METHOD OF LOWERING BLOOD SUGAR IN VERTEBRATES

BACKGROUND OF THE INVENTION

1. Field of the invention

This invention relates to the lowering of blood sugar level in vertebrates. It relates to the com¬ pounds that can be used to increase release of insu¬ lin by the beta cells of the islets of Langerhans. This occurs in diabetes mellitus (Type II) when blood sugar values rise above normal levels, i.e., hyper lycemia.

The invention is directed to providing pharma¬ ceutical preparations that will increase release of insulin.

Diabetes is a very common disease, occurring in all age groups. However, the late onset diabetes (Type II) results from inadequate insulin release and becomes increasingly common the older the population becomes.

UBSTITUTE SHEET

2 _α Prior Art ²

"Monoa ine oxidase is a flavoprotein oxidase of pur¬ ported CENTRAL METABOLIC IMPORTANCE CONVERTING NEUROACTIVE AMINES INTO INACTIVE ALDEHYDES The lavin linked mono¬ amine oxidase is localized in the OUTER MITOCHONDRIAL MEM¬ BRANE OF ANIMAL CELLS. Walsh pp. -02, -03.

"Actions: Monoamine oxidase is a complex enzyme sys¬ tem widely distributed throughout the body. Drugs that in¬ hibit monoamine oxidase in the laboratory are associated with a number of clinical effects. Thus, it is UNKNOWN WHETHER MAO INHIBITOR PER SE, OTHER PHARMACOLOGICAL ACTIONS, OR AN INTERACTION OF BOTH IS responsible for the clinical effects observed. Therefore, the physician should become familiar with all the ef ects produced by drugs in this class. PDR (Physicians' Desk Reference 1983) p, 1516.

Two classifications of a ine oxidases were presented in 1959. That of Blashko, et al used the response to carbo- nyl inhibitors to distinguish between the activities of the various a ine oxidase. That of Zeller, et al, used semi- carbazide inhibitors. The use of inhibitors to classify the amine oxidases reflected difficulties encountered in purifying these enzymes and studying the structure of their active sites.

"A. Occurence Monoamine oxidase (MAO) has been found in all classes of vertebrates so far examined (1970) : mammals, birds, reptiles, amphibians and teleosts (l6l)„ The enzyme occurs in many different tissues, particularly in glands, plain muscle, and the nervous system (162).

CHEMICAL EFFECTS OF MONOAMINE OXIDASE

"SPECIFICITY

"The enzyme isolated from a number of sources exhibits low specificity. In general, primary, secondary, and tertiary amines, tryptamine derivatives and catechol- amines are oxidized (1,5) • The enzyme isolated from human placenta, however, will only attack primary amines and with simple alkyl amines increase in chain length results in increased affinity (7)_" Barman p. 180.

"Inhibition of MAO leads to a very pronounced increase in the levels of norepinephrine in the sympathetic nerv¬ ous system and of the monoa ines serotonin, norepine¬ phrine, and dopa ine in the monoamine-containing neurones of the CNS..„ .Large amounts of amine now accumulate in the cytoplasm. The storage sites rapidly become filled to capacity with the transmitter. This enhanced accumu¬ lation of neuroamines within the neurones is presumed to be the basis for the antidepressant action of the MAO inhibitors....It should be added that the presence in the urine of large amounts of unmetaboli-zed serotonin and 3-0- methylated catedholamines is characteristic of patients on MAO inhibitor antidepressants." Bevan pp_ 183 _» 18^.

These urinary compounds indicate clearance of the above amines from the blood and is consistent with an in¬ creased turnover rate of increased amounts of each amine.

"The flavoprotein responsible .for the oxidative deami- nation of the catecholamine (monoamine oxidase) is found in a wide variety of tissues and is located primarily in the outer membrane of mitochondria." Frisell p. 628.

HEET

CHEMICAL EFFECTS ON MONOAMINE OXIDASE

Halogenated compounds enter the body frequently from the environment_ The anaesthetics halothane and methoxy- flurane are cases in point.

"Incubation of the volatile general anaesthetics halothane or methoxyflurane (labelled with Cl) with hepatic micro- somes, NADPH, and oxygen is accompanied by extensive DECHLORINATIONo

"Similarly thyroxine and triiodothyronine undergo deiodi- nation by hepatic icrosomal enzymes (8)." Bacq p. 577•

"Dimino and Hoch (1972) found a considerable enrichment of iodine in liver mitochondria of rats injected with T^, . These mitochondria were more dense than those of untreated animals and appeared to contain iodine TIGHTLY BOUND TO THEIR INNER MEMBRANES (9). ...Direct effects of T^ on isolated mitochondria have been known for some time, but they occur only at HIGH, UNPHYSIOLOGICAL CONCENTRATIONS and their significance i ^' s doubtful, (9) _° " Lash p. 332.

"The actual biochemical mechanism of thyroid hormone action on neural tissue is poorly understood.";

"It is evident that a single regulatory reaction has not been found to explain the multiple effects of thyroid hormones<.

"Although the activities of more than 100 enzymes have been shown to be affected by thyroxine administration it appears that all are not influenced to the same degree. (10) ." Frisell p. βθδ.

MANGANESE METABOLISM

"The early studies of Greenberg (65) with radioman- ganese indicated only 3-^% of an orally administered dose is absorbed in rats ₀ The absorbed manganese quickly ap¬ peared in the bile and was excreted in the feces. Experi¬ ments since that time with several species including man indicate that manganese is almost totally excreted via the intestinal wall by several routes. These routes are inter¬ dependent and combine to provide the body with an efficient homeostatic mechanism regulating the manganese levels in the tissues (16,90,129). The relative stability of man¬ ganese concentrations in the tissues to which earlier ref¬ erence was made is due to such controlled excretion rather than to regulated absorption. (27)." Underwood p. 18^.

It is important to realize that each of these tis¬ sues in the intestinal tract are actually using the same system to take in and to dispose of manganese. Whis is being described above is the flow of manganese into mito¬ chondria and out again» It is a reflection of the mito- chondrial pool, which is a very labile pool. Manganese is carried in the plasma bound to protein. Very little of it is cleared by the kidneys.

"Injected radiomanganese disappears rapidly from the bloodstream (23,90). Borg and Cotzias (28) have re¬ solved this clearance into three phases. The first and fastest of these is identical to the CLEARANCE RATE OF OTHER SMALL IONS, SUGGESTING THE NORMAL TRANSCAPILLARY MOVEMENT, the second can be identified with the ENTRANCE

OF THE MANGANESE INTO THE MITOCHONDRIA OF THE. TISSUES, AND

THE THIRD AND SLOWEST COMPONENT COULD INDICATE THE RATE OF

NUCLEAR ACCUMULATION OF THE ELEMENT The kinetic patterns for blood clearnace and or liver uptake of manganese are almost identical indicating that the two manganese pools- BLOOD MANGANESE AND LIVER MITOCHONDRIAL MANGANESE - RAPIDLY ENTER EQUILIBRIUM. A high proportion of body manganese must, therefore, be in a dynamic mobile state. Underwood p. 185. e- .

"The turnover of parenterally administered ^J Mn has been directly related to the level of stable manganese in the diet of mice over a wide range (27)„ A linear rela¬ tionship between the rate of excretion of the tracer and the level of manganese in the diet was observed and the con- c-u. centra ion of ^ Mn in the tissues was directly related to the level of the stable manganese in the diet. THIS PRO^ VIDES FURTHER SUPPORT FOR THE CONTENTION THAT VARIABLE EXCRETION RATHER THAN VARIABLE ABSORPTION REGULATES THE CONCENTRATION OF THE METAL IN TISSUES." Underwood p. 185.

"Little is known of the mechanism of absorption of manganese from the gastrointestinal tract, or of the means by which excess dietary calcium and phosphorus reduce man¬ ganese availability....The effect of variations in dietary calcium and phosphorus on the metabolism of Mn in rats has been studied further by Lassiter and associates (100) . These workers found that the fecal excretion of parenteral- ly administered Mn was much higher and the liver reten¬ tion lower on a 1.0$ calcium diet than on a 0.6 - calcium diet.

dieto It appears, therefore, thaτ calcium can influence man¬ ganese metabolism by affecting retention of absorbed manganese as well as by affecting manganese absorption. Variations in dietary phosphorus had no comparable effects on the excretion of intraperitoneally administered ^ Mn, BUT THE ABSORPTION OF ORALLY ADMINISTERED ^Mn WAS IMPAIRED. Underwood. p„ 186.

During 1970 a rash of books drew attention to energized translocation or transport and to the changes in conformation of the membranes of the mitochondria. There were extensive correlations devised with the mitochondrial oxidative phos- phorylations. By 1975 some of this was discounted by claims that many solutes crossed the mitochondrial membrane without active transport. A number of postulates evolved including proton, phosphate and other mechanisms for these transfers.

In muscle and nervous tissue there are differences of sixty millivolts or more between the inner and outer surfaces of cell membranes. A Ca/Mg pump explains a wide variety of data. There seemed initially to be good data for high reso¬ nant phosphate compounds activating the cation pumps of mito¬ chondria. Such a pump is affected by changes in concentra¬ tion of calcium and it is also modulated by magnesium. Mn goes in and out of mitochondria readily. It dose so by active translocation and in the company of alkaline earth metal cations. Other metals participate but to a lesser degree. A Ca/Mg pump operating in tandem with Na/K ATPase pumps not only fits the cell membrane, but it also would have a place in the mitochondrial scheme of things.

It has long been suggested that mitochondria represent primitive bacteria originally ingested when cells developed phagocytic functions. The effective oxidation processes of the ingested cells are cited as the cause of the symbiosis developing. The corollary of that suggestion is the need that developed to correlate flow of high resonant compounds between the original cell and the mitochondriao This theory suggests that metabolic disease might well occur at the site of such a complex metabolic adjustment between the metabolism of two different cells_ This mechanism of.regulation is con¬ sistent with that theory.

The added point must be made that the high efficiency ascribed to mitochondria as sources of high resonant bonds highlights the need for a central control mechanis _ Such a mechanism must collate the energy production of the mitochon¬ dria with the energy metabolism of the cells, organs, and in¬ deed the entire organism. Calcium would seem a logical choice as the modulator of a system interactive between eu- karyotic cells and mitochondriao This is consistent with the present presentation.

This mechanism or system of control has been called a mechanism of regulation,. Listing the sequence of components described includes cation, ATPase pump, Mn, deiodinase, thy¬ roid hormones, monoamine oxidase and amines. ALL ARE FOUND IN CLOSE PROXIMITY IN THE MITOCHONDRIA.

The term tertiary structure refers to the manner in which the polypeptide chain is bent or folded to form compact, tightly folded structure of globular proteins (Fi ure 3-2). The more general term conformation is used to refer to the combined secondary and tertiary structure of the peptide chain in proteins. The term quaternary structure denotes the manner in which the individual polypeptide chains of a protein having more than one chain are arranged or clustered in space. Most larger proteins, whether fibrous or globular, contain two or more polypeptide chains, between which THERE MAY BE NO COVALENT LINKAGES (Fig. 2-2). In general, the polypeptide chains of proteins usually have between 100 to 300 amino acid units (mol wt 12,000 to 36,000). A few proteins have longer chains, such as serum albumin (about 550 residues) and myosin (about 1,800 residues) , However, any protein having a molecular weight exceeding 50,000 can be suspected to have two or more chains.

"Proteins possessing more than one chain are known as oligomerie proteins; theircoraponent chains are called protomers. A well-known example of an oligomerie protein is hemoglobin, which consists of four polypeptide chains, two identical alpha- chains and two identical beta-chains. Each chain has about l_ ₊ 0 amino acids. The four chains fit together tightly to form a globular assembly OF GREAT STABILITY, despite the fact that THERE ARE NO COVALENT LINKAGES. Oligomerie proteins usually contain an even number of peptide chains. .

There may be anywhere from two to twelve subunit chains among the smaller oligomerie roteins to dozens or even hundreds among the larger proteins. Tobacco mosaic virus particles have over 2,000 peptide chains.

"Since oligomerie proteins contain two or more polypeptide chains, which are usually not covalently attached to each other, it may appear oproper or at least ambiguous to refer to oligomerie proteins as "molecules" and to speak of their "molecular weight. Howwer, in most oligomerie profceinS the separate chains are so tightly associated that the complete particle usually behaves in solution like a simple molecule. Moreover, ALL THE COMPONENT CHAINS OR SUBDNITS OF 0LIG0MERIC PROTEINS ARE USUALLY NECESSARY FOR THEIR FUNCTIONS."

To bring this further into perspective in physiological terms and enable us to match structural details with observed changes in vital functions, we had best enlarge still further upon the "subunits'J" This is discussed on pp. 184.-185 of Lehninger as follows:

"This mechanism for hemoglobin oxygenation is directly applicable to regulatory enzymes. The binding of the first substrate molecule to one subunit of a ho otropic enzyme enhances the binding of a second substrate molecule to a second subunit because there is a conformational change in the first subunit which is transmitted nechanically or sterically to the second subunit. In all cases studied to date, regulatory enzymes have been found to be rather large molecules containing subunits; pres"Jπκ*EL , the existence of

interacting units is necessary for their function.

"Note that the term "subunit" IS AMBIGUOUS and may have TWO DIFFERENT MEANINGS WHEN APPLIED TO OLIGOMERIC PROTEINS. Hemoglobin contains four structural subunits or protomers, i.e., the two alpha and two beta chains, but two functional subunits, i.e., the two alphabeta half molecules. ISOZYMES

"Recent research has revealed another way in which the activity of some enzymes may be controlled THROUGH FEATURES OF THEIR MOLECULAR STRUCTURE. A number of different en¬ zymes have been found to exist in multiple molecular forms WITHIN A SINGLE SPECIES, or EVEN WITHIN A SINGLE CELL. Such multiple forms can be detected and separated by gel electrophoresis of cell extracts; they are therefore dis¬ tinct molecular species differing in net electrical charge_ Multiple forms within a single species or cell are called isozymes (or isoenzymes) .

"Lactate dehydrogenase, one of the first enzymes in this class to have been studied extensively, exists in five dif¬ ferent major forms, or isozymes, in the tissues of the rat (Figure 9-12) ■> These have been obtained in pure form. Al¬ though all five isozymes of lactate dehydrogenase catalyze the same reaction overall, they have DISTINCTLY DIFFERENT

Km VALUES for their substrates; the biological signifi- cance of these differences will be described in Chapter 15 and 18o The five isozymes all have the same particle weight, about 13^*000, and all contain four polypeptide chains, each of ol wt 33,5000.

Diabetes mellitus was named for its freα _^ uency of urination

(polyuria) by a Greek physician Aretaeus about 70 A.D. The disease was apparently written about by the ancient Chinese. It is a major cause of death throughout the world.

It has been described as due to inability to burn glucose.

It has been.explained as due to burning fat instead of sugar.

Organic acids accumulate to produce the acidosis characteristic of the disease. Not being able to burn all the fat needed to meet the needs of the body has been given as an explanation of the accumulation of acetoacatic acid, beta-hydroxybut^ric acid, and acetone when acidosis or ketosis develops.

Of course, these explanations are facile. In order to know which have merit, it would be necessary to identify the specific chemical reaction(s) that are abnormal in the disease.

In the cytosol, breakdown of glucose (glucolysis) produces pyruvate. Products of pyruvate enter the citrate synthase system in the tricarboxylic acid cycle of the mitochondria. Either oxaloacetate or 'active acetate ¹ are formed from pyruvate. These are the two compounds necessary to form citrate, "...the cycle is at the center of aerobic metabolism,. _{• •} " Frisβll, p. 530. It is the citrate synthase reaction that is slowest of all in the cycle. It is, therefore, the rate-limiting step. Such a reaction qualifies as a controlling reaction in a metabolic cycle.

Somewhere in this aerobic oxidation system exists the means of preserving the chemical balance of the many metabolic pathways necessary to maintain vital functions of the organism. Diabetes, then, can be defined as a disease in which proper proportions of products of the TCA cycle are not maintained. The question is where in the myriad possibilities for imbalance does the derangement in a chemical reaction occur which results, in diabetes mellitus?

Pyruvate from the breakdown of glucose provides oxaloacetate and acetyl CoA for the citrate synthase step of the Krebs cycle. There is a pool of each of these substrates. In addition, o course, there are pools of citrate molecules, alpha-ketoglutaratβ molecules and of molecules of succinyl CoA, succinate, fumarate, and alate.

The size of each of these pools is determined in part by the preceding enzyme substrate in the cycle, BUT ONLY IN PART.

For instance, the oxaloacetste pool is directly increased by the breakdown of asparagine and aspartic acid. The fumarate is increased by the degradation of the aromatic amino acids phenylalanine and tyrosine. The pyruvate from the breakdown of alanine, threonine, glycine, serine, and cysteine becomes available for formation of both the oxaloacetate pool and the acetyl CoA pool The great portion of the acetyl CoA pool, however, is derived from the breakdown of fatty acid chains by beta oxidation in the liτ_olytic cycle.

Besides the citrate synthetase or condensing enzyme, the tricarboxylate cycle has ^' some seven other enzyme reactions. Three different forms of tricarboxylic acid exist in the cycle. The first of these, citric acid, is formed by the citrate synthase system. Citric acid is part of a three member tautomeric isomerization, which includes aconitate and isocitric acid. The interconversions are assisted enzymatically by aconitase. Isocitrate is drained off by isocitrate dehydrogenase for forming of alpha-ketoglutarate. The latter in turn is converted into succinyl CoA by alpha- ketoglutarate .dehydrogenase.

The succinyl CoA is acted upon by succinyl CoA synthetase. While it is producing a molecule of guanosine triphosphate (GTP), succinyl CoA synthetase releases the succinic acid from the succinyl CoA. The resonance of the CoA sulfur bond is thus transferred to GDP in forming the GTP.

From the succinic acid two hydrogen atoms are removed by succinate dehydrogenzse to produce a double bond. The compound formed is fumarate with hydrogen atoms trans to one another at the double bond. A water molecule is added across the double bond. This is achieved by the activity of fumarate hydratase (fumarase) and forms mal&te. Finally, when two hydrogens are removed from malate by malate dehydrogenase we have oxaloacβtβte again and have completed a full cycle around the Krebs (TCA) cycle. We have traversed a tautomeric isomerizεtion with a hydratase and two dehydrogenases followed by a synthetase, -a. dehydrogenase, another hydratase and a final dehycrogenase.

"Amino acids whose carbon chains can be oxidized more or less directly to acetylCoA can also yield acetoacetate, a KETONE BODY. Formation of acetoacetate from such amino acids is particulsrly evident in fasting animals and accordingly, they are labeled KETOGENIC. These amino acids are lβucine, lysine, isoleucine, tryptophan, phenylalanine, and tyrosine. If we define a ketogenic acid as one WHOSE CARBON CHAIN CAN GIVE RISE ONLY TO ACETOACETATE, there are only two ketogenic amino acids - leucine ∑_nd lysine. The other four - isoleucine, tryptophan, phenylalanine, and tyrosine - can also give rise to glucose precursors and are, therefore, both ketogenic and glucogenic.

"Amino acids that can serve as precursors of phoβphoenolpyruvate (therefore, of glucose) are called GLYCOGENIC OR GLUCOGENIC. These amino acids are: alanine cysteine(ine) histidine serine arginine glutamate methionine threonine aspartate glutamine proline tryptophan asparagine glycine — hydroxyproline vsline" p. 2Λ.5 Frisell (1982)

We can realize from this that the acetyl CoA pool is a crossroads substrate for the TCA cycle and that it enters at the rate-limiting citrate synthase step in the cycle.

With this by way of introduction, we have two more substrates of the TCA cycle to consider. One of these is the alpha-ketoglutarate substrate and the other is its product from the action of alpha-ketoglutarate dehydrogenase. That product is succinyl CoA, the next substrate in the Krebs cycle.

When glutamate dehydrogenase is inhibited, less trans- amination occurs. This results in less breakdown of amino acids, those which have transa inase enzymes. Although this is true of the inhibition of the polymeric form of glutamate dehydrogenase, the monomeric forms proceed to oxidatively de- aminate the three branched chain amino acids. Deamination of the methionine chain presumably occurs at the L-homoserine hydro-lyase (deaminating) conversion to 2-oxo-butyrate. This enzyme is also named homoserine dehydratase with HOH being added and NH- and HOH being products of the reaction.. How¬ ever, methionine and also 2-aminobutyrate are listed as sub¬ strates of glutamate dehydrogenase ₉ presumably in the mono¬ meric form. This raises some question as to the route of degradation of methionine in its ultimate production of suc¬ cinyl CoA. The cytosol is a thick suspension. The matrix of the mitochondria may be an even thicker suspension with a very high protein content. In view of the monomers of glut¬ amate dehydrogenase occurring in such milieu, it would be sonsistent to think of methionine breakdown not involving the B-6 assisted step in this context„

Lysine and threonine are the two essential amino acids not transaminatedo In fact, they are not broken down readily„ Lysine is used for forming organic electrolytes, the poly- amines, and for the synthesis of carnitine, necessary for carnitine acyl transferase activity needed to trans er branched chain fatty acids into the mitochondriao

The overall effect of the above enzymatic steps pre¬ sumably would be to increase the demand for the branched

chain amino acids and succinyl CoA can assume prime importance in determining the overall rate of the TCA cycle. A number of substances are degraded to succinyl CoA. These include:

1. isoleucine and valine via methylmalonyl CoA;

2. branched chain fatty acids via propionyl CoA which in turn is changed into methylmalonyl Co;

3. methionine and tryptophan via alpha-ketoglutarate to the propionyl CoA„

The methylmalonyl CoA rearranges through- the action of a mutase to form succinyl CoA. This conversion requires B-12 for the enzyme to be active. The reaction on propionyl CoA itself requires biotin. ij-o pyrimidine breakdown products including those from thymine also contribute to the succinyl CoA pool_ The point has been made in detail before that the polymeric glutamate dehydrogenase feeds into this pool indirectly through the alpha-ketoglutarate (2-oxo-glutarate) _ When the polymeric form is inhibited, the monomeric forms increase breakdown.of the above amino acids to produce succinyl CoA as if replacing that which was lost when the polymeric form was inhibited.

SUPPLEMENTARY STATEMENT OF THE PRIOR ART

At the symposium celebrating the.hundredth anniversary of Langerhans work various substances promoting insulin secre¬ tion were discussed_ These included arginine and leucine. Their mechanisms of action have been much studied.

A wide variety of work has been undertaken with cell culture techniques and the use of analogues of insulin secre- togogues among others. The alpha and beta cells of the islets of Langerhans derive embryologically from the small intestinal mucosa<> These cells are differentiated to pro¬ duce and to secrete the oligopeptides glucagon and insulin respectively. It has been clearly demonstrated that diabetes mellitus is essentially a disease of lack of adequate levels of insulino Rarely, however, there is a patient in whom there is an overproduction of glucagon.

The leucine metabolism within the beta cells, there¬ fore, is of special interest_ The question arises whether activation of the production of insulin and the secretion of insulin within the beta cell results from the metabolites of leucineo Do these initiate the cyclic nucleotide response that results when the latter occupies the allosteric site of a protein kinase?

In any event, the release of insulin can be demon¬ strated to be associated with compounds that are intimately involved in the metabolism of the tricarboxylic acic cycle.

SUMMARY OF THE INVENTION

The present invention provides a method for lowering blood glucose in vertebrates with hyperglycemia. The use of amino acids having hypoglycemic actions in various ratios each wi.ch the other and each with manganese in effective ratios increases insulin release in NIDDM (Type II) diabetes mellitus. The present invention differs in its relation to effective amounts given in that these amounts are constantly changing so that there is a pattern of changing administraτion in terms of frequency, amount and individual requirements. By this multifactorial method of treatment modulation of effect on the blood glucose level is facilitated into a predictable, effective treatment program.

18

DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with this invention, patients with NIDDM (Type II) diabetes mellitus have usually been treated with diet, exercise and a hypoglycemic agent such as sulfonylurea medication. The above program and medication can be continued as this method of treatment is initiated.

Following an introductory period of observation, the hypoglycemic agents named in the invention should be introduced stepwise. The introductory period permits evaluation of the fasting blood sugar. When the hypoglycemic agents are introduced, a fasting blood sugar is taken. The desired amount of the agent is then given with food and a subsequent fasting blood sugar determined ϊh a day or two to observe for any change in blood glucose.

For example, tryptophan in the amount of 100 mg may be given initially. If there is no change, this should be increased stepwise until a drop in the blood glucose can be -orrelated with the amount given.

For leucine an initial amount of 250 or 500 mg may be used and increased up to 1000 or 1500 with a similar correlation of drop in blood glucose with the amount given.

For valine no such correlation is anticipated. However, its relation to leucine would warrant an effort to correlate a combination of leucine and valine in equal amounts with drop in blood glucose.

The indirect actions of arginine and ornithine are best explored after a treatment schedule has been undertaken. The effects of these are sometimes dramatic,but the blood glucose level may quickly return to its former level.

Initially for manganese a small dose of one or two mg (manganese content) manganese gluconate can be given separately. Both the subjective response and the glucose •level are monitored. If initial amounts tolerated are large, the increments of one or two mg can be increased to increments of five to ten mg. Usually more than 25 g are not required at any one time, and these levels are likely to decrease quickly. There is no need to activate special methods of disposing of manganese by giving large amounts. The amount reached for positive correlation with glucose level dropping is likely -to fall progressively over a few weeks or days. Increments of five to ten mg used initially may just as well become decrements of five to ten mg quickly and the spacing of the manganese becomes greater and greater. It is best to be prepared to promptly drop to lower amounts at the earliest indication that the amount required is decreasing.

Combinations of the hypoglycemic agents appear to have synergistic effects. This reflects the different modes of action of each.

As amounts are adjusted downwards, increase the interval between treatments as well as decreasing the amounts given. This is the cumulative effect, as if filling a hole. The clinician must be aware of the implications of the above. It is best that the medication be administered personally during the initial period of adjustment which varies from patient to patient. It is unwise to initially provide the patient with a standard daily dosage on the assumption that the effects will be predictable.

Initial amounts of sulfonylurea should be stabilized initially be ore beginning introduction of these hypoglycemic agents. When this is done and the amounts of manganese tolerated has been determined, Combinations of the various agents can be used.

It. is important to recall that when a drop in blood sugar occurs, the dose of the sulfonylurea must be decreased. Continue this until no ^' sulfonyl urea is being given. At that time an estimate as to further treatment will be easier to make.

The combined use of manganese with one or more of the other hypoglycemic agents named here is likely to more frequently be accompanied by a drop in the blood glucose than with the other hypoglycemic agents alone. In a sense, the manganese may be used to modulate the downward drift in th blood glucose level. No effort should be made to force the level below the normal range, which may be estimated at 110 to 1^0 milligrams/100 milliliters.

During injury, infection or forms of stress for various reasons, here will be variations in the amounts to be ^' iven of each of these. No manganese sh uld be given during fever.

Ratios should be calculated in mg as between leucine/Mn, tryptophan/Mn, and leucine to tryptophan, leucine/tryp ophan.

Seldom will as much as 500 mg of tryptophan be required, and that sh uld decrease quickly. Amounts of leucine may be as much as 1500 mg or more. Leucine may be thought of as a uel.

Tryptophan may be thought of as primitive hormone precursor.

Manganese may be thought of as modulator of the level of metabolism. Valine may be thought of as a precursor of leucine if needed.

Example 1

Patient A.H.

Period of chronic muscle strain in type II diabetes mellitus and essential hypertension.

Treatment Periods: daily to one to three week intervals.

Treatment: Fasting blood sugar drawn periodically

(70 of time). Hypoglycemic agents given 80% of time when seen. Antihypertensive agents given 70% of time,. Manganese gluconate given 30% of time when seen in doses ranging from 2 to 6 milligrams Mn„

Blood glucose: varied from (90-100) to (170-180) mg 100 ml, usually from 100 to 150.

Blood pressure: varied from 130/82 to 18 /80 mm. of mercur „

Systolic pressure: varied from 130 to 184-.

Diastolic pressure: varied from 70 to 90.

Pulse: varied from 68 to 92.

Pulse pressure: (= systolic pressure - diastolic pressure) varied from 48 to 106 mm. of mercury ₀

Medication ratios: As between hypoglycemic agents varied from 2.5/1 to 30/1 All dosages in milligrams. Dosages of hypoglycemic agents varied from 50 to 1500.

The hypoglycemic agent with the lowest ratio to manganese ranged from 17/1 to lOO/l when given concurrently ₀

The hypoglycemic agent with the highest ratio to manganese ranged from I67/I to 750/1.

Treatment intervals for Mn varied from daily to 2 weeks or more.

Clinical response: gradually improving muscle strain.,

Higher glucose levels occurred in assoication with episodes of pain and associated loss of sleep„ Eighty per cent of fasting blood glucose values fell between 100 and 150 mg 100 ml.

SUBSTITUTE SHEET

Example 2

Patient E.G. Late middle-age

Period of stress in late-onset (type II) diabetes mellitus NIDDM

Treatment Periods: daily to every to 5 days.

Treatment: Blood sugar drawn fasting and hypoglycemic agents given each morning seen with manganese gluconate 6-25 mg/treatment„

Blood sugar: varied from 175 mg to 120 mg over six week interval.

Objective of treatment: to restore blood sugar to normal range with 110 to 140 mg acceptable.

Clinical response: feeling well despite levels of blood sugar patient essentially symptom-free ? occult infection..