Complete Text (Part 6)

a fasting stomach and the blood and urine sugar are followed together, the blood being withdrawn at 1, 2, 3, 1 and 5 hours after taking and the urine being examined at the same time. Alimentary i^lycosiiria • Food — Intestines — Liver Hyperglycaemia — Glycosuria Diabetic Glycosuria Glycosuria C. N. S. — Adrenal . ' ' ' Sugar accumu- lation in blood 1 Tissues I ^— Pancreas impairment — -Liver — Epinepliriu I I These condition can occur under nervous excite- ment, fear, anx- iety and diabetes. 72 QuaJitativG test for glucose. Trommer's test: To 2 parts of urine add 1 part of 10% :N'aOH. To this add 10% solution of CuSOl till a bluish precipitate results and remains after shaking (add the CuSO^ drop by drop). Warm the solution (do not boil), and in the presence of sugar a red- dish or yellowish precipitate will form, spreading graduall}' downward and leaving a decolorized su- ]3ernatent liquid. Reaction : H2O + XaOH + 1 drop CuSO, = Cu ( OH) ^ CuSO, + 2NaOH = :\X.SO, + Cu(0H)2 Cu(0H)2 + heat = CuO (black) + H.O 2CuO + sugar + heat — reduction Yellow = CU2OH (alkalinity low) Red = CuoO (alkalinity high) l^he color depends upon the degree of alkalinity. Uric acid, creatinin glycuronic acid, albumin, allan- toin, mucin, lactose, maltose and alkapton have the propert}^ of holding Cu(0H)2 in solution and reduce it to lower salts by heating. A false reduction by these substances can be avoided by warming and not boiling, for these substances reduce only upon boiling, and it tends to occur on cooling. They give a dirty yellowish green color to the previous blue solution. Dilute urine for more accurate de- termination. Requirements necessary for positive reaction: 1. Prompt formation of a precipitate on warm- ing, settling to the bottom and leaving a clear solution above, before cooling. 2. Avoid adding CuSO^ to excess, which gives black j)recipitate and masks reaction. 7^ P*. Any discoloration after prolonged boiling should be neglected. 4. Albumin 'should be removed before perform- ing test if it is present in large amounts. Qualitative tests for sugar. Fehling's test : Solution A. CuSO, 34.65 gm. H2O qs. ad. 1000 cc. Solution B. Rochelle salts 173 gm. NaOH 50 H2O qs. ad. 1000 cc. Equal parts of solution A and B are mixed, which gives a clear blue solution. Boil mixture in a test tube and add urine not over 1/2 by volume. Sugar gives reduction in from 5 to 10 seconds. If no re- duction occurs, bring the solution back to boil. Prolonged boiling gives misleading results. Sensi- tive to .08%. Errors : Normal Constituents — Uric acid, crea- tinin, blood pigments, giycuronic acid and phenol derivatives ; also reduced by chloroform and formal- dehyde. Drugs — Trional, sulphonal, camphor, mor- phia, salicylates, benzoic acid, antipyrene and phe- nacetine. Benedict's test. Benedict's solution : CuSO, Sodium citrate Sodium carbonate HoO qs. ad. 17.3 gm. 173.0 gm. 200.0 gm. (crystals) 1000.0 cc. Method of preparing: Sodium citrate and sodium carbonate crystals are dissolved in 700 cc. of hot Wiiter. Filter while hot and add a proper amount of solution of CuSO^ to make 1000 cc. This solu- tion is permanently stable, lasting 5 to 10 years, 74 and the test is more delicate than Fehling's. Sensi- tive to .02— .05%. Technique of the test : To 5 cc. of the reagent add 5 dro]>s of urine. Boil vigorously for 2 or 3 minutes and let cool spontaneously. A positive reaction is indicated by a reddish or reddisli yellow precipitate. If less than ."% is present a positive reaction is likely to occur only as the solution cools. In the absence of sugar the solution remains clear or slight- ly turbid. It is not reduced by normal urinary con- stituent?. Haine's test. Same as former, except that sodium carbonate is replaced by glycerin. Xot as sensitive as Benedict's. Xylander's test. Copper is replaced by bismuth subnitrate. Solution : Bismuth subnitrate 2 gm. 10% NaOH 100 cc Rochelle salts 4 gm. Keep solution in a brown bottle and don't expose to light. Technique of test: To 9 parts of urine add 1 part of reagent. Put in water bath for 5 minutes ; boiling is ijermissible. A positive test is indicated by a black ])recipitate of bismuth. In urine with only a small percentage of sugar the precipitate takes on more of a brownish color somewhat darker at the bottom. Advantages of the test : It is not reduced by nor- mal urinary constituents or alkapton. Disadvantages of test: It is reduced by hexoses, pentose, glycuronic acid, marked indicanuria and urobilinuria. It is also reduced by sulphonal, trional, rhubarb, senna, cascara and large doses of urotropin and quinine. 75 Galactose. Conditions which show galactose in the urine: 1. Infants nursing at breast and having gastro- intestinal disturbance. 2. Used in test for hepatic function (formerly). Disaccharides : 1. Lactose, which splits up into I ^, , ,^ J Galactose 2, Maltose, which splits up into I „, 3. Saccharose, which splits up into I Levulose ) Glucose Lactose : Physiologically it occurs during the puerperium and sometimes throughout the period of nursing. It also occurs in persons on an exclusive milk diet. The tolerance for it is low, 80 to 100 grams. It never occurs in diabetes. It does not ferment with yeast unless it is first broken down to glucose and galactose by ferments or bacteria, the former of which is fermentable by yeast. To obtain osazones the urine should be evaporated down in order to make it more concentrated, extracted with an alco- hol, evaporate extract, take up in a small amount of water and apply phenylhydrazine test. Their melting point is 200 degrees. Kubner's test for lactose: To from 5 to 8 cc. of urine add a large excess of basic lead acetate, boil several minutes and filter. To the filtrate add XHIrOII till a precipitate forms, redissolves and a permanent precipitate recurs, which is a brick red color. The filtrate also takes on a reddish color. A red solution with a yellow precipitate indicates glucose. Lactose is to be expected in urines giving slow reduction and D rotatory. 76 JfaUosr. There are ouly a I'eAN' cases ou record of its occur- rence in the urine. It is associated with interstitial lesions in the pancreas. It should be suspected in urine when rotation is greatly in excess of the amount of sugar present, which is determined by titration. To determine its presence, hydrolyze the urine for one hour with dilute acid. Neutralize and test by titration and with polarimeter. Saccharose. This sugar is hot known in pathological condi- tionr. Chronic maligners often put this sugar in the urine in order to get into a hospital for the winter. The urine usually has a high specific grav- ity, and is of a syrupy nature. Hydrolyze for an hour with ^^eak acid. Neutralize with Xa2C03, titrate and polarize. Fcn)t€)itatio)i : It is said that only those sugars which have 3 or a multiple of o carbon atoms will ferment, which fact does not hold good, only 2 fermentable sugars occurs in urine — glucose most frequent, levulose next. I'cchniquc: Controls, 1. Normal urine -j- yeast = O 2. Normal urine + yeast -- dextrose = positive 3. Specimen of urine alone = O Test. 4. Specimen of urine -|- yeast = -- according to presence of sugar. Do not mix by violent shaking Avhen adding yeast, and thus admit small bubble?. No. 3 can be omitted by first boiling No. -1 . Allow to stand for from 3 to I hours at 371/4 degrees. Sensitive from .1 to .05%. Phenylhydrazine test : Given by all hexoses, pentoses, glucose, levulose iiiul iiiaiiuose, but tlie melting point of the ciystals is different. Teclmiqne: l^lienylliydnizine 5 drops Urine 4 cc. Clacial acetic 0.5 cc. Boil and shake 2 minutes, and while still warm add 4 to 6 drops of 20% NaOH. Do not add enough to make alkaline. Boil from 30 seconds to one min- ute longer. Cool spontaneously. If sugar be pres- ent the solution will become turbid and deposit a yelloAv precitate which indicates crj^stals of osazone. This test is extremely sensitive and is the court of last appeal for the determination of a sugar. The crystals have a characteristic appearance arranged in sheathes. To purify the crystals the filter paper is punctured and the precipitate washed into a theater containing 60% alcohol. Heat and dissolve •the crystals; evaporate down and crystals reappear. Repeat several times. The crystals are insoluble in chloroform, water and ether. They are slightly soluble in absolute alcohol, fairly soluble in hot gla- cial acetic acid, but best solvent in 60% alcohol. Uric acid, ghxuronic acid, oxalic acid and acetone may give positive tests. This test should not be done on a urine containing albumin. Dextrosazone 78 Monosaccharides. Glucose, levulose and galactose. Leviilosuria. There are 3 cliuical groups. 1. Alimentary. 100 grams is the normal limit of tolerance. Churchman (See J. H. H. Bui.) found that 26% of normal persons show levulosuria in 100 grams of the sugar, while some patients with actual hepatic disease do not. Churchman's conclusions on the Strause levulose liver test are: 1. The test is modified by extrahepatic fac- tors of sugar metabolism (renal, adrenal and pancreatic complications). 2. There are difficulties in the test (nausea and vomiting ) . 3. The clinical value of the test is insufficient to warrant its continuance. 2. Diabetic. A good many diabetics have a tolerance for levulose. The occurrence of levulosuria constantly with glucose indi cates a rather serious prognosis. It also gives an error in calculating the glucose excretion. 3. Idiopathic. There are only 8 of these cases reported in the literature. Those patients who show continual levulosuria generally have some complication in the glands of internal secretion. Three of the cases re- ported had hypophj^seal trouble and others had trouble with gonads. Seliwanoff's test for levulose : Urine 10 cc. Concentrated HCl 5 cc. Kesorcin few Xls. Bring the solution to a boil or put in boiling watei 79 for .')() secoiul>'. First a reddish blush a])pears, and upon standing and allowed to cool a granular red- dish precipitate forms. Mannose, maltose and glu- cose in large amounts will give the test, also. Pentosuria. Kinds : 1. Alimentary. Follows heavy ingestion of pen- tose containing food, such as apples, plums, cherries, etc. It probably occurs more fre- quently than recognized. It is character- ized by optically active xylose and arabi- nose. 2. Diabetic. Frequent in severe types. It is characterized by L xylose, Avhich probably comes from broken-down pancreatic nucleo- protein. 3. Essential or ideopathic (Dr. Janeway, Am. J. Med, Sci., Sept., 1906). Features : 1. Constant excretion irrespective of the diet. 2. Hereditary tendency. 3. Patients excrete it for long periods without evil effect. 4. Sugar excreted in this disease optically inac^ five. 5. Amount excreted is usually small (.2 to .6%). One case with 1% reported. C). Specific gravity somewhat increased. 7. Amount of urine not excessive. 8. Acidity high. 9. Pentoses fed come through in the urine as such. 10. Pentose may be formed from glucose, but not determined. 11. Glycuronic acid excretion continues as normal. 80 Tests for pentose. Phloroglucin (Tollen's). To urine add a small amount of HCl saturated with phroglucin. Heat in water bath. A deep-red color indicates a positive reaction. Watch for the initial shade of red to appear and then cease heating. AVhen extracted with aniyl alcohol and examined spectroscopically a band is seen between D and E. Errors : Test given also hj lactose, galactose and glycuronic acid. Orcin. To urine add HCl and a few crystals of orcin. Heat in water bath. The development of a green color and the formation of a greenish pre- cipitate indicates a positive test. Extracted with amyl alcohol gives a band betAveen C and D. Bial's method : Solution : 30% HCl 300 cc. 10% Ferric chloride 30 drops Orcin 1 gm. About 4 or 5 cc. of this solution are heated to boiling, and urine is added drop by drop (not over 1 cc). A positive reaction is indicated by a clear emerald green color. This test is best of the three. Glycuronic acid. Glycuronic acid is an oxidation product of sugar metabolism and is not concerned with protein meta- bolism. It is not a forerunner of diabetes and does not represent anything but detoxication. Its osazone has the loAvest melting point of any met with. It has an aldehyde group, which accounts for its reduc- ing properties. Pure glycuronic acid is dextro-rota- tory. Test for gl^^curonic acid (Tollen's) : To 5 cc. of urine add % cc. of 1% alcoholic solu- tion;of naphthoresorcin and 5 cc. of con. HCl. Put in water bath and allow to stand 15 minutes. Allow 81 to cool at room temperature for -L minutes, and then cool under tap. Add ether, which extracts a blue- violet color, indicating a positive test. Examined spectroscopically, an absorption band occurs at b, but is not specific. Clinical significance : 1. Glycuronic acid combines with toxic sub- stances and appears in the urine as glycu- ronates. Glycuronic acid combines readily with the coal tar products, morphin, cocain, etc. ; therefore, after such medica- tion lools: for glycuronates in the urine. 2. Copper and bismuth reduction absent or atypical. 3. No fermentation occurs. 4. Glycuronates in fi'esh urine are levo-rotatory, and osazones are formed with difficultv (m.p = 114 to 115 deg.). 5. They are easily confused with pentoses be cause reactions are same, but can be differ^ entiated by optical activity, glycuronates being levo-rotatory and after hydrolysis become dextro-rotatorv. 82 SCHEME FOR DETECTION OF AN UNKNOWN REDUCING BODY IN THE URINE. I. Feliling's test: A. Negative, no sugar present. B. Positive, proceed with II. Njlander's test: A. Negative, eliminates confusing substances in I. B. Positive, sugar is probably present, so proceed with III. Fermentation test: A. Positive, sugar present may be glucose or levulose. B. Negative, reducing substance may be: Lactose, maltose, saccharose, pentose, glycuronic acid. These become positive with hydrolysis. IV. Polariscopic examination : A. No. I, II and III positive: Urine D-rotatory : indicates glucose. Urine L-rotatory : indicates levulose. B. No. 1, II positive and III negative : Urine D-rotatory indicates: Lactose maltose, saccharose, alimentary f^en- tosuria. Urine inactive indicates pentose. Urine L-rotatory indicates glycuronic acid. V. Phenylhydrazine : A. Osazones easily obtained with dextrose, levulose, pentose, maltose. B. Osazones obtained only by special proce- dure with lactose. C. Osazones obtained after hydrolysis with glycuronic acid or saccharose. 83 Glycuronic acid osazones have melting point of 114 to 115 degrees. Pentose osazones have melting point of 156 to 160 degrees. Eemaining osazones have melting point of 200 to 205 degrees. 84 QUAXTITATIYE DETEKMIXATIOX OF SUGAE. Urinary output and specific gravity. (Xaimyn.) This method is inaccurate, but in the absence of other means gives some information as to the amount present. Amount of Urine. Specific Gravity. % of Sugar. 2000 cc. 102S to 1030 2 to 3% 3000 cc. 1028 to 1032 3 to 5% 5000 cc. 1030 to 1035 5 to 7% G to 10.000 1030 to 1012 G to 10% ^'/K'(:•//fc gravity-icrincntation method. This method is based upon the principle that fermentation by yeast results in lowering the specific gravity. A small amount of albumin can be neglected and the urine should be acid in reaction; if not so, make acid with acetic. Technique: 50 cc. of a 21-hour specimen of urine are taken and the specific gravity is determined. Add 2 cc. of a thick yeast emulsion and allow to stand from 21 to IS hours at 37 deg. C, until the reduction test is negative. Again make specific gravity determination. The ditference between the first and second determination multiplied by 231 coetiicient equals the per cent of glucose. This metliod is accurate to .1%, but sugar present should not be less than .5%. Fermentation metliod. A. Einhorn's method. Mix a sufficient quantity of urine to fill the special fermentation tube with a piece of yeast the size of a pea. Incubate for 21 hours at room temperature or 6 hours at body temperature. Eead the amount of gas given off and its equivalent percentage of sugar on the scale. Eun controls : Activity of yeast, fermentable substance in yeast, and urine alone without yeast. B. Lohnstein's method. This is the most accurate. To % cc. of urine add .2 cc. of yeast solution (1 85 part yeast to 3 parts water) and layer this solution on the mercury in a special fermentation apparatus. Tilt the apparatus until the mercury in the upright column lies even with the zero point on the scale. Turn the cork so as to shut off the air and incubate from 5 to 6 hours and read. Titration metliod. 1. Benedict's method. Advantages : 1. Single solution. 2. Permanent. 3. Sharp end point, degree. Solution : 4. Accurate to a marked Pure CuSO^ NaCOg crystaline Na or K citrate KSCN (accurate) 200 gm. 18 gm. 200 gm. 125 gm. 5. 5% solution KSCN 5 cc. 6. H2O qs. ad. 1000 cc. Dissolve Nos. 2, 3 and 1 in about 800 cc. of boiling HgO and filter. To filtrate add No. 1 dissolved in hot H2O. Allow to cool and add ^o. 5 and Xo. G. Technique : 25 cc. of the reagent are placed in a porcelain dish and 10 to 20 gm. of NaoCOg added to make the end reaction more clear. Run the urine diluted 1 to 10 in from a buret, rapidly at first, Avhich gives a white precipitate, and then 2 or 3 drops at a time, boiling 30 seconds between each addition. Add distilled water if the solution becomes low. The end reaction is the permanent (lisapperance of the blue color. Calculation: 25 cc. of the reagent equals .05 gm. of