THE LIPOTROPIC HORMONE OF THE PANCREAS
Researchers who have dedicated themselves to diabetes observed that obesity in the city is much more frequent that in the country, and that obesity is the most important etiologic factor after inheritance.
The sedentary life of the city and the comforts greater than those of the country are the main determining factors for our observing a greater number of obese people in the cities.
Studies made in the nutrition laboratories of the Carnegie Institute, show that after the age of 50, diabetes is rarely acquired if the weight is below normal.
The more accentuated the obesity, the greater is mortality by diabetes.
Studies made by Joslin and Himsworth show that the consumption of sugar in each country does not have any relation to the number of diabetic patients does not have any relation. Two examples demonstrate this. Japan has very high sugar consumption and very low proportion of diabetics. In the United States, for a long time sugar consumption has stayed at the same level, however the number of diabetics has increased considerably. The same researchers noticed that instead of greater sugar consumption, the proportion of fats in the food of the United States has increased alarmingly, caused by the substitution with sugar for fats in the diet of diabetics.
These data made us realize that fats constitute an important etiologic factor in the presentation of diabetes. Only hypotheses not yet well defined have been presented to give the explanation of these facts. Newburgh expresses the hypothesis, not confirmed, in which fatty hepatic infiltration happening in the obese, causes a diminution of the power of this organ (liver) to form and to store glycogen, originating hyperglycemia in this fashion, first transitory and soon permanent, until becoming diabetes. Studies of other researchers support the theories of Newburgh. From 65% to 75% of obese people without diabetes turn out to have abnormal glucose tolerance tests. These people are being called pre-diabetics. And 50% of noticeably obese individuals develop diabetes. Disorders to the glucose tolerance test begin after 11 years of obesity stay positive after 18 years of obesity, and a diminution of glucose tolerance always remains.
These considerations have lead to study the second point, which is the study of the role of fats in the physiopathologic disorders present in diabetes. IN SPITE OF THE NUMEROUS STUDIES DONE TO KNOW THE PHENOMENA HAPPENING IN THE METABOLISM OF DIABETICS, NO ONE HAS SUCCEEDED IN UNIFYING THE CRITERIA, not even in the most substantial aspect, and for this reason, only hypotheses, which were mostly justified, have been proposed after numerous studies done by dedicated researchers.
The theory supported by Minskouski, is that the oxidation of glucose is insufficient — to a greater or smaller degree. The opposite is Von Noorden’s overproduction hypothesis, which maintains that in the diabetic, an increase in glycogenolysis with overproduction of sugar from sources different than carbohydrates exists, mainly lipids and protids.
According to the first hypothesis, the insulin would work by causing the correct use of glucose in tissues, and in agreement with the second, inhibiting the exaggerated glycogenesis. As much in the case of the decrease of the consumption of glucose, as in the case of its excessive production, the metabolism of fats becomes a factor of fundamental importance, since in the first case the organism is losing all the energy of proteins, where fats become the most important source of energy.
In the second case, the fundamental disorder is indeed in the lipid metabolism, since the diabetic is losing great amounts of fat in the urine after it is transformed into glucose. As it is in the liver, where the metabolic processes of fats begin, therefore this organ has a role of the utmost importance in diabetes, since as we know, there is a balance in the metabolism of glucids. Their formation occurs in the liver and their utilization takes place in all the tissues.
The works of Mann and Magath, in hepatectomized dogs demonstrating that this organ is the only source of glucose, gives us the verification of this. Confirming the foregoing are studies done by Bridge, Winter, and Joslin. They correlate the action of insulin and the respiratory coefficient, confirming that it is in the liver that the main disorders of the metabolism in diabetics are rooted. On the other hand, as it is already demonstrated, those disorders affect essentially fats.
The intimate mechanism of the oxidation of fats in the liver, as well as of its disturbances in diabetics, has not been clarified. Only theories have been launched, of which the most important are: Knoop maintains that fatty acids undergo successive oxidation, losing two carbon atoms in each, until the formation of acetic acid and a ketonic molecule for each fatty acid molecule. In support of this theory, Shaeffer recognizes that the ketones can only be oxidized by the simultaneous oxidation of a certain quantity of glucids.
Another theory maintains that fats are converted into carbohydrates in the liver. Serious works and studies have been carried out and it appears that there is something reasonable to be thinking in this fashion; however all the doubts about this theory, cannot be erased.
More successful is THE THEORY OF THE INSUFFICIENT UTILIZATION OF FATS, since this explains that, in spite of the losses of energy by the lack of combustion of proteins and carbohydrates, the proper energy is assured by the energy originated during the oxidation of fats, and at the same time explains the influence of carbohydrates in the production of ketonemia according to the well-known expression that “fats are burned in the sugar furnace.” Experiments in pancreatectomized cats, determining in their livers the quantities of oxygen, ketones, acetic acid, and fatty acids, lead to conclusions opposite to the previous theories. Resuscitating with this the old theory of Hurtley, according to which fats undergo multiple and alternating oxidations, producing in each stage of their combustion a ketone molecule without producing acetic acid in any of these stages. Slices of liver performed in the same animals demonstrated that there was no acetic acid as it should have, according to the theory of Knoop; however there is an amount of ketones much larger than that which, according to the theory of Knoop, had to exist, very similar to what was to be expected according to the theory of Hurtley.
Measuring the quantity of oxygen, it would be insufficient to cause the combustion of fatty acids, according to the theory of Knoop and necessary to transform them into ketones. On the other hand, Stadie refutes the theory of Shaeffer, from the ketogenic-antiketogenic relation, demonstrating that the ketones can enter in combustion and are used by the organism without the intervention of carbohydrates or of insulin. From these experiments the following conclusions can be reached: The diabetic, who for reasons of lack of insulin is incapable to use carbohydrates to fill his metabolic needs, supplies himself energy at the expense of fats. Part of these needs is filled by the initiation and completion of the oxidation of fats in the muscles. Nevertheless, a considerable fraction, estimated as a third part of half of the caloric needs supplied by fats, is obtained by a preliminary oxidation of these fats in the liver until the formation of ketones. From this oxidation they form neither acetic acid nor glucose. These ketones produce energy by their free use in the periphery, without insulin and the simultaneous oxidation of carbohydrates. This reserve mechanism is incapable of delicate regulation. Thus when the demand for calories, that the fats must provide, exceeds a certain level, approximately 2.5 grams of fat per kilogram and per day, excess ketones form in the liver. This excess is excreted as acetone in the urine. If this excessive fatty catabolism continues without stopping, acetonemia and coma ensue.
All these works firmly support the hypothesis of Hurtley called: “The mechanism of the oxidation of carbon atoms.”
It is not possible so far to sustain the theory of Shaeffer about the oxidation of fats accompanied unavoidably to that of the glucids, since the ketones are used directly by tissues without the intervention of glucids or of insulin. This reserve mechanism constitutes in the diabetic the main source of energy to which the organism resorts in greater proportion as its capability of oxidizing glucids improves.
According to all the aforementioned, it is observed that the lipid metabolism, as much in its hepatic stage as in its tissue stage, acquires an extraordinary importance; being altered or modified to fill energy demands, after having lost the normal sources.
The putting into play of those reserve mechanisms happening in diabetes, and the modifications occurring in the liver during the combustion of fatty acids and ketones, are also translated in alterations into another of the aspects of the lipids’ metabolism: lipid deposits in the tissues and most especially in the arteries. This aspect of lipid metabolism is one the most important factors in the physiopathologic processes of diabetes and is the cause of some of its more serious symptomatologic manifestations.
The discovery of insulin was at first believed to solve the totality of the problems of diabetes. They thought that when giving the lacking hormone to the dogs in the research, all their disorders would be corrected and their life would be extended for an indefinite period of time. But unfortunately, as we observed daily, their course, which is always progressive, has only stopped in some cases. The dogs without their pancreas submitted to a strict diet and with an insulin treatment in appropriate doses, only managed to maintain in those conditions for two or three weeks. Later, strange phenomena were observed. Thus as time passes, glycemia decreases, and the same happens to the insulin needed. In the beginning, 20 units are habitually applied. After four weeks it is necessary to reduce the dose to five units. And still this dose causes in them severe crises of hypoglycemia that can be lethal.
This fact, considered separately, could be interpreted as an improvement in the glucid metabolism. In reality it is not, because at the same time other symptoms of greater importance appear. Blood lipids that in the first week increase considerably to numbers much lower than normal, simultaneously produce hepatic functional disorders revealed by the Bromosulphophtaleine test[sometimes called Bromosulphtaleine or Bromosulphaleine test]. The animal loses appetite, emaciates considerably, and dies. In the autopsy there is invariably a liver of increased volume, up to two or three times its normal size, with phenomena of fatty infiltration and degeneration in the arterial walls, reaching atheroma. These observations confirmed by other researchers lead to the conviction THAT A FACTOR DIFFERENT FROM INSULIN INTERVENES in these animals, causing serious alterations in the lipid metabolism, which the insulin is not able to remedy. It was thought that the lack of external secretion of the pancreas was the cause, because of its content in lipases. McLeod and his collaborators made a pancreatectomized dog ingest great amounts of raw pancreas, finding with surprise that hyperglycemia and glycosuria increased, that the animal needed enormous amounts of insulin, which improved its appetite and its general state, extending its life almost for an indefinite time. In the autopsy, they verified the diminution of the liver to its normal volume and the disappearance of the phenomena of fatty infiltration and degeneration, having demonstrated therefore, THAT THERE IS A FACTOR IN THE PANCREAS ACTING ON THE METABOLISM OF LIPIDS, avoiding its excessive deposit in the liver and the arteries. It allows the combustion of fatty acids and its possible glucose transformation, which enables insulin to work, increasing glycogenesis and glycolysis, restoring therefore the metabolic phenomena to normalcy.
Other researchers discarded the theory that the external secretion of the pancreas relates to the lipases. They believed that it was the choline contained in the pancreas that was causing the protective action against fatty infiltration. After a series of experiments they demonstrated that phospholipids and choline supplied to the dogs without their pancreas caused the same phenomena as supplying raw pancreas, whenever these substances are given in sufficient amount. Other authors confirmed this. The action of phospholipids and choline not only prevents and corrects fatty infiltration in the pancreatectomized dogs, but also in other animals, when trying to provoke it experimentally by the administration of cholesterol and phosphorus. From here the lipotropic action of the choline and that of the phospholipids was observed.
Danovan, Vermenlen, Van Prohaska, and others made similar experiments and reached different results. They discarded the possibility that the pancreatic juice is the cause of the lipotropic action, since fatty infiltration is not avoided by providing large quantities of pancreatic juice to the pancreatectomized dogs. And the total elimination of pancreatic juice by means of fistulas made in dogs does not produce hepatic fatty infiltration. Secondly, they prepared extracts of pancreas, free of lecithin and choline, that are as effective as the raw pancreas, in preventing and curing the fatty changes in the liver of the operated dogs, allowing at the same time the survival of these animals.
The fresh pancreas is first extracted with neutral alcohol, filtering until dry and extracting it with ether. This ether extract contains the fat of pancreas, therefore, practically all the choline and all the lecithin. Nevertheless this extract has no therapeutic value, because it does not have any appreciable lipotropic action. However, the fraction free of fat extracted with alcohol, is effective, and has a perfectly appreciable lipotropic action. When administered orally in amounts of one to two grams per day, it manages to cure fatty infiltration in the liver of dogs without their pancreas. Dissolving it in water and precipitating it by saturation with ammonium sulfate can purify this fraction free of fat. The precipitate can then be dissolved in glacial acetic acid. When adding ether, it produces a new precipitate that has all the lipotropic properties of the raw pancreas and is active orally and subcutaneously in smaller amounts of 15 centigrams per day. This product does not contain fat and is free of appreciable amounts of choline.
This substance, which plays an important and almost vital role in the metabolism of fats, is processed by the pancreas and is distinct from insulin. It was baptized with the name LIPOCAIC, from Greek: I burn fat.
The experimental bases that showed this second hormone of the pancreas, have lead us to think that their secretion is given by the ALPHA CELLS of the same islets of Langerhans, whose function until now is not known, since the BETA CELLS are those that process the insulin, as has been already well demonstrated.
Discriminating the ones from the others is possible. Between other experiments the granules of ALPHA cells are soluble in water, those of BETA cells in alcohol. In addition the tinctorial qualities and their distribution in the islets of Langerhans is completely distinct.
Summarizing, of the numerous works executed with LIPOCAIC: the pancreatectomized dogs, in spite of a dietetic treatment and the administration of insulin, died in the course of four weeks as a result of disorders of fat metabolism. These consisted mainly of the diminution of blood lipids to less than half of the normal level; fatty infiltration and degeneration of the liver and, as noticeable consequence, insufficiency of this organ, demonstrated among other tests by the Bromosulphophtaleine test; fatty infiltration of the arteries and production of atheroma; diminution of glycemia and glycosuria and increasing intolerance of insulin, which is translated into undernourishment and death of the animal.
If the animals submitted to these experiments are subjected at the same time to a suitable diet when the insulin is only applied in doses of five units, and are administered orally two grams daily of LIPOCAIC, a clearly favorable change in the animal is observed. An increase of glycosuria and glycemia begins to be noticed, a greater tolerance of insulin, until administering greater doses as they were using during the first days of the experiment; an increase of blood lipids that quickly rises to levels greater than normal; but after this initial phase, the level of blood lipids, after five weeks of treatment with LIPOCAIC, fluctuates around the value found before the operation. Simultaneously, and with greater sluggishness, hepatic fatty infiltration is reduced until returned to the normal structure of the liver. The Bromosulphophtaleine test becomes normal after six weeks, the dog improves in its physical condition: the appetite increases, it recovers the weight lost, and it can survive several years in these conditions.
These experiments conducted on operated dogs were then done on rabbits. Those which received cholesterin (cholesterol) in sufficient quantity to cause fatty infiltration, hyperlipemia, hypercholesterolemia at the same time that they were given LIPOCAIC, obtained diminution of hyperlipemia and suppression of fatty infiltration of the liver.
Some clinical facts obtained in different clinics support the experiments observed on animals; nevertheless in other clinics the results have not been constant and this has given rise to controversies. But anyway, the conclusions reached by the objectors to the theory of the LIPOCAIC are:
a. — The results of experiments in which pancreas, liver, and fresh meat of ox are added to the diet, do not suggest that the pancreas contains a new specific factor that affects the deposit of fat in the liver of white mice.
b. — LIPOCAIC presented the lipotropic effect that could be expected from its choline content.
c. — It seems improbable that an amount of choline equivalent to the total lipotropic value of the dose of pancreatic extract (lipocaic) used in the experiments by Dragstedt, has any influence on the fat of the liver of the dog without pancreas, being a very small dose.
d. — The lipotropic effect of the pancreatic extract was the same as that of the casein in the mice.
Against these last opposite arguments to LIPOCAIC, it is maintained:
1. — Practically, two grams of choline per day or more are required to obtain beneficial effects. In one hundred grams of pancreas, which is an effective dose, there are only about 250 milligrams of choline.
2. — The action of the pancreas is specific. The liver and the brain, which contain as much or more lecithin and choline, do not exert any beneficial action.
3. — In the extracts made from pancreas, the active substance is in the alcoholic extract lacking fat, whereas the fractions soluble in ether, containing all the lecithin of the pancreas and almost all the choline, are completely inactive.
4. — It has been possible to obtain an extract of pancreas exerting the typical action of that gland in a daily dose of 60 to 100 milligrams of dried substance; this material is fat free, contains no more than one or two percent of free choline, and is as effective by oral administration as subcutaneously.
Because of all the research performed to date, it can be accepted that the pancreas has a specific action on the metabolism of lipids, distinct from the one caused indirectly by insulin. The Alpha cells of the islets of Langerhans are the source of this hormonal secretion that is totally different from the insulin secretion produced by the Beta cells.
These experiments give a good orientation to the problem of the metabolism of lipids; research is still being conducted to corroborate the existence of the lipotropic hormone secreted by the Alpha cells of the islets of Langerhans, LIPOCAIC.
HOW THE ENERGETIC FUNCTIONS OF GLUCOSE TAKE PLACE
Phosphagen is decomposed by muscular contraction, into a molecule of phosphoric acid and another of creatine, supplying the necessary energy to make the contraction of the muscle. But on the other hand the organism uses equal energy to remake the creatine-phosphoric combination. One derives from the oxidation of a glucose molecule; soon the decomposition of the glucids serves to remake the phosphagen molecule. The role of phosphates is of extreme importance in the metabolism during muscular contraction. All this degradation of the glucids is done in the presence of an enzyme that is formed by the pyrophosphates of adenylic acid in the presence of magnesium salts. Adenylic acid is a nucleoid composed of a purine base (adenine), a pentose, and an orthophosphoric acid molecule. The adenylpyrophosphoric acid plays a double role: it serves as coenzyme, as we have just seen, and gets its energetic action by the transformation it undergoes in muscular contraction. By a series of hydrolysis steps, giving off heat, the adenylpyrophosphoric acid disperses itself as a molecule of adenylic acid and another one of pyrophosphoric acid. After this, it splits into two molecules of orthophosphoric acid attaching them to hexose derived from glycogen, constituting hexose diphosphate.
The heat given off by this reaction is 170 calories per gram of released phosphoric acid, allowing phosphagen to hydrolyze into creatine and phosphoric acid, and thanks to these reactions the adenylpyrophosphoric acid molecule can be reconstructed. By these reactions, the adenine is transformed into hypoxanthine finally giving ammonia. These chemical changes by deamination are reversible, taking place in remarkable form in muscles tired by exercise. Because of these reactions, we observe, in all the elimination channels, an increase of nitrogen during any muscular exercise.
The phosphagen molecule is reconstructed by phosphoric acid coming from the decomposition of hexose-phosphates, thanks to the released energy from the oxidation of pyruvic acid.
The thermochemical reaction of muscular contraction can be summarized the following way: adenylpyrophosphoric acid is where all the reactions begin. When being decomposed into phosphagen, it gives the true energetic reaction, and the restoration of other phosphorous compounds, at the expense of the degradation of glucids.
FLEXIBLE ROLE OF GLUCOSE
Glucose and galactose form lactose; these, united with a water molecule that gets lost, constitute the biological phenomenon that takes place for the formation of lactose (sugar of milk); also the organism directly produces lactose from glucose. In general chemistry this form of constitution of lactose is impossible, only the organism can make this transformation.
Pregnancy and breast-feeding cause the growth of the mammary gland, and also the considerable increase of blood dextrose (hyperglycemia). Mammary glands synthesize lactose, since suppressing these glands demonstrates that although there is hyperglycemia lactose is not produced. Later these glands form the lactose of milk.
Starting at the sixth or seventh month of pregnancy, a considerable increase of glucose appears in the blood. This glucose is of hepatic origin. Possibly by the influence of a hormone secreted by the fetus or the placenta, an increase of dextrose takes place in the liver. From the eighth month of the pregnancy, under combined hormonal influences, milk is manufactured in the form of COLOSTRUM (the mother's first milk). Between the sixth and the seventh month of pregnancy there is glycosuria. At the moment of childbirth there is galactosuria, and after the weaning lactosuria. These three forms of sugar elimination happen in the urine and are physiological.
The lactose of milk plays a role flexible, because in addition it is going to form galactolipids, component elements of the nervous tissues of all animals and all other bodies formed with protids composed of amino acids.
Briefly we have seen how glucids perform energetic functions, which are the best ones known, the mixed and the plastic.
Soon all the elements taking part, either definitively or in transient form, in the metabolism of glucids, are deeply affected when searching, either with therapeutic objectives, as it happens in Cellular Therapy, or in pathological states.
The ternary compounds of carbon, hydrogen, and oxygen, whose relation between these last two is in the same proportion as in water, were called HYDRATES OF CARBON; imagine the formula in these general terms: Cm(H2O)n, in which m can be the same as or different from n. The hydrates of carbon are particular compounds that contain sugar, that is to say, they are compounds in which sugar could be formed or whose molecule was constituted solely by sugar.
But it was discovered that other different compounds that can yield sugar do not correspond to the same formula; in virtue of which this appellation of hydrates of carbon was abandoned and were given the name of “sugars” or “saccharogenic substances;” even this denomination did not include certain substances of the same family, such as those then called “glycosides.” Thus it is for multiple reasons, that at the International Convention of Chemistry, encompassing all these compounds, it was agreed to call them GLUCIDS, including all the compounds of organic chemistry located in its molecule, being in free state, or being in combination of one or several molecules of OSES, that is to say one or several non-hydrolyzable sugar molecules.
This group of compounds is formed by elements of great importance, since they play two important roles in animals: ENERGETIC, to give to the organism most of its energy for growth and metabolism, and STRUCTURAL, used to build compounds that are going to form the cells of the organism.
The linear chain compounds formed of carbon, hydrogen, and oxygen, whose carbons minus one of the main chain have a primary or secondary alcohol function, and this carbon has an aldehyde or ketone function; because of this, they are divided in two groups: aldoses and ketoses.
GLUCOSE is one of the most widespread OSES, it contains six atoms of carbon; one of these has the aldehydic function, and the other five, a primary alcohol function.
All the OSES have the following qualities; they are reducers, fermentable; the specific property to be combined with hydrazines; they can be united with hydrocyanic acid to form nitriles (this property is being used for the search of glucose in urine); they have in their asymmetric molecule carbon atoms which rotates the polarization of polarized light.
From the works of Claude Bernard it was demonstrated that blood contains glucose in a proportion of 0.80 to 1 gram per liter. According to Lépine, glucose that is dosed immediately as soon as the blood is extracted is the “actual sugar.” But there is another sugar that it is not possible to determine the dose of immediately after the blood is extracted, and it is the “virtual sugar.” These variations in the measuring have caused controversies and this point about “virtual sugar” has still not been clarified.
The glucose that is commonly measured is the free blood glucose that is in a state of TRUE SOLUTION and is dialyzable. Another sugar exists, in addition to the two indicated. It is the proteinic sugar, it seems that it is or comes from the glycogen contained in the white cells or comes from the OSES of certain conjugated proteins, like mucopeptides.
From all this we observed that in fact there are three existing glucoses in the blood: The glucose or “actual sugar,” the “virtual sugar,” and the “proteinic sugar.”
For these reasons, when studying the glucids of the blood, we must distinguish: GLUCIDEMIA, that represents the totality of glucids, reducing or not, hydrolyzable or not, and free or not, and GLYCEMIA that represents the concentration of glucose in the blood.
The classic methods of measuring are founded on the reduction property of glucose. They are those of Folin, Wu, and Benedict. In these works the method used for diverse studies was the first method of measuring.
We must consider that the blood glucose is converted by glycolysis into two lactic acid molecules. For this reason, in spite of the different preservatives, fluoride or sodium oxalate, glucose measurement must take place as soon as possible; otherwise the measuring is no longer accurate.
The pathological causes of hypoglycemia include several mechanisms. We can have hypoglycemia by glandular disorders, such as adrenaline hypo-secretion, as in Addison disease. Tuberculosis of the suprarenal capsules produces hypoglycemia. The normal operation of this gland increases blood glucids; these patients are very sensitive to the effects of insulin.
The exaggerated secretion of insulin brings about the loss of blood sugar; this is very frequent in tumors of all types that are observed in the pancreas; with greater reason if these tumors are malignant. In many occasions the doctor does not have the time sufficient to arrive at the diagnosis. Before doing it, the patient perishes by hypoglycemia. Operations have been done on a pancreas with tumors and it has sometimes been possible to save the life by removing them. This SYNDROME OF HYPERINSULINISM, previously rare or little known, is now very frequent; only a great experience leads to this diagnosis. The insular adenomas and carcinomas (tumors in the islets of Langerhans) are those that most commonly produce these symptoms.
By single clinical examination, it is very difficult to make the diagnosis. The location of the pancreas, deep behind the stomach, and the impossibility to see it on X-rays, forces a surgical intervention to make simultaneously the diagnosis and the treatment. Some chronic pancreatitis can produce blood glucose loss but in these cases the digestive disorders dominate those of hypoglycemia. In fact it includes the lesion, not only of the islets of Langerhans but also of most of the pancreas.
Lesions of the pancreas are now recognized as one of the main causes of HYPOGLYCEMIA. Malignant or benign tumors, inflammatory lesions of various origins, malformations (as a rule congenital), and hypertrophies or hyperplasias of the islets of Langerhans, all these causes produce INSULAR HYPOGLYCEMIA by hyperinsulinism.
There are hypoglycemias attributed to the nervous system or to the endocrine system, but the truth is not known and the cause of these hypoglycemias remains a mystery. Various medications acting on the pancreas or the other glands do not have any effect. The reason why it was believed that these hypoglycemias obey to a general cause where other factors take place is so far unknown. We think, nevertheless, that either the islets secrete a greater amount of insulin, or that the organism, by special circumstances, is more sensitive to the action of this hormone in small doses.
What has been observed, in this type of hypoglycemias, is that individuals with this disorder are nervous (vagotonic imbalance), with clear endocrine disorders like menstrual dysfunction, certain hyperthyroidism, low blood pressure, etc. Clinically it is very difficult to make a diagnosis with respect to the real cause of hypoglycemia; neither does the laboratory help in this diagnosis.
It is said that endogenous hyperglycemias caused by adrenaline, asphyxia, ether, morphine, or the puncture of the fourth ventricle, are not improved by the hypoglycemic action of insulin. When the hypoglycemic action of insulin is below 20mg, the hypoglycemic effects of adrenaline are not sufficient, and then it is necessary to resort to glucose injections.
MEASUREMENT OF GLUCOSE IN THE BLOOD
ANTECEDENTS: In the beginning of our studies we took three blood samples: one before the injection of the insulin, that was considered normal; another one in the state of hypoglycemia, before initiating the application of the intravenous injection with appropriate medications appropriate for the case; and a third sample when the patient had left the hypoglycemic state and was considered again in a normal condition.
In these conditions, the first two samples were always constant, a normal one and a hypoglycemic one. In certain cases, unusual hypoglycemias were noted showing between 65 and 80mg of glucose per 100cc of blood, and we have attributed these to the fear of the treatment by certain patients. The third samples, taken after the heat of the first part of the treatment always gave notable readings of hyperglycemia. So we have abandoned it, because it gave data from 200 to 450 and up to 500mg of glucose per 100cc of blood. We attributed these high readings to the fact that to keep from disturbing the patient with a new puncture, we took this third sample minutes after the application of the medications, using as vehicle a hypertonic glucose solution, and this left the needle full of this solution. Although a few drops of blood were allowed to drip so that the needle would get washed, readings of slight hyperglycemia were never obtained.
In these conditions, we kept taking the first sample or normal, before the initial insulin injection, and the second, in full insulin shock, before applying medications.
Preparation of the filtrate of blood free of protids: Method of Folin-Wu.
This method is based on total precipitation of blood protids by tungstic acid, formed by double decomposition between sodium tungstate and sulfuric acid, filtered by dry filter and the filtrate contains all the blood components that are determined by this system and which are:
Non-proteic nitrogen, urea, uric acid, creatine and creatinine, glucose, amino acids and chlorides. To be able to make all these determinations, around 10cc of blood sample are needed, but since in the beginning we are only interested in the glucose, we work with a sample of 1 to 2cc.
The samples were gathered in sterile assay tubes containing a drop of saturated solution of sodium oxalate, which had been evaporated by placing the tubes on an electric grill in horizontal position to have a better distribution of the anticoagulant agent. And as soon as the blood was received, they were shaken to dissolve the oxalate in the blood, and to avoid therefore the coagulation that always occurred when this detail was omitted. We also used, although not very often, potassium or sodium fluorides, as well as formaldehyde in the cases where, at the place of the treatment, the samples were going to be kept at room temperature from one to two hours and even longer, because we didn't always have the use of a refrigerator to preserve them. In most of the analyses that we gave later we preferably used the oxalate as anticoagulant.
During the process of obtaining this filtrate free of protids, in the beginning, we used the modification of Hadens to the classic technique of Folin-Wu, which consists of adding to the blood (one volume), 8 volumes of 12N sulfuric acid, to shake and later add 1 volume of tungstate of sodium at 10%, shake well and filter, but frequently we collected poor data. In theses cases we used the classic technique, which consists of hemolyzing one volume of sodium tungstate at 10%, shaking, and later adding one drop at the time, with constant agitation, one volume of sulfuric acid 2/3 normal. If the operation is well conducted, there is no foam formation and after a rest of five minutes, when the color of the mixture has changed from bright red to dark coffee. If this change in color does not occur, it is an indication that the coagulation is incomplete. We can still take advantage of the sample by adding more sulfuric acid drop by drop and shaking energetically after each addition. This occurs generally when an excess of oxalate is used. Given the small proportions of the tests, the sample was filtered by double filter according to the García Junco technique and the filtrate was picked up in another assay tube. The filtrate is completely transparent from the first drops and with it proceeds to the...
DETERMINATION OF GLUCOSE -- Method of Folin-Wu
This method consists of warming up the filtrate free of protids, obtained as previously described, with a cupric alkaline solution, in a special tube that has a bulb in the inferior part, of 4cc capacity, then a narrowing, that must have, more or less, 4cm in length and that serves to avoid the oxidation of the reacting mixture, and then follows the wide tube. The tubes that we used had three markings: at 6, 12.5, and 25cc. The cupric oxide, which is formed by reduction of the cupric salt, precipitates in the form of a brick-red powder. It is treated with a reagent solution of phosphomolybdic acid, obtaining this way a blue color due to the reduction of the molybdenum, and that within the limits of the procedure is proportional to the initial quantity of glucose. We compare it in a colorimeter with witness solutions or glass patterns, which came with the colorimeter we were using.
The technique to follow is to put 2cc of the free tungstic protein filtrate in the special tube of Folin-Wu (if they expect very high data it is suitable to put 1cc of filtrate and 1cc of water) and 2cc of alkaline copper solution reagent. The free level of the mixture should reach the narrow part of the tube. If you do not have glass patterns, put in other tubes a sufficient quantity of glucose type solution to make the comparisons with the colorimeter. The tubes are warmed up 8 minutes in a Bain Marie (water bath). Then add 2cc of phosphomolybdic acid solution and they are diluted, normally, up to the 25cc mark, in which case the reading is direct. If the test is not very blue it is suitable to dilute only up to the 12.5 mark and to multiply the result by two. We had extremely low results in the blood of hypoglycemics, so we left it in that volume, multiplying the result by 4.17. The Klett colorimeter we were using has in the inferior part a movable tape to shorten the calculation and the data thus obtained referring to a normal test, is corrected in the manner previously mentioned.
We tried in some cases the methods of Benedict, but the difficulty of continuous preparation of the reagent makes it impractical for routine determinations. The substitution of tartrates by alanine for the formation of the cupric compound also makes it expensive for our means, since the price of this reagent is much higher and it is not always available. The data collected by this method are lower than the ones obtained by the method of Folin-Wu, since the reagent of Benedict does not react with the non-sugar reducers of the blood, such as glutathione and the thioneinethat is included in the data of Folin-Wu, being about15 to 30mg per 100cc.
We have not included the formulas of the reagents here because they can be found in all the specialized books on blood analysis. We used the “Practical Physiological Chemistry” of Phillip B. Hask, eleventh edition.
The normal average quantity of glucose in the blood of the inhabitants of Mexico is 82mg per 100cc, staying the same, despite the quantity of carbohydrate foods that we ingest. (See graphs # 1 and 2). The hemo-gluco-regulator apparatus is constituted by the endocrine glands: suprarenal, thyroids and hypophysis (pituitary), that increase the glucose level in the blood; and the islets of Langerhans, the parathyroid, and the ovaries, that have the opposite action. By a different mechanism, also intervene: the neuro-vago-sympathetic system and the central nervous system (puncture of the fourth ventricle, that Claude Bernard called diabetic, in 1855). This experiment lead also led to the discovery of the intervention of the liver in glycemia, by a mechanism different from the previous ones.
Glucose, condensed in glycogen form, is stored in the liver. It comes mainly from carbohydrate foods. We must emphasize that not only carbohydrates give this substance, but also fats and albuminoids, as was established in previous paragraphs, and which we have observed clinically.
If we give only fatty or albuminoid foods to an individual whose blood glucose has been lowered to half its normal level by means of insulin, the symptoms of hypoglycemia begin to disappear in his organism after a greater length of time than required for carbohydrates. This demonstrates that using those foods, glucose is also formed and, therefore, glycogen.
The action of adrenaline on hepatic cells is demonstrated by the influence exerted, preventing the destruction of glucose and accentuating, at the same time, glycogenolysis. This action is probably carried before a glycogenolytic enzyme that is also glycolytic according to the conditions in which glycogen is found. In order to carry out the conversion of glucose to lactic acid, the enzyme must be fixed to the glycogen. The glycogen of colloid nature would be the stimulating coenzyme, carrying on its surface the stimulated coenzyme. All these phenomena take place under the disseminating action of adrenaline, which, when injected, diminishes the glycolysis in the blood and in the hepatic cells.
The suppression of the suprarenal capsules induces hypoglycemia. By direct action on hepatic glycogen, and by consecutive vasotonic influence to the central nervous system, adrenaline regulates the metabolism of carbohydrates in the liver as in the other organs. The metabolism of lipids and protids is related to the metabolism of glucids. Adrenaline then carries out a primary role in the general metabolism; it is hyperglycemic, glycosuric by glycogenolysis and by defective use of glucose. Adrenaline is shown opposing the action of insulin against glycogen.
By their internal secretion, the suprarenal glands are anti-assimilator organs, antagonistic to the pancreas. Adrenaline is, then, an anti-enzyme of assimilation. It is, also, disassimilating, since it accentuates oxidation; it plays the aid-enzyme role of disassimilation.
The pancreas is an organ of assimilation by its external secretion and also by its internal secretion, since the later allows the use of foods digested by it. At strong dose, it is an antidisassimilating agent for the exaggerated increase of oxidation: it is, then, an anti-enzyme of disassimilation. Insulin, in insufficient doses to cause clinical hypoglycemia, does not bring any remarkable variation in the elimination of carbonic anhydride and water, according to the formula:
C2 H12 O6 + 6O = 6CO2 + 6H2O
Oxidation is done by intermediary stages and in the presence of catalysts and true enzyme. It is also said that the following products have an important role in this dissociation: hexose phosphate, phosphoric acid, and mainly, the calcium ion. The conversion of glucose into lactic acid liberates energy; the opposite occurs in the resynthesis of glycogen from lactic acid, absorbing a certain amount of oxygen and energy.
The metabolic action is not only of the glucids, but also, in general, of all the components of the organism: fats, albuminoids, etc., etc.
Not all carbohydrates are transformed into lactic acid; most of the missing sugar undergoes transformations still unknown.
As will be observed, this glycolytic activity of disintegration and synthesis of the glucids is intimately united to the reaction of the humoral medium; also, it demonstrates the preponderant role of carbohydrates in cellular nutrition.
If the dose of insulin is very strong, glycogenolysis reduces the level of glycogen, hypoglycemia settles in, CO2 is eliminated and the temperature drops progressively, because the excess of insulin flocculates and smoothes out the glycogen to such point that glycogen synthesis cannot take place, and only glycolysis exists. The level of glycogen is then minimal, and it cannot increase for lack of new glycogen. If oxidation is insufficient, the lactic acid just formed paralyzes the coenzyme and stops the metabolism of carbohydrates, as well as oxidation, increasing hypothermia, and even capable of causing death.
The liver functions are: regulatory, biliary, hematolytic, hematopoietic, glycogenic, glucocemic, adipogenic, fibrinogenic or coagulant, ureopoietic, antitoxic, etc. All these manifestations that seem to be different functions of the hepatic cell, are intimately united, and they are only the result of cellular operation.
The antitoxic action of the liver is proportional to the glycogen riches of the organ. The influence of glycogen in detoxifying is indirect, because it provides glucose that turns into glucuronic acid, which, in addition to sulfo-conjugation, decontaminates indirectly, in proportion to the amount of glucose that it is susceptible to provide. Consequently, a liver that does not contain glycogen does not work on poisons.
This action is verified in the following fashion: toxic substances have, or can acquire in the organism, an alcoholic or phenolic function that, together with the glucuronic or glucose of the liver, forms glycosides. These undergo oxidation on their terminal alcoholic chain and produce conjugated glucuronic acids that are very little toxic, soluble, and easily eliminated by renal secretion. Glycogen, in addition to being a nutritional power reserve, is an antitoxic material reserve.
We can consider, each in its turn, the four functions of glucose:
A muscular energetic, since under certain conditions it is transformed into lactic acid, indispensable for muscular contraction, a thermogenic, for which part of the glucose is dissociated in lactic acid, providing heat for the combustion of this acid probably passing to acetic acid and finally to CO2 and H2O. The antitoxic function, for glucuronic conjugation, and, since glucose is able to participate in the synthesis of fats, protids, and gluco-protids, it has a flexible metabolic function. Glucose has oxidizing power, although this property is less pronounced than that of glycogen, which it does energetically, thanks to its power of absorption. The energetic oxidizing action of glycogen explains cellular respiration, that is to say, the one that represents the gaseous exchanges between the blood and the tissues. The oxygen carried by the blood to the cell, enters into chemical activity at the atomic level. Glycogen-oxidase explains, therefore, the inner respiration and it is the extension of the level of glycogen guiding respiratory exchanges. From this function of oxidase-glycogen ensues its antitoxic role, by virtue of which all the detrimental oxidizable substances are destroyed, remaining in lactic acid form as an intermediate step.
According to McLeod, glucose and mannose are the most important antihypoglycemic medications. Later follow fructose, galactose, and maltose. But the improvement that these “Oses” produce is only temporary. And arabinose, saccharose, and lactose do not have any effect on the hypoglycemic action of insulin.
These facts demonstrate that indeed, of all the “Oses” known, glucose is the only one that arrests and improves the hypoglycemic symptoms caused by insulin.
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