERYL GLY
********
0–1


TIGLY GLY
0.0
0–1


BETA MET CROT GLY
********
0–1


GLYCINE
9496
 0–500


ALANINE
7063
 0–130


SARCOSINE
80.5
0–8


BETA-ALANINE
0.0
0–2


B-AMINOISOBUTYRIC
525
 0–50


SERINE
10517
 0–85


PROLINE
917.5
0–8


HYDROXY PROLINE
12808
 0–75


HYDROXY LYSINE
1407.6
0–1


ASPARTIC ACID
1866.1
0–2


ASPARAGINE
0.0
0–2


N-AC ASPARTIC
0.0
 0–20


ORNITHINE
1826.4
0–5


GLUTAMIC ACID
364.9
0–6


GLUTAMINE
0
 0–210


PIPECOLIC ACID
0.0
0–1


LEUCINE
1200.1
0–9


KETO LEUCINE
913.8
0–1


VALINE
1532.7
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
871.7
0–5


KETO-ISOLEUCINE
0.0
0–1


LYSINE
34440
 0–35


HISTIDINE
1307
 0–225


THREONINE
1240
 0–45


HOMOSERINE
0.0
0–1


METHIONINE
********
0–3


CYSTEINE
10527
 0–160


HOMECYSTEINE
0.0
0–1


CYSTATHIONINE
0.0
0–1


HOMOCYSTINE
0.0
0–1


CYSTINE
0.0
0–5


PHENYLALANINE
896
 0–20


TYROSINE
1136
 0–22


TRYPTOPHAN
575
 0–25





This sample contained 0.00 uMoles Creatinine/7.20 ml.













TABLE 34







METABOLIC SCREENING LABORATORY URINE ORGANIC


CONSTITUENTS FRACTION VIII, BEAR URINE JZ4091


CONCENTRATION: THIS SAMPLE CONTAINED


0.00 uM CREATININE/mL












PEAK

LIB
FIT
AREA
AREA %


#
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















14
13, JZ4051
2321
783
0.61
2309.70


18
13, JZ4051
2321
759
2.92
11073.36


62
SILANE, TRIMETHYLPHENOXY-
1122
877
0.63
2396.66


69
1,3 PROPANEDIOL DI-TMS
1675
925
2.01
7601.11


74
LACTIC ACID DI-TMS
1510
907
0.65
2452.00


114
104, NJ3031
2131
850
3.43
12980.22


185
BETA-LACTATE DI-TMS
1654
773
0.42
1575.81


189
2-HYDROXY PENTANOIC ACID DI-TMS
141
918
1.13
4290.31


291
291
0
0
1.55
5864.71


354
DIMETHYL MALANIC ACID DI-TMS
171
954
0.82
3110.44


362
TRIMETHYLSILYL ETHER OF GLYCEROL
273
938
0.99
3754.66


622
3-METHYL 2-PENTENEDIOIC ACID DI-TMS
224
892
0.62
2366.22


687
3-METHYL BUTANOATE GLYCINE CONJUGATE TMS
74
628
0.47
1788.05


696
3-METHYL 2-PENTENDIOIC ACID DI-TMS, Z-
222
840
0.47
1778.00


752
GLYCINE, N-3-METHYL-1OXOBUTYL-N-TMS-, TRIMET
255
942
3.62
13706.16


808
METHYL D3 CREATININE TRI-TMS
1466
743
16.38
62054.19


848
848, JZ4021
2317
887
3.09
11698.73


1104
1104
0
0
3.57
13521.55


1123
1112, M20021
1823
765
0.67
2526.55


1158
3,4 -DIHYDROXY BENZENEACETIC ACID TRI-TMS
531
834
0.54
2054.74


1196
1189, JZ4051
2322
961
3.87
14654.56


1211
1189, NU3061
2118
697
19.22
72808.71


1232
L-GLUTAMIC ACID, N-ACETYL-N-TMS-, BIS-TMS EST
587
526
2.22
8414.89


1241
P-HYDROXYPHENYL LACTIC ACID TRI-TMS
578
941
9.80
37151.69


1287
HYDROXY PROLINE DI-TMS
1610
424
0.72
2710.46


1370
PALMITIC ACID TMS
335
639
1.07
4055.54


1413
1481, NU3091
2124
403
0.46
1761.13


1506
PARA-HYDROXY HIPPURIC ACID DI-TMS
377
901
1.04
3941.33


1596
PSEUDO URIDINE PENTA-TMS
1779
953
7.00
26509.32


1642
1631, M15041
1802
795
8.81
33369.32


1740
TREHALOSE PER-TMS
1850
781
0.44
1655.34


1746
SUCROSE OCTA-TMS
1080
892
1.40
5286.62





*The named compound matches the sample peak with a reliability given by “FIT”/1000.













TABLE 35







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION IX, BEAR URINE JZ4101:9










mM/M
Nrml



CREATININE
Range













Organic Acids




LACTIC ACID
856
 0–75


PYRUVIC ACID
52
 0–20


GLYCOLIC ACID
7
 0–50


ALPHA-OH-BUTYRIC
1.9
0–1


OXALIC
0.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
415.0
 0–11


5-HYDROXYCAPROIC
0.0
0–1


OCTANOIC
0.0
0–1


BETA-LACTATE
0.0
0–8


SUCCINIC ACID
4
 0–20


GLUTARIC ACID
0.0
0–2


2-OXO-GLUTARATE
0
 0–210


FUMARIC
7.1
0–5


MALEIC
0.0
0


MALIC ACID
0.0
0–2


ADIPIC ACID
33.7
0–7


SUBERIC ACID
536.8
 0–11


SEBACIC ACID
1.1
0–2


GLYCERIC ACID
0
0–4


BETA-OH-BUTYRIC
12
0–3


METHYLSUCCINIC
0.0
0


METHYLMALONIC
0
0–5


ETHYLMALONIC
137.0
0–4


HOMOGENTISIC ACID
0.0
0–1


PHENYLPYRUVIC ACID
110.6
0–1


SUCCINYLACETONE
0.0
0–1


3-OH-ISOVALERIC
1.8
0–2


PHOSPHATE
317
  0–3000


CITRIC ACID
136
 0–450


HIPPURIC ACID
35604
  0–2000


URIC ACID
4
 0–360


Nutritionals


KYNURENIC ACID
297.6


FORMIMINOGLUTAMIC
0.00
0–3


4-PYRIDOXIC ACID
0.0
0–9


PANTOTHENIC ACID
37
 0–30


XANTHURENIC ACID
18.4
0–1


KYNURENINE
19.8
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
0.00
0–3


D-AM LEVULINIC
20.0
 0–18


3-METHYL HISTIDINE
32
 0–75


NIACINAMIDE
0.0
0–1


PSEUDOURIDINE
22608
 10–220


2-DEOXYTETRONIC
2
 0–75


P-HO-PHEN-ACETIC
18
 0–12


XANTHINE
6
 0–18


UROCANIC ACID
49
0–3


ASCORBIC ACID
2
 0–160


GLYCEROL
352
0–9


Carbohydrates


THREITOL
0
 0–40


ERYTHRITOL
0
 0–55


ARABINOSE
9
 0–30


FUCOSE
41.0
 0–12


RIBOSE
41.0
 0–12


XYLOSE
3
 0–70


FRUCTOSE
14
 0–115


GLUCOSE
232
 0–110


GALACTOSE
1239
 0–200


MANNOSE
35
 0–70


N-AC-GLUCOSAMINE
6.5
0–3


LACTOSE
145
 0–60


MALTOSE
140
 0–40


XYLITOL
0.0
 0–15


ARABINITOL
0.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
6.4
 0–10


GLUCURONIC ACID
38.1
 0–50


GALACTONIC ACID
421
 0–60


GLUCONIC ACID
4.9
 0–35


GLUCARIC
2.9
0–5


MANNITOL
4.1
 0–15


DULCITOL
1.0
 0–10


SORBITOL
7.7
 0–10


INOSITOL
3.9
 0–12


SUCROSE
483
 0–75


Neurotransmitters


GABA
8.8
0–1


HOMOVANILLIC ACID
6221.3
 0–10


NORMETANEPHRINE
53.6
0–1


VANILLYLMANDELIC
30.3
0–6


METANEPHRINE
156.8
0–2


5-HIAA
4791.4
0–6


MHPG
0.0
0–1


ETHANOLAMINE
211
10–90


Amino Acids and Glycine Conjugates


PROPIONYL GLY
8.7
0–1


BUTYRYL GLYCINE
0.0
0–1


HEXANOYL GLYCINE
39.1
0–1


PHENYL PROP GLY
0.0
0–1


SUBERYL GLYCINE
0.3
0–1


ISOVALERYL GLY
1852.0
0–1


TIGLY GLY
4.7
0–1


BETA MET CROT GLY
36.8
0–1


GLYCINE
614
 0–500


ALANINE
3
 0–130


SARCOSINE
1.2
0–8


BETA-ALANINE
0.0
0–2


B-AMINOISOBUTYRIC
232
 0–50


SERINE
403
 0–85


PROLINE
35.4
0–8


HYDROXY PROLINE
1036
 0–75


HYDROXY LYSINE
14.3
0–1


ASPARTIC ACID
105.0
0–2


ASPARAGINE
0.6
0–2


N-AC ASPARTIC
41.4
 0–20


ORNITHINE
153.8
0–5


GLUTAMIC ACID
53.2
0–6


GLUTAMINE
40
 0–210


PIPECOLIC ACID
0.0
0–1


LEUCINE
62.3
0–9


KETO LEUCINE
533.3
0–1


VALINE
60.8
 0–18


KETO-VALINE
0.0
0–1


ISOLEUCINE
49.9
0–5


KETO-ISOLEUCINE
0.0
0–1


LYSINE
16777
 0–35


HISTIDINE
452
 0–225


THREONINE
69
 0–45


HOMOSERINE
0.0
0–1


METHIONINE
254.1
0–3


CYSTEINE
2504
 0–160


HOMOCYSTEINE
0.0
0–1


CYSTATHIONINE
0.5
0–1


HOMOCYSTINE
4.3
0–1


CYSTINE
16.5
0–5


PHENYLALANINE
216
 0–20


TYROSINE
73
 0–22


TRYPTOPHAN
404
 0–25





This sample contained 0.02 uMoles Creatinine/7.20 ml.













TABLE 36







METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS


FRACTION IX, BEAR URINE JZ4101


CONCENTRATION: THIS SAMPLE CONTAINED 0.00 uM CREATININE/mL












PEAK

LIB
FIT
AREA
AREA %


#
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















7
6, JI4081
2189
745
0.67
179.37


10
13, JZ4051
2321
739
0.18
47.76


19
13, AK2011
2044
737
1.17
312.52


66
SILANE, TRIMETHYLPHENOXY-
1122
896
0.29
77.67


71
ETHYL AM1NE DI-TMS
22
549
1.79
479.46


78
PROPENE GLYCOL DI-TMS
50
922
0.16
41.63


107
107, JZ4011
2301
849
0.14
37.91


117
104, NJ3031
2131
851
3.34
897.03


122
119, JQ4011
2243
902
0.13
34.73


186
BETA-LACTATE DI-TMS
1654
777
0.41
110.10


293
2-HYDROXY HEXANOIC ACID DI-TMS
1682
784
3.73
1000.76


362
TRIMETHYLSILYL ETHER OF GLYCEROL
273
909
0.50
134.35


383
SILANE, TRIMETHYL 1-METHYLBUTOXY-
1112
493
0.11
30.42


540
539, JZ4041
2320
930
0.29
78.34


613
613
0
0
0.24
63.30


622
3-METHYL 2-PENTENEDIOIC ACID DI-TMS
224
833
0.33
88.13


642
613, JZ4101
2370
711
1.24
332.62


687
BENZENEACETIC ACID, ALPHA - - TMS-OXY, -TRIM
246
889
1.24
332.20


696
3-METHYL 2-PENTENDIOIC ACID DI-TMS, Z-
222
891
1.16
41.93


753
HEXANEDIOIC ACID, 3-METHYL-, BIS-TMS-ESTER
258
663
1.64
440.24


781
HEXANEDIOIC ACID, 3-METHYL-, BIS-TMS-ESTER
258
793
0.18
49.23


798
METHYL D3 CREATININE TRI-TMS
1466
717
0.11
30.11


809
METHYL D3 CREATININE TRI-TMS
1466
701
12.34
3310.78


821
ORTHO-HYDROXYPHENYLACETIC ACID DI-TMS
247
929
0.60
161.70


852
2-HYDROXY 3-PHENYL PROPIONIC ACID DI-TMS
287
921
7.95
2132.51


861
848, JZ4021
2317
685
0.18
47.45


879
HEPTANEDIOIC ACID, BIS-TMS- ESTER
259
905
1.33
355.68


903
PARA-HYDROXY BENZOIC DI-TMS
202
868
0.45
119.54


913
PARA-HYDROXYPHENYLACETIC ACID-DI-TMS
1485
927
0.13
35.95


925
PARA-HYDROXYPHENYLACETIC ACID-DI-TMS
1485
835
13.82
3707.87


930
938, DQ3041
2164
757
0.10
28.08


975
975
0
0
1.18
316.77


986
985, JZ4021
2318
899
0.29
78.99


991
991
0
0
0.15
38.94


1001
OCTANEDIOIC ACID, BIS-TMS-ESTER
306
744
0.36
95.83


1087
HOMOVANILLIC ACID DI-TMS
331
946
2.49
667.03


1103
1104, JZ4091
2369
930
0.43
114.58


1116
1116
0
0
0.53
142.93


1125
1112, M20021
1823
763
4.82
1292.51


1146
HIPPORIC ACID TMS ESTER
103
903
1.02
273.29


1184
1189, JZ4051
2322
954
0.31
82.08


1192
1189, JZ4051
2322
890
0.33
89.21


1200
1189, NU3061
2118
705
0.72
194.06


1211
1189, NU3061
2118
704
5.65
1515.93


1234
L-GLYTAMIC ACID, N-ACETYL-N-TMS-, BIS-TMS EST
587
494
3.37
902.66


1243
P-HYDROXYPHENYL, LACTIC ACID, TRI-TMS
578
951
0.75
201.16


1259
PROPANEDIOIC ACID, TMS-OXY-, BIS-TMS ESTER
594
238
0.52
139.80


1273
HYDROXY PROLINE DI-TMS
1610
349
0.17
46.73


1280
IH-INDOLE-2-CARBOXYLIC ACID, 5-ETHYL-1-TMS-
343
646
0.29
76.62


1289
991, JZ4101
2372
460
1.53
409.12


1332
1332
0
0
0.13
35.00


1354
1354
0
0
0.13
35.22


1364
MANNO-ONIC ACID, LACTONE TETRA-TMS
732
454
0.30
81.30


1371
PALMITIC ACID TMS
335
670
0.91
245.18


1414
1481, NU3091
2124
464
0.60
160.27


1426
SILANE, TRIMETHYL 3-PHENYLPROPOXY-
1158
500
0.19
50.80


1451
BETA AMINO BUTYRIC ACID DI-TMS
89
761
0.22
58.41


1481
TRYPIOPHAN TRI-TMS
1965
477
0.55
146.22


1486
1472, VST031
2031
771
4.74
1271.10


1509
5-HYDROXY INDOLE ACETIC ACID TRI-TMS
592
943
3.19
856.94


1520
STEARIC ACID TMS
434
787
0.14
36.29


1573
6-HYDROXY-HEPTANOIC DI-TMS
1690
275
0.30
79.25


1596
PSEUDO URIDINE PENTA-TMS
1779
746
5.92
1587.71


1628
1472, VST031
2031
799
0.26
69.56


1641
1631, M15041
1802
826
0.87
234.00


1673
1472, VST031
2031
650
1.73
464.94


1680
1676, JD2011
2001
624
0.33
87.91


1746
SUCROSE OCTA-TMS
1080
847
0.31
83.08





*The named compound matches the sample peak with a reliability given by “FIT”/1000.













TABLE 37







QUANTIFIED TARGET PANEL URINE ORGANIC COMPOUNDS


FRACTION X, BEAR URINE JZ4111:8










mM/M
Nml



CREATININE
Range













Organic Acids




LACTIC ACID
19433
 0–75


PYRUVIC ACID
950
 0–20


GLYCOLIC ACID
196
 0–50


ALPHA-OH-BUTYRIC
14.8
0–1


OXALIC
36.0
 0–25


4-OH-BUTYRIC
0.0
0–1


HEXANOIC ACID
60.0
 0–11


5-HYDROXYCAPROIC
12.6
0–1


OCTANOIC
37.4
0–1


BETA-LACTATE
234.1
0–8


SUCCINIC ACID
135
 0–20


GLUTARIC ACID
0.0
0–2


2-OXO-GLUTARATE
0
 0–210


FUMARIC
21.9
0–5


MALEIC
0.0
0


MALIC ACID
18.8
0–2


ADIPIC ACID
30.4
0–7


SUBERIC ACID
4707.2
 0–11


SEBACIC ACID
3.0
0–2


GLYCERIC ACID
30
0–4


BETA-OH-BUTYRIC
321
0–3


METHYLSUCCINIC
0.0
0


METHYLMALONIC
0
0–5


ETHYLMALONI
103.0
0–4


HOMOGENTISIC ACID
0.0
0–1


PHENYLPYRUVIC ACID
347.5
0–1


SUCCINYLACETONE
2.2
0–1


3-OH-ISOVALERIC
1.8
 0–21


PHOSPHATE
814
  0–3000


CITRIC ACID
46
 0–450


HIPPURIC ACID
5949
  0–2000


URIC ACID
40
 0–360


Nutritionals


KYNURENIC ACID
6.2


FORMIMINOGLUTAMIC
0.60
0–3


4-PYRIDOXIC ACID
0.0
0–9


PANTOTHENIC ACID
3
 0–30


XANTHURENIC ACID
2.6
0–1


KYNURENINE
70.3
0–1


QUINOLINIC
0.0
0–6


OROTIC ACID
28.54
0–3


D-AM LEVULINIC
541.3
 0–18


3-METHYL HISTIDINE
216
 0–75


NIACINAMIDE
62.7
0–1


PSEUDOURIDINE
10351
 10–220


2-DEOXYTETRONIC
41
 0–75


P-HO-PHEN-ACETIC
254
 0–12


XANTHINE
14
 0–18


UROCANIC ACID
255
0–3


ASCORBIC ACID
1
 0–160


GLYCEROL
11477
0–9


Carbohydrates


THREITOL
7
 0–40


ERYTHRITOL
7
 0–55


ARABINOSE
25
 0–30


FUCOSE
379.6
 0–12


RIBOSE
219.1
 0–12


XYLOSE
8
 0–70


FRUCTOSE
808
 0–115


GLUCOSE
432
 0–110


GALACTOSE
19
 0–200


MANNOSE
406
 0–70


N-AC-GLUCOSAMINE
28.8
0–3


LACTOSE
349
 0–60


MALTOSE
237
 0–40


XYLITOL
27.6
 0–15


ARABINITOL
16.0
 0–30


RIBITOL
0.0
 0–10


ALLOSE
61.7
 0–10


GLUCURONIC ACID
239.8
 0–50


GALACTONIC ACID
400
 0–60


GLUCONIC ACID
11.2
 0–35


GLUCARIC
9.0
0–5


MANNITOL
31.5
 0–15


DULCITOL
10.6
 0–10


SORBITOL
55.4
 0–10


INOSITOL
13.6
 0–12


SUCROSE
1788
 0–75


Neurotransmitters


GABA
24.8
0–1


HOMOVANILLIC ACID
11673.5
 0–10


NORMETANEPHRINE
17.0
0–1


VANILLYLMANDELIC
2.6
0–6


METANEPHRINE
3.1
0–2


5-HIAA
1026.9
0–6


MHPG
1.2
0–1


ETHANOLAMINE
679
10–90


Amino Acids and Glycine Conjugates


PROPIONYL GLY
16.6
0–1


BUTYRYL GLYCINE
0.0
0–1


HEXANOL GLYCINE
444.9
0–1


PHENYL PROP GLY
243.3
0–1


SUBERYL GLYCINE
4.4
0–1


ISOVALERYL GLY
144.3
0–1


TIGLY GLY
5.7
0–1


BETA MET CROT GLY
353.8
0–1


GLYCINE
2601
 0–500


ALANINE
1316
 0–130


SARCOSINE
15.4
0–8


BETA-ALANINE
31.3
0–2


B-AMINOISOBUTYRIC
538
 0–50


SERINE
2443
 0–85


PROLINE
244.2
0–8


HYDROXY PROLINE
3372
 0–75


HYDROXY LYSINE
127.6
0–1


ASPARTIC ACID
499.6
0–2


ASPARAGINE
0.2
0–2


N-AC ASPARTIC
13.5
 0–20


ORNITHINE
442.4
0–5


GLUTAMIC ACID
6.0
0–6


GLUTAMINE
220
 0–210


PIPECOLIC ACID
0.4
0–1


LEUCINE
337.8
0–9


KETO LEUCINE
1066.2
0–1


VALINE
417.4
 0–18


KETO-VALINE
1.7
0–1


ISOLEUCINE
274.6
0–5


KETO-ISOLEUCINE
80.6
0–1


LYSINE
2599
 0–35


HISTIDINE
203
 0–225


THREONINE
377
 0–45


HOMOSERINE
0.0
0–1


METHIONINE
20.8
0–3


CYSTEINE
3059
 0–160


HOMECYSTEINE
1.0
0–1


CYSTATHIONINE
5.6
0–1


HOMOCYSTINE
59.7
0–1


CYSTINE
9.4
0–5


PHENYLALANINE
233
 0–20


TYROSINE
190
 0–22


TRYPTOPHAN
130
 0–25





This sample contained 0.03 uMoles Creatinine/100 ml.













TABLE 38







METABOLIC SCREENING LABORATORY URINE ORGANIC CONSTITUENTS


FRACTION X, BEAR URINE JZ4111


CONCENTRATION: THIS SAMPLE CONTAINED 0.03 uM CREATININE/mL












PEAK

LIB
FIT
AREA
AREA


#
CONSTITUENT'S BEST MATCH FROM LIBRARY*
ENTRY
vs 1000
%
OF CREAT















6
6, JI4081
2189
675
0.71
314.00


9
10, STN031
1893
719
0.65
288.12


12
13, JZ4051
2321
561
0.48
215.50


20
10, M13011
1782
719
2.07
921.84


36
35, JZ4011
2300
847
0.22
97.76


51
42, M20021
1816
726
0.19
83.08


59
49, AK2011
2047
833
0.19
83.56


68
SILANE, TRIMETHYLPHENOXY-
1122
847
0.73
324.60


72
ETHYL AMINE DI-TMS
22
513
2.08
923.09


80
LACTIC ACID DI-TMS
1510
874
1.34
594.49


88
BORATE TRI-TMS
186
618
0.06
26.27


108
107, JZ4011
2301
847
0.20
90.08


118
104, NJ3031
2131
744
2.49
1108.84


123
119, JQ4011
2243
907
0.30
131.95


166
SILANOL, TRIMETHYL-, CARBONATE 2:1
1429
647
0.07
32.24


186
BETA-LACTATE DI-TMS
1654
781
0.54
241.79


224
92, NA3011
2070
757
0.07
29.54


252
251, JZ4011
2302
848
0.09
39.70


294
4-METHYL 2-HYDROXY PETANOIC ACID DI-TMS
178
807
5.30
2356.51


297
2-HYDROXY HEXANOIC ACID DI-TMS
1682
786
3.49
1551.67


301
291, JZ4091
2368
775
1.56
693.60


336
ETHANOLAMINE TRI-TMS
181
907
0.13
59.44


349
PEAK 459, A02011
1855
511
0.06
26.28


365
TRIMETHYLSILYL ETHER OF GLYCEROL
273
824
1.90
844.99


386
TETRADECANOIC ACID TMS
251
510
0.12
52.53


398
GLYCINE TRI-TMS
1539
869
0.44
197.40


503
SERINE TRI-TMS
322
957
0.51
228.07


540
539, JZ4041
2320
886
0.37
166.09


613
613, JZ4101
2370
855
0.41
182.98


642
1364, JZ4011
2312
370
0.69
307.69


686
BENZENEACETIC ACID, .ALPHA. - -TMS-OXY -, TRIM
246
874
0.19
83.47


753
HEXANEDIOIC ACID, 3-METHYL- BIS-TMS- ESTER
258
758
1.53
678.67


773
SILANE, DIMETHYLPHENOXY TRIMETHYL-
1150
332
0.12
55.52


781
HEPANEDIOIC ACID, BIS-TMS- ESTER
259
624
0.14
60.31


798
METHYL D3 CREATININE TRI-TMS
1466
715
0.04
18.49


809
METHYL D3 CREATININE TRI-TMS
1466
707
4.53
2013.68


822
ORTHO-HYDROXYPHENYLACETIC ACID DI-TMS
247
907
1.04
460.14


856
2-HYDROXY 3-PHENYL PROPIONIC ACID DI-TMS
287
872
7.69
3420.08


880
HEPTANEDIOIC ACID, BIS-TMS- ESTER
259
866
0.95
420.88


907
PARA HYDROXY BENZOIC DI-TMS
202
873
4.41
1959.38


914
PARA-HYDROXYPHENYLACETIC ACID DI-TMS
1485
628
0.94
418.25


928
PARA-HYDROXYPHENYLACETIC ACID DI-TMS
1485
811
9.47
4211.72


938
1234, JZ4061
2333
444
0.07
32.28


946
HEXANOYL GLYCINE DI-TMS
1656
724
0.19
83.16


971
975, JZ4101
2371
813
0.23
100.98


976
975, JZ4101
2371
877
2.17
964.67


987
985, JZ4021
2318
756
0.18
81.73


992
991, JZ4101
2372
814
0.20
88.90


996
SUBERIC ACID DI-TMS
1633
520
0.05
21.95


1003
OCTANEDIOIC ACID, BIS-TMS- ESTER
306
726
2.12
940.43


1010
1062, NJ3051
2135
474
0.37
163.67


1015
561, LB1031 VALPROIC ACID METABOLITE, MSL
1973
527
0.55
246.28


1031
SILANE, TRIMETHYL PHENETHYLTHIO-
1161
389
0.23
102.67


1046
SEBACIC ACID, BIS-TMS- ESTER
393
612
0.36
160.75


1060
975, JZ4101
2371
704
0.04
19.97


1068
HYDROCINNAMIC ACID, P-TMS-, TRIMETHYLSILYL ES
288
688
0.28
126.21


1081
1160, JG4021
2179
315
0.37
164.16


1088
1062, NJ3051
2135
770
1.35
599.54


1095
1332, JZ4101
2374
598
0.39
172.38


1103
1104, JZ4091
2369
784
0.06
26.57


1116
1116, JZ4101
2373
861
0.86
382.04


1124
1112, M20021
1823
804
0.34
149.94


1133
877, JK4071
2237
414
0.28
125.70


1138
975, JZ4101
2371
386
0.41
181.50


1145
HIPPURIC ACID TMS ESTER
103
779
0.13
59.11


1157
ORNITHINE N5, N5 TETRA-TMS
1536
836
0.13
57.72


1164
FRUCTOSE PENTA-TMS
881
660
0.18
79.07


1169
TETRADECANOIC ACID TMS
251
789
0.17
75.71


1175
METHYL ALPHA-GLUCOSIDE TETRA-TMS
790
410
0.30
134.71


1187
24, AK2011
2045
508
0.23
103.04


1199
1189, JZ4051
2322
828
3.17
1408.37


1213
1189, NU3061
2118
676
6.41
2850.85


1222
SEBACIC ACID, BIS-TMS- ESTER
393
521
0.07
31.48


1227
META-HYDROXYPHENYL ACETIC ACID DI-TMS
248
274
0.21
91.70


1234
ACETIC ACID, PHENOXY-, TRIMETHYLSILYL ESTER
66
481
0.60
265.32


1255
GALACTOSE PENTA-TMS
878
571
0.69
304.74


1263
996, JZ4061
2329
391
0.08
37.07


1279
1H-INDOLE-2-CARBOXYLIC ACID, 5-ETHYL-1-TMS-,
343
445
0.11
49.11


1288
INDOLE 2-ACETIC ACID 1-TMS, TMS-ESTER
316
858
2.51
1117.19


1302
GL1021, 678
1964
451
0.32
140.03


1309
1H-INDOLE-3-ETHANAMINE, N, 1-BIS-TMS-5- TMS-OX
547
565
0.27
119.16


1334
3-HYDROXYTETRADECENEDIOIC ACID I
1708
420
0.13
59.54


1344
IH-1NDOLE-5-CARBOXYLIC ACID, 1-TMS-, TRIMETHY
266
441
0.38
170.74


1355
D-MANTMOPYRANOSE PENTA-TMS
892
905
0.43
192.76


1371
PALMITIC ACID TMS
335
892
0.77
340.90


1398
GALACTURONIC ACID PENTATMS
915
629
0.07
31.57


1406
1246, JZ4061
2334
434
0.24
108.11


1411
1032, M15041
1796
335
0.04
19.48


1423
988, NE3031
2088
407
0.13
57.19


1443
1300, JZ4071
2356
465
0.09
37.89


1455
DODECENEDIOIC ACID DI-TMS, CIS?
1695
433
0.07
31.96


1489
1472, VST031
2031
694
4.88
2167.24


1502
OLEIC ACID, TRIMETHYLSILYL ESTER.
1614
677
0.13
56.05


1509
5-HYDROXY INDOLE ACETIC ACID TRI-TMS
592
889
0.36
159.16


1520
STEARIC ACID TMS
434
728
0.55
244.65


1529
982, N03031
2142
405
0.12
53.30


1537
3-HYDROXYDODECANEDIOIC ACID-TMS-3
1776
708
0.05
20.19


1546
996, G11021
1958
448
0.27
118.50


1558
HEPTANEDIOIC ACID, 4-OXO-, BIS-TMS ESTER
305
381
0.12
54.63


1562
1472, VST031
2031
635
0.07
32.52


1596
PSEUDO URIDINE PENTA-TMS
1779
690
2.10
933.44


1603
988, OK1041
1990
574
0.09
40.28


1609
1472, VST031
2031
552
0.04
19.08


1612
251, JZ4011
2302
365
0.06
24.80


1620
D-GALACTOSE, 2-AMINO-2-DEOXY-3, 4, 5, 6-TETRAKIS
746
406
0.07
33.22


1628
1472, VST031
2031
729
0.55
246.19


1652
1472, VST031
2031
713
0.14
62.64


1664
1631, M15041
1802
567
0.09
41.81


1674
1669, P17031
1984
687
2.27
1011.28


1680
1472, VST031
2031
463
0.08
33.58


1686
1189, JZ4051
2322
252
0.06
25.53


1692
1073, RT1051
2040
395
0.05
22.18


1701
2-HYDROXYTETRADECENEDIOIC ACID
1704
385
0.08
36.13


1728
533, LB1031 VALPROIC ACID METABOLITE, MSL
1972
409
0.04
19.96


1746
SUCROSE OCTA-TMS
1080
888
0.73
324.31


1795
LACTOSE OCTA-TMS
1854
785
0.08
36.36


1839
1785, YD1011
1875
414
0.06
25.81





*The named compound matches the sample peak with a reliability given by “FIT”/1000






Further Purification of MNC in Fraction VI Using HPLC

Fraction VI was further purified using HPLC. After lyophilization and reconstitution in methanol, aliquots of Fraction VI were loaded onto a HPLC using a C18 column. A gradient of 0.1M ammonium formate and a 9:1 mixture of acetonitrile/water was the solvent system used for further separation of Fraction VI. Four peaks were visualized using a UV-Vis detector. Based on the increased absorbance at 220 nm, 230 nm, and 280 nm, four fractions were collected.
Peak 3 was further purified by HPLC using an isocratic solvent system. A representative tracing from HPLC of repetitive injections of Peak 3 recorded at wavelengths of 220 nm, 230 nm, and 280 nm. Both peaks were collected and labeled as 3A and 3B respectively.
Peak 4 was further purified by HPLC using a gradient system. It was detected by increased UV absorbance readings at 220 nm, 230 nm, and 280 nm. Peak 4 was separated into two peaks and collected as Fractions 4A and 4B.
Submission of HPLC Fractions for Analysis by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS)
Fractions labeled as 3A and 3B were submitted to NMR and MS using chemical ionization and electron ionization. The molecular weight of Fraction 3A is estimated to be 279. Interpretation of the NMR spectra suggests a phenolic compound.
Fraction 3B has a molecular weight of 209 with an empirical formula consisting of C10H11NO4. The substance para-hydroxyphenylacetylglycine has a similar molecular weight of 209. However, NMR data do not support the theory that para-hydroxyphenylacetylglycine exists in the MNC complex. An ester structure found by NMR in the MNC complex is not found in the structure of para-hydroxyphenylacetylglycine. Also, para-hydroxyphenylacetylglycine has been only detected in Fraction VI.
Data from NMR support the conclusion that Peak 4 contains both an indole structure and a phenol structure.
Summary
1. MNC from Fraction VI has been further purified using gradient and isocratic HPLC into compounds 1, 2, 3A, 3B, 4A, and 4B.
2. The molecular weight of compound 3B is known at 209 (C10H11NO4).
3. One structure with a molecular weight of 209 has been found in Fraction VI. It has been identified as para-hydroxyphenylacetylglycine.
4. However, a unique compound with a phenylester structure and having an empirical formula of C10OH11NO4 best corresponds to the data accumulated from NMR.
5. Thus, a unique substance (which is part of the MNC complex associated only with the denning phenomenon) is found in Fraction VI. This unique substance also contains significant biopotential for stimulation of osteoblasts.
ANTICIPATED TREATMENT RESULTS
Based upon studies with guinea pigs, bone cultures, black bears, and polar bears, the anticipated results of BDI treatment in humans follow.
Osteoporosis
Successful treatment of females or males suffering from osteoporosis or prevention of bone loss in them or in astronauts will be due to stimulation of osteoblasts (the cells that form bone), inhibition of resorption activity of osteoclasts, or simultaneous effects of osteoblasts and osteoclasts.
Thus, BDI becomes a potent, naturally occurring component to not only prevent osteoporosis but to increase size and strength of bone and successfully treat the debilitating condition of osteoporosis.
These changes may be evaluated by a general medical examination and optional diagnostic evaluations including radiographic assessment, measurement of the density of vertebral and other bones, prevention of bone fractures, and special assessment of skeletal remodeling activity.
Kidney Disease
Patients with chronic kidney disease or end stage renal failure may be treated so that the recycling of excess urea back into protein would result in the symptoms of kidney failure being reduced or abolished, to the extent that dialysis or kidney transplantation would not be needed.
Burns and Trauma
The prevention of excessive loss of protein from non-involved muscle and other tissues would treat patients with severe bums and trauma.
Muscle Atrophy
This treatment may maintain muscle mass in humans as they age and may prevent loss of muscle tissue in astronauts.
Obesity and Other Eating Disorders
The interfacing of increasing deposition of healthy lean tissue while eating less would have a pronounced favorable effect on the treatment of obesity in human beings. When the effective dose of BDI is adjusted for safety and to a degree that it promotes less food intake to a point of complete absence while preserving lean tissues, treatment of one of the most resistant disorders of human beings may be accomplished.
An anticipated treatment result, based on studies of hyperphagic black bears, would be to stimulate food intake in humans suffering from poor food intake such as anorexia nervosa.
General Health
In humans, the overall effects of BDI are expected to enhance general health while substantially reducing cost of health care.
PREDICTABILITY AND CORRELATABILITY OF COMPARABLE RESULTS IN HUMANS
While in vivo tests have not been made with regard to bone remodeling by the bear derived isolate of claim 1, in vitro tests have been done. Such in vitro tests are set forth in a recent April 1994 draft publication by the FDA. The publication is entitled “Guidelines for Pre-Clinical and Clinical Evaluation of Agents Used in the Prevention or Treatment of Post Menopausal Osteoporosis”. The draft was prepared by The Division of Metabolism and Endocrine Drug Products of the FDA, as indicated in April of 1994. The following shows a comparison between the guidelines (Page 4, Section IV) and results achieved with BDI.











Suggested FDA
BDI Test


Guidelines
Results


















1.
At least one biochemical
1.
BDI isolated from summer fasting urine



marker of bone resorption.

inhibits the production of tartrate





resistant acid phosphatase in mouse





calvaria organ cultures. Tartrate





resistant acid phosphatase is produced





by osteoclasts and serves as a sign of





bone resorption (Lau, et al., 1987;





Delmas, 1988).


2.
At least one biochemical
2.
When added to an organ (bone) culture



marker for bone formation.

of mouse calvaria, BDI isolated from





winter denning urine or from summer





fasting urine produced a statistically





significant production of alkaline





phosphatase which represents





stimulation of osteoblasts (Aurback,





Marx, et al., 1992; Delmas, 1988, 1993;





Mundy, Roodman, 1991; Parviainen,





Pirskanen, 1991; Stein, Lian, 1990,





1993; Quarles, Yokay, et al., 1992).


3.
That alkaline phosphatase
3.
When BDI was broken down into ten



is the suggested biochemical

individual fractions, fractions V, VI,



marker for bone formation.

and VII proved to be the most potent in





stimulating statistically significant





production of alkaline phosphatase by





osteoblasts located in the bone of





mouse calvaria.


4.
A suggested biochemical
4.
Rather than using an indirect method to



marker of bone resorption

assess bone resorption, our studies have



is urinary pyridinium crosslinks.

shown that BDI inhibits resorption in





two ways - the conversions of bone





marrow monocytes into osteoclasts, and





by the inhibition of osteoclasts already





functioning in bone resorptive cavities.


5.
Measurement of serum osteocalcin



(a specific marker of bone formation)



is encouraged.









The foregoing results confirm in vitro bone remodeling consistent with the FDA outlined guidelines. Ongoing in vivo studies have confirmed the following.












Pre-Clinical in vivo Studies










1.
Study conducted in an
1.
When compared with the untreated,



in vivo model such as the

osteoporotic ovariectomized rat,



ovariectomized, osteoporotic rat.

ovariectomized rats that had been





treated with DBI showed a 16-fold





increase in bone mineral density of





the femoral bone and a 4-fold





increase in the vertebral bones





when compared with bone mineral





density of humans receiving





therapeutic estrogen therapy over





the same or trial period.


2.
Histomorphometry or measurement
2.
Histomorphometry of the femoral



of serum osteocalcium (a specific

and vertebral bones from the DBI



marker of bone formation) is

treated, ovariectomized,



encouraged.

osteoporatic rats is now underway.









In addition, the subject matter of claim 1 has the ability to modulate the urea to creatinine ratio in urine of the guinea pig to values of 10 or less. Thus, tests were affirmative, and indicative of an increased ability of the guinea pig to recycle urea (Table 16). Bone mineral density in ovariectomized rats increased when those rats were treated with the subject matter of claim 1.
Nelson, Jones, et al. (1975) showed that urea is continually produced in the denning bear. Since the bear doesn't urinate, urea levels in blood, if unchecked, would result in high levels of urea (uremia) and death. Ahlquist, Nelson, et al. (1984) and Wolfe, Nelson et al. (1982, 1982a) showed that uremia is prevented by recycling the newly formed urea almost immediately back into protein from which it came. Nitrogen from urea was split off and attached to glycerol released from stored fat in adipose tissue. The newly formed amino acids were then incorporated in proteins such as albumin and fibrinogen.
Nelson, Beck, et al. (1984) showed that the rapid recycling of urea resulted in a decline of the level of urea in blood. When expressed as a ratio of urea to creatinine, the ratio decreased from 20 or more to less than 10. Such ratios were only found in denning bears who were not drinking or urinating. In catheterized urine specimens of denning bears, Nelson, Wahner, et al. (1973) showed when urea recycling was in process, the urea to creatinine ratio in urine was also reduced to values less than 10.
When BDI was injected into guinea pigs, urine U/C was decreased to values less than 10 indicative of similar urea recycling in guinea pigs as shown by denning bears. A strong indicator of suitability of bear originated materials for pharmacologic use in humans is the use of the bile salt produced by the bear, ursodeoxycholic acid (UDCA).

    
    
        1. UDCA is safe and effective therapy for patients with cholesterol gall stones (Rubin, Kowalski, et al., 1994).
        2. UDCA currently offers the best combination of efficacy and lack of side effects in treatment of primary biliary cirrhosis and reduces the need for liver transplants (Lim, Northfield 1994; Poupon, Poupon, et al., 1994).
        3. UDCA improves liver function in primary sclerosing cholangitis of the liver (Jazrawi, De Coestecker, et al., 1994).
        4. UDCA is a safe, well-tolerated, and efficacious treatment of refractory chronic graft versus host disease of the liver occurring in patients receiving bone marrow transplants (Fried, Murakawi, et al., 1992).
        5. UDCA is a bear derivative acceptable and approved to be administered to humans.
    
    


Accordingly, it is extrapolated that if one bear derivative is administered pharmaceutically to humans as a pharmacological product, another bear derivative will be similarly acceptable. This acceptability is reinforced by the cited tests with guinea pigs.
In summary, the conclusion reached after many years of study, observation of the phenomenon of bears, and predicated upon numerous publications set forth in the bibliography filed with this application, the predictability and correlatability to comparable results when administered to humans is present within the confines of the current disclosure.
OTHER INVESTIGATIONS
In addition to those described, investigations relating the close proximity of the BDI isolate with other normally appearing metabolic substances suggests that they are required to achieve action. Thus, BDI, the bear derived isolate alone, may require other metabolites to exert its action. Further portions of the entirety of the isolate may be combined or absorbed into these substances to exert action. This equivalency may be a function of these interactions and substantially produce the same result.
Summary of Present Discovery and Areas for Further Research
Already achieved as set forth above is the discovery of how the bear forms bone, even though existing in a state similar to post-menopausal women. The discovery reveals that BDI inhibits bone resorption by inhibiting the maturation of osteoclasts from bone marrow monocytes and by directly inhibiting functioning osteoclasts. The discovery has confirmed that a unique feature of BDI is that rather than inhibiting osteoblasts as current drugs do (and thus reducing bone production), BDI independently stimulates osteoblasts to form bone. Even though the bear inhibits osteoclasts, at the same time it independently stimulates osteoblasts to form bone. This novel, unique approach of direct osteoblast stimulation by BDI has been shown in cell and organ bone cultures. When current drugs on the market inhibit bone resorption by osteoclasts, osteoblast numbers and activity are also inhibited. BDI's unique abiltiy to directly stimulate osteoblastic proliferation is demonstrated. Moreover, BDI directly stimulates fibroblastic activity which involves the matrix formation and production of bone stimulating factors. Again, no drugs on the market have this action. Finally, BDI stimulates bone formation in the ovariectomized rat, a model similar to post-menopausal women.
GC/MS has established the identifiable ingredients present in BDI. Using countercurrent chromatography (CCC), fractions were developed that separated BDI into semi-purified fractional components that affect osteoblasts, osteoclasts, and fibroblasts. These discoveries include the potent Fractions V, VI, and VII that stimulate osteoblast and fibroblast proliferation and bone formation by osteoblasts. This is to the exclusion of the inhibition of osteoblastic activity of BDI found in Fraction III. Moreover, the discoveries of the constitutents of Fractions V, VI, and VII by first producing them by CCC and then by determining their composition and concentration by GC/MS has led to further investigations. This includes the fact that bone resorption inhibiting activity of BDI is found mainly in the first three fractions of BDI as produced by CCC. Also, Fraction III inhibits osteoblasts directly.
Additionally, the potency of Fractions V, VI, and VII on forming bone in the osteoporotic rat can be calculated from the in vivo rat studies, the in vitro organ cultures of mouse calvarial bone and the cell cultures of osteoblasts.
Future Investigations
What is thus required is the following:
The combined potency of Fractions V, VI, and VII of BDI needs to be determined. This may result in the discovery of a unique substance that orchestrates all of the bone forming activity of BDI or in the fact that BDI represents a novel and unique combination of previously known as well as recently discovered new compounds. This substance or combination will be tested using in vitro and in vivo methods. This novel and unique substance or combination of substances will be synthesized and tested for bone forming activity in a model of the post-menopausal human, ovariectomized rats.
Other Bear Species
The effects of BDI as related to urea recycling extend from the black bear to include grizzly and polar bears. Both of these species demonstrate urea recycling as shown by a low blood urea to creatinine ratio when not drinking water or eating snow. No other mammal has this ability. If not drinking water, or if water is withheld, all other animals show an increase in blood urea and dehydration. Their urea to creatinine ratio rises above 20 and death will occur if water is not taken. Because of the effective urea recycling process, when not drinking or eating, black, grizzly, and polar bears protect their lean body mass, behave normally, and can be physically active. Since BDI induces denning phenomenon in guinea pigs (including urea recycling), BDI can be predicted to be similar in effects if obtained from urine or blood from grizzly or polar bears.
SCOPE OF THE INVENTION
It will be understood that within the scope of the invention as expressed in the appended claims, various changes in the details and materials which have been herein described and illustrated in order to explain the nature of the invention, may be made by those skilled in the art within the principle and scope of the invention as expressed in the appended claims.

## Claims

1. An anti-osteoclastic pharmaceutical composition of matter having the characteristics of the deproteinated urine or blood serum isolate of fasting bear which bear has not eaten for two weeks or more, which, when administered to a mammal other than a denning black bear, exhibits overall enhanced bone formation whether by enhanced osteoblastic activity, or diminished osteoclastic activity, or enhanced fibroblastic activity, or any positive combination of the foregoing, wherein the net result is enhanced bone remodeling.

2. An ursus-like pharmacological substance which is the deproteinated isolate of the urine or blood of a fasting bear which, when injected into a mammal other than a bear, produces phenomena as exhibited in a denning black bear which bear neither eats, drinks, urinates, nor defecates for lengthy periods of time, said phenomena including stimulation of bone production in mammals, including humans, at risk to develop osteoporosis, regeneration of protein from nitrogenous waste products at a rate faster than protein breakdown, and producing anorexia.

3. A composition of matter being an ursus-like pharmacological isolate having the characteristics of a urine sample concentrate taken from a fasting bear, which bear has not eaten for two weeks or more, which urine sample concentrate remains after deproteinating such sample and thereafter purifying the same by chromatographic treatment.

4. A composition of matter according to claim 1 comprising the deproteinated urine or serum of a fasting bear which has not had food for two weeks or more and capable of producing when injected in a guinea pig:
enhanced bone remodeling.

5. A composition of matter according to claim 1 comprising the deproteinated urine or serum of a fasting bear which has not had food for two weeks or more, and capable of producing when injected in an ovariectomized rat:
enhanced bone formation.

6. A pharmaceutical composition according to claim 1 for stimulating osteoblastic activity as shown by alkaline phosphatase production, said composition comprising an active agent obtained by the steps comprising:
(a) obtaining the serum or urine of a fasting bear;
(b) deproteinating said serum or urine;
(c) drying said deproteinated serum or urine;
(d) separating the product of step (c) into fractions by chromatography,
(e) drying the fractions obtained in step (d);
(f) testing the fractions for alkaline phosphatase stimulating activity in an in vitro bone culture.

7. A pharmaceutical composition according to claim 1 for stimulating osteoblastic activity as shown by alkaline phosphatase production, said composition comprising an active agent obtained by the steps comprising:
(a) obtaining the serum or urine of a fasting bear;
(b) deproteinating said serum or urine;
(c) drying said deproteinated serum or urine;
(d) (1) separating the product of step (c) into fractions by means of countercurrent chromatography using a 1-butanol:water:acetic acid (20:20:1) mixture, wherein the organic phase of said mixture is used as a stationary phase and the aqueous phase of said mixture is used as a mobile phase, wherein the first 100 ml eluted is Fraction I and each successive 100 ml to be eluted is a subsequent Fraction and continuing step (d) (1) up to the collection of Fraction VI.

8. A pharmaceutical composition as in claim 7, wherein the aqueous phase of a 1-butanol:water:acetic acid (20:20:1) mixture as a mobile phase is passed through the product of step (c) at a rate of 4 ml/minute for 25 minutes for each of Fractions I thorough VI.

9. A pharmaceutical composition as in claim 7, wherein said composition containing an active agent is obtained by the further steps comprising:
(d) (2) after collection of Fraction VI, collecting Fractions VII and VIII by passing the aqueous phase of said 1-butanol:water:acetic acid (20:20:1) mixture as a mobile phase through the product of step (c) remaining after step (d) (1) at a rate of 10-ml/minute for 10 minutes for each of Fractions VII and VIII.

10. A pharmaceutical composition as in claim 9, wherein said composition containing an active agent is obtained by the further steps comprising:
(d)(3) after collection of Fractions VII and VIII, collecting Fraction IX by replacing the 1-butanol:water:acetic acid (20:20:1) mixture with methanol:water (1:1) and passing the mobile phase thorough the product of step (c) remaining after step (d) (2) at a 10 ml/minute for 10 minutes for collection of Fraction IX.

11. A pharmaceutical composition as in claim 10, wherein said composition containing an active agent is obtained by the further steps comprising:
(d)(4) after collection of Fraction IX, collecting Fraction X, by replacing the 1:1 methanol:water mixture with methanol and passing the mobile phase through the product of step (c) remaining after step (d) (3) at a rate of 10 ml/minute for 10 minutes followed by forced air for collection of Fraction X.

12. A method for regulating bone remodeling comprising:
(a) obtaining the serum or urine of a fasting bear;
(b) deproteinating said serum or urine;
(c) drying said deproteinated serum or urine;
(d) separating the product of step (c) into fractions by countercurrent chromatography;
(e) during the fractions obtained in step (d);
(f) testing the fractions for osteoblast activity as shown by alkaline phosphatase production;
(g) exposing the bone to be regulated to an effective amount of a fraction having osteoblast activity as shown by stimulation of alkaline phosphatase.

13. A pharmaceutical composition according to claim 1 for inhibiting osteoblastic activity as shown by alkaline phosphatase production, said composition comprising an active agent obtained by the steps comprising:
(a) obtaining the serum or urine of a fasting bear;
(b) deproteinating said serum or urine;
(c) drying said deproteinated serum or urine;
(d) separating the product of step (c) into fractions by chromatography;
(e) drying the fractions obtained in step (d);
(f) testing the fractions for osteoblastic inhibition as evidenced by alkaline phosphatase inhibition in an in vitro bone culture.

14. A composition according to claim 1 functioning to reduce osteoblastic alkaline phosphatase comprising at least one active compound extracted from the serum or urine of a fasting bear, said at least one active substance being capable of functioning as an inhibitor of osteoblastic activity as shown by diminution of alkaline phosphatase production.

15. A composition according to claim 1 functioning to reduce osteoclasts as demonstrated by a reduction in production of tartrate resistant acid phosphatase comprising at least one active compound extracted from the serum or urine of a fasting bear, said at least one active substance being capable of functioning as an inhibitor of osteoclastic activity as shown by diminution of tartrate resistant acid phosphatase.

16. A pharmaceutical composition according to claim 1 comprising deproteinated whole urine or blood taken from a denning black bear combined with a pharmaceutical carrier, wherein said bear neither eats, drinks, urinates, or defecates for lengthy periods of time wherein said composition has the following properties:
soluble in water, methanol, and 1-butanol,
insoluble in less polar organic solvents including ethyl acetate, chloroform, toluene and hexane,
stable at room temperature for four days or more,
heat resistant to 65° C., and
stable when frozen in a light resistant container under nitrogen gas, and wherein said composition is an effective amount to inhibit osteoclast activity and/or stimulate osteoblast activity.

17. The pharmaceutical composition of claim 16, wherein said composition gives a pink spot with ninhydrin at an Rf value of 0.74 to 0.80 on a silica plate with 1-butanol:acetic acid:water (4:4:1).