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After coolmg the contents of the centrifuge tubes are transferred quan titatively mto Hagedom-Jenaen tubes. Whea large centrifuge tubes are used fig 24 rthe titrarion-maybe perfenned directly m the centrifuge tubes.

E-xact potassium values can only be obtained when tho scrum is completely free of hemoglobin. Principle of the mdhod Calcium is precipitated as calcium oxalate in serum or plasma.

Example ThlcMuliate used up by the blank 1 09 ml thloaolfate used up by the aample 0. In order to prevent any precipita tion of magnesium when ammomum oxalate is added, drops of acetic acid are added until the green color changes back to red.

Three ml of ammonia solu tion arc added the tubes ogam centnfuged and the supernatant removed as described above. This procedure is repeated tmee more The precipitate must not be stirred up durmg tho process of washmg Now the precipitate is dissolved m 0.

The phoephorua content of the precipitate la detenmned and from this value tho magnesium content of tlie ongmol aample a cal ciliated.

ReagenU 1 cold saturated ammonium oxalate solution 2 2 per cent ammonium phosphate Bolution 3 concentrated ammonia MflJnly according to W Donnie Journ Biol Chem 51 One mb of 10 N HiSOi ts slowlj added to the hot sample to dissolve the preerpilate After cooling it a mode up to the mati.

Now the sample is filtered through a Pregl filter fig 25a while still hot To avoid the formation of a precipitate along the walls of the filter tubing the apparatus is nnsed uith hot water immediately be- fore use.

A high magne- sium lev cl can bo encountered in mvo'Uicnla and during pcnodic paml VMS of tho extremities which maj be connected with an occasiona! To each flask 3 ml of sodium acetate and bromme-glacml acetic acid is added from a droppmg flask until the brown color bromme persists.

After each determination the apparatus must be washed with re- distilled ivater, the water being sucked through the inner tube of the condenser with a water pump If organs are to be digested 1 ml of concentrated nitnc acid is added to the digestion acid and after the water has evaporated the sample is treated as described above.

Pfld of reagent 2 the following reagents are used reagent B HNOi concentrate p. The digestion is modifled os foUoxrs With a capillary pipet 0 Calojlation Thiosulfate value for the sample rmnus value obtained for the blank multiplied by 0 gives mg iron m the unknown sample.

Procedure One ml of HCl 1 is added to 2 ml of scrum or plasma slight hemolysis will not mtorfero m a "mnll Erlonmoy er flask or test tube, well nuxod and allowed to stand for ten mmutes.

After the addition of 2 ml of tnchlomcctio acid 2 the flask is left standing for another ten mmutes whereupon the mixture is transferred to a centnfugo tube and centnfugod The supernatant is filtered through a small iron free filter mto a graduated 10 ml gloss tube.

Between 35 and 35 ml of filtrate ore obtamed 0 05 mL of phenantrohne eolation DETEHinNATlON OF THE KATIONB 3 0 1 ml of hTdroquilione Bolution 4 and 0 05 ml of mtrophenol 6 are added to the tube xveU imxed and ammonia 6 la added alowiy drop by drop At first the pink color of the phenantrolme eomixrand appears, after a few more drops the definite deep yellow color of the mdicator can be observed At this point sulfuno acid 7 ifl added drop by drop untfl the yellow color diaappeors and the pmk color is again viaible.

The color ivill remain amstant for several hours and the reeding need not bo done immediately Two ml of standard solution 0 in a centrifuge tube are mixed with 1 mL of HCl 1 and 2 ml of tnchloracetio acid 2 , then centrifuged and filtered The some amount of this filtrate os serum filtrate obtamed M treated exactly as described above.

Values bdow C6 7 per cent and above y per cent for males and below 5o y per cent and above y pier cent for fomales must be considered pathologic Tho scrum level of iron is lowered m all ane mias with iron deficiency, often m jxTmciouB anemia during remission and m various infectious diseases It is elevated m hcanolytic processes, shortly after transfusions and after massive therapeutic iron dosage, often m iHsrnicious nnfTnin.

If very bttle scrum ts available only 2 ml of serum may be diluted with 10 ml of water and 8 ml of trichloracetic acid is added For the onaJyEiB 6 ml of flltxnto corresponding to 0.

It 10 well shaken until the red color of the solution begins to fade "Ilicn it is cooled with shaking under r unnin g water until the color turns green The supematant is decanted and the romairung mercury is washed several tunes with distilled water the nwh water bemg added each tune to the decanted supernatant The bquid is mode up to mL with dist il led water The solution should be dear, othennse it must be allowed to stand until the precipitate has settled and 13 again decanted b carbonate-free NaOH m a porcehun dish resting on a cork plate Gm of NaOH ore dissolved In Gm of water with Accordlnf to a soggoetioa of Rassol L.

After this timo the solution roust be colorlm. It a then cooled and dflutod w ith water to tho 35 mL mark and Nessler roogent 6 It added to tho 60 ml mark with cooling, and the colon are compared In the colonmotcr CalcuUUwn For the Dubosquo colonmoter reading of standard X 30 ,.

With a capillary pipot 0 1 ml or 0. After coolmg the precipitate b filterod off Whatman Nr 42, 7 cm diameter Now 0 ml of filtrate arc transferred into the Pamns-Wagner distillation apparatus, the funnel is washed with 0 ml of totraborato solution, and the liberated NHj distilled twice, each time over a penod of four minutes, care being token that during the first four minute penod the coo denser tube reaches well below the surface of the liquid In the receiver flask.

If small normal amounts of urea ore expected and also m research work it is recommended to usd lass hypobromite, 2 ml of hypobromite for 46 mg per cent of mtrogen.

Caladaium Thiosulfate used up by the blank mmus thiosulfate used up by the sample multlpbed by OXHOfl atomic weight of mtrogen 14 dmded by 3 X gives urea mtrogen content of the sample m mg If Merenca is multiplied by S2 mg.

To one tube is added 0. If the color of the unknown sample is more intense than the color of the standard the sample is placed into the wedge and the standard is placed into the cup and the calculation is done as follows X Should the color of the sample be more intense than that of the standard, the sample is diluted Avith distilled water, the dilution taken mto consideration and calculated scconiingly If a high urea value is expected, only 0 01 mL of blood is used 0 49 ml of buffer ureaae mixUire are employed m this case and the final calculated value is multiplied by 2 Under normal conditions the urea nitrojen content is mg per cent fastmg value, houra after last meal The urea level m blood depends upon the protem mtahe.

There- fore urea values can oily be compared under mmilnr dietary con ditKms. B inamirato meaauremeDt of unne v ojumc. Consequently analyses can be ran off at the rate of about one every four or five mmutea.

One blank analysis run at tbo beginning, serves for an entire senes of onalj'scs. This is followed by 6 mk of tnchloracetic add 4 The mixture is ivcU slinkcn and filtered.

Procedure Each analysis is performed in several steps 1 the reagents, i. Examj U reading of standard 20 mm.

Then the tubes are pbced mto a boilmg nater bath for two mmutea The tubes are cooled rapidly with running water and the colors are com pared m the Dubosque colorimeter Caladaiton readmg of standard X canc entratioD of standard X readmg of sample — unc acid content m mg per cent Exomple 20 mnu IK mm.

In this concentration of one aad the color IS mteneo and can be read easily Caladalwn a for the Dubosque cdonmeter reading of standard X 8.

One ml of this solution corresponds to 0 03 mg of unc acid IIiIb solution wfll keep mdefinitely when stored m a dark bottle and m a cool place 4 cold saturated sodium carbonate solution, approxnnately 20 per eexA.

If the sample is expected to contain much creatimno, 5 ml of standard solution are oaed TTie mftnmfil color Intensity is reached after minutes and the colors are compared m the Dubos- que cdorunoter with the standard set at 20 or m the Helllge colon meter The reading should be perfemned latest fifteen min utes after the picnc cod reagent has been added CalcultUian a for the Dnbosquo colonmeter Reading of atandard X conecntratioo of standard reading ol unknown empfe " creMmine content of sample m mg If the above figure B multiplied by 6 ml of filtrate corrosponding to 0,5 ml of scrum analysed the creatlmne content of the sample 18 obtained expressed in mg per cent.

Pnnaple of the method Blood or seram is deprotemued with tn chlorecetio aad A known amount of thymol or bronunated thymol 18 added to the protem free filtrate For the standard tubes Br thymol IS used as it has been shown that the thymol mdican com pound deecmpoees after a much shorter tune than the correspoctding Br thymol compound.

At tbo same time trichloracetic amd and the alcohol of the Br thymol m the presence of concenlrsted HCl form a com pound which appears as ofly drops m the reaction mixture and which absorbs quantitatively any dye formed the isolation of the dye by extraction with chlorofonn is avoided In this manner The oil drops win also appear in the nbeence of thjTnoI, i.

In uremic coma values up to 2 7 mg per cent have been noted 3 eclampsia m the bcgmnmg of the disease a rapid rise up to 0 0 mg per cent, fast declmo mth the disappearance of the acute symptoms, 4 nephritis of pregnancy values of 0 A further nse is noted with mcrcasod seventy of tho disease In contrast to eclamp- sia a high mdican level wnll persist for many weeks after dehvery, oven at times when tho urea level has returned to normal Xanthoprotein Reactiov Pnnctpfe of the method Tho aromatic compounds are nitrated with mtno acid and the resulting nitrocompounds form a yellow brown solution m NaOH, IleageTiU 1 20 per cent trichloracetic aad 2 concentrated HNOj specific gravity 1 4 3 pure NaOH, 33 per cent Procedure Serum fa mixed with equal aunounta of 20 per cent tnchloracotic acid, and filtered To tho clear protem free filtrate one-fourth of its volume of concentrated IINOj is added and heated to slow boiling for i mrauto After cooling approximately J of tho volume of NaOH 3 la added In.

Ucagtrde Seo nonprotem nitrogen p Procedure Two hundredths of a rafUniter of eorura or plasms is drawn m a long capillary plpot Sahli pipet , the tip is wiped carefuDy and the contents oro debiered into a micro-lvjoldahl flask.

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