Micro Assay of Vit in Clinical Chemisty

8/3/2019 Micro Assay of Vit in Clinical Chemisty

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The M icro bio log ic A ssay o f V itam in sin C lin ic al Chem is try

D on ald D . V an S lyke Lectu re, 1958

Harry Sobotka

HE STUDY OF PRESENCE AND QUANTITY OF VITAM INS in the body fluidshas gained ever-increasing importance for clinical m edicine. M ostvitam ins have been discovered in the course of investigations of thedeficiency disorders to which their absence gives rise, and some havetheir names actually derived from the diseases caused by their ab-sence. The practice of their quantitative analysis is not confined tothese conditions, but it has been found that the amount in which theyoccur in various fluids and tissues, and the rate at which they areabsorbed, bound, retained, excreted, or otherw ise metabolized, bearssignificant relationships to a variety of pathologic states and is apt toillum inate their etiology (1).

Rather than trying to give a form al definition of vitam in, we shallshortly survey the principles of their biochem ical function. Let it besaid at the onset that m any of the water-soluble vitam ins of the Btype have been correlated with certain enzymes or groups of enzymes;they form indeed the prosthetic group of these enzymes, the coenzyme(Table 1). Their character of vitam ins is based on the inability of theanim al organism to synthesize the non-protein moiety of vital en-zymes. In these instances, know ledge has advanced to the point wherewe may answer w ith more or less assurance the question, W hat con-nection is there in a given avith .m inosis between the morphologiclesion and physiologic dysfunction, their m etabolic etiology, usually

From the Departm ent o f C h em i st ry , Moun t Sinai Hospital, New York, N . Y .Delivered before the N ew York M etropolitan Section of the Am erican Association of

C linical Chem ists, February 11, 1958.Received for publication February 17, 1958.C op yr ig ht # {1 74 }958 , by Th i Asic AssoowrzoN 07 C LIN IO AZ . C ES NIST S, INC.

93


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94 SOBOTKA C lin ka l C 1 i.in kfrTable 1. v ITAMINs AS COFACTORS IN ENZYMATIC R EA CT IO NS A ND M ErA BO LIC SYSTEMS

Via.,in Enzymatic function

Thiam ine CocarboxylaseRiboflavin (Ily co ly sis (c yto ch rome o xid as e)Nicot inamide DehydrogenasesPyridoxal Transam inase, am ino acid decarboxylase, tryptophan metabolismPanto thenic acid Coenzym e A , 2-carbon transfer, citric acid cycle, con jugation of

acidsbile

Biotin F atty acid m etab olismFolinic acid N ucleic acid m etabolismCyanocobalnmlne Nucleic acid metabolism ; methyl transfer, choline synthesisThioctic acid Oxidative decarboxylation, conjugation of thiam ine

centering on an enzymatic defect, and the prim ary cause, namely theabsence or inavailability of the vitam in?Methods of chem ical analysis have been applied to the water-solu-

ble vitam ins, the vitam ins of the B group, and are serving as controlfor the anim al test-e.g ., in the industrial preparation of these fac-tors. W herever the chem ical constitution of a vitam in has been clari-fied, chem ical analysis has of course become the prim ary checkingmethod in production and in the clinical laboratory.

I shall discuss today m icrobiologic methods for a number of de-ficiency factors, which have been developed in our laboratory and else-where for dilnicochem ical problems. M ore than 10 years ago, I hadbecome interested in the m icrobiologic assay of deficiency factors. W edeveloped a method for the deteTm ination of inositol in patientsserum by means of a strain of S ac ch arom yc es c arisb erg en sis, basedon A tkin s modification of W oolley s m ethod (2).

In the course of subsequent work on thermophilic algae and bacffliwe had to use synthetic media; these experiments led first to thestudy of nutritional requirem ents and trace elem ents and eventuallyto the development of m icrobiologic assay methods w ith thermophificand mesophilic bacilli (3) and w ith protozoa.1

Table 2 gives a survey of the vitam ins studied, of the deficiencysymptom s produced by their absence, and of other pathologic condi-tions where they have been suspected to be involved.

W e are using various m icroorganisms, bacilli, cocci, and protozoa,I am greatly indebted to Dr. Seymour Hutner for his counsel as protis tologist, to th e

staff of our vitam in a ss ay l ab or at or y for their devoted assistance, and I w ish to thankespecially my collaborator Dr. Herman Baker for hi s ingen ious approach to the theoreticalan d experimental aspects of this work.


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Vol. 4, No. 2 M ICROBIOLOG IC ASSAY OF VITAMINS 95Table 2. WATER-SOLUBLE VITAMINS AND HUMAN Diszasz

ViAn.4nCondition w4t

d4cioncy Area of i nvoZveawntThinm ine Beriberi N eurologic conditionsR iboflav in Cheiosis Dermatologic conditionsNicotinamide Pellagra Derm atologic conditions, dementiaPyridoxin group Infantile convulsions D iabetes, infection , toxemia of pregnancyPanto thenic acid Neurologic; endocrine malfunctionB iotin D erm ato logic conditions ()Cyanocobnlnrniu Pernicious anem ia N eurologic an d hematologicFolic acid group Pernicious anem ia N eu ro lo gic a nd h em a to lo gicT hio ctic ac id . H ep atic d iseaseInositol Deficiency of fat metabolism

but our technics deviate in several points from those of traditionalbacteriology. The media we are using are strictly synthetic. Theselection of grade and brand of the inorganic constituents used inbulk are based on extended comparative studies and are designed toexclude known and unknown trace elem ents at or above those m inuteconcentrations, where they still could influence the outcome of thetest. Sim ilar considerations apply to the organic constituents of themedia, which are rigorously screened and confined to compounds ofknown constitution and good uniform ity such as oligosaccharides andam ino acids. W e can not accept, for example, proteins such as serumalbum in, however well they may have been purified, since their con-stitution is unknown indeed, and the nature and amount of bound,adsorbed, engulfed, or otherw ise connected factors rem ains eitherundeterm ined or variable. Such factors m ay themselves be vitam ins,or lipids, or, most important, trace elem ents.

The latter are introduced individually and each of them is testedfor its function as a required factor for the m icroorganism in ques-tion. In many instances we are lucky to be able to discern the specificpurpose of their presence, usually as the known component of anenzyme system . As w ith bulk ingredients, w e have assured ourselvesof the purity of the compound we introduce from traces of other traceelem ents, so to speak-trace elements of the second order.

M ost of the trace elem ents are heavy metals; the absolute require-m ent of them in the medium places us in a quandary: Too high concen-tration.s w ill prove toxic to the m icroorganism , too low concentrationsw ifi not support grow th beyond a rather low level. The way out ofthis dilemma consists in the addition of a chelating agent which form s


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96 SOBOTKA Clinical Chemistrycomplexes w ith the various heavy metal ions. In this m anner theionization of the various cations is repressed and kept below the toxiclevel. A t the rate at which ions of a given species are incorporated ina grow ing bacterium , some atom s dissociate from the complex andbecome free cations in proportions governed by the dissociation con-stant of the complex. W hen, on the other hand, free ions are liberatedby lysis of the m icrobes, they are chelated to maintain the equilibriumand no toxic concentration of the free ion can accumulate.

Substances like citric acid serve this purpose well enough, but, asthey themselves m ay enter into the metabolism of the m icroorganism ,they threaten to vanish, and their chelating effect w ith them . The useof nonmetabolizable chelating agents, especially of ethylenediam inetetracetic acid (Versene) and the related Versenol has removed thisdifficulty. The numerical values of the dissociation constants of theircomplexes w ith various trace elem ents render these compoundsideally suited to act simultaneously as buffers for a variety of m etalions.

W hat has just been said about nonmetabolizable chelating agentsholds equally for nonmetabolizable pH buffers. Superior to phos-phate and other conventional buffers, which could be incorporated orconsumed as nutriment, substances have been selected such as trans-aconitic acid, triethanolam ine, and dihydroxyethylethylenediam inewhich are not m etabolized and insure stability of pH .

METHODSThe media may be made up from the individual ingredients eachtim e when required, or the solid components m ay be well m ixed inquantities, totalling several kilograms and then used by dissolvingappropriate amounts of such a solid m ix with the addition of thefew liquid com ponents.

The medium is then distributed, usually by automatic pipet hold-ing, say, 5 m l., into a number of squat glass Erlenmeyer flasks of 10or 25 m l. capacity . A t this point a volatile preservative may be added,which w ill later completely evaporate on autoclaving. R ising quanti-ties of ingredients to be tested for their effects on bacterial grow thare added to the flasks. In the case of clinical analysis, system aticdilutions of suitably treated blood, serum , urine, cerebrospinal fluidetc. are added, usually in the ratio 1:3:10:30:100, etc., w ith extra dilu-tions interpolated in a critical range. Finally, all flasks are properlym arked and covered w ith glass or alum inum caps. A ll these opera-


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Vol. 4, No. 2 M ICROBIOLOGIC ASSAY OF VITAMINS 97tions are carried out w ith a m inimum of exposure to atmospheric con-tam ination, but w ithout flam ing and w ithout the use of cotton plugs,as these have been found to contain biotin , certain lipids, and otherunw anted factors.

The flasks are then placed in a Pyrex glass tray of 20 by 36 ems. andare then sterilized at 118#{176}or 30 mm. in the autoclave to avoid con-tam ination with spore form ers. A ring of condensed water betweenflask and cap protects the contents against contam ination.

Upon removal from the autoclave, the flasks are inoculated, usingthe customary sterile precautions. In the case of thermophilic cul-tures to be incubated at 55#{176}r higher, the bottom of the tray is cov-ered w ith a layer of 0.5-1.0 cm . of water to keep the volumes in theflasks constant. The tray is then covered w ith an inverted tray ofidentical dim ensions, and both trays are sealed together hermeticallyby plastic tape. They are now placed in an incubator of the propertemperature or, in the case of chlorophyll containing algae, in a boxw ith overhead illum ination which has been selected for optim al in-tensity and wave length. W e do not find it necessary to acceleratealgal grow th by shaking.

A fter a given incubation period, e.g . 24 hours or 5 days, the testsare read by determ ining the optical density (O .D .). The contents ofthe individual flasks are read in cells of dim ension 12 by 12 by 48 mm .in a W elsh Densichron. The reading of bacterial cultures rarely ex-ceeds unity . The algal cultures run to optical densities of over 3.0;for higher values appropriate dilutions are m ade and the O .D . of theoriginal solution is obtained by computation.

Together w ith each group of unknown samples, a standard test,consisting of a series of dilutions of the pure vitam in, is set up, inocu-lated and incubated under identical conditions. From its optical den-sities one prepares a standard curve (Fig. 1). In a successful run, theO .D . s of the unknown sets w ill fall on curves of sim ilar shape andamplitude. The position along the abscissa at which an unknowncurve coincides w ith the standard curve perm its the calculation of thevitam in content of the unknown sample.

RESULTSW e shall now relate some of the results obtained in specific in-

stances and refer to special technical points as we go along.


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B

4Fci3 /////

I 3 10 30 100 300 1000 3000

98 SOBOTKA Clinical Chemistry

ijg./mI.Fl%. 1. Growth of L oc to ba os flu a i ci ohm an ns i (L), Ochr o rn o naa ma lh c rn en si s (0), ugiena

grac#{252}ssB) w ith increasing am ounts of v itam in B12, The o rdin ate g ives optical density ofthe cultures. The dotted line (L) gives the curve for L ac to ba ci il us i es chm an n# {2 48 }ith th eordinates adjusted to the level of Eugiena.

CYANOCOBALAMINA vitam in that is effective in quantities 1000 times sm aller than any

other vitam in (w ith the exception of biotin) is cyanocobalam in, orvitam in B12, the extrinsic factor of Castle. The quantities in whichit occurs in the human body fluids and tissues are proportionatelym inute and many orders of m agnitude below the detectability by themost delicate physicochem ical methods. Its natural history has notbeen fully disentangled, but we know that its absorption through thewall of the gastrointestinal tract depends in general on the presenceof the intrinsic factor, a specific mucoprotein. The part it plays inhematopoiesis, or, more w idely speaking, in nucleic acid synthesis,appears to be of a multiple nature and interlocks w ith the role of thefolic acid complex. A tentative scheme of these interrelations is de-picted in Fig. 2.

A s has been so well demonstrated in the cases of mutants whichhave lost a specific enzyme, one finds that either a vitally importantenzyme, when absent in a m icroorganism s own arsenal, m ay beadded, or one may compensate for its absence by supplying a product


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Vol. 4, No. 2 M ICROBIOLOGIC ASSAY OF VITAMINS 99D iet or In testinal F lora Gastric M ucosa

Folic Acid Vitam in B1, Intrinsic Factor(A sco rbic A cid)

-B it-C om plex in BloodFolinic AcidIUracil- -+Thymine1 + Desoxyribose(M eth yl D on or) I

Thymidine(Desoxyriboside)a nd P ho sp ha teIT hym idylic A cid(Desoxyribotide)N ucleic A cid(B lo od C ells)

F ig. 2 . Function of vitam in B12 and folic acid in nucleic acid synthesis.

in the metabolic chain, follow ing the step for which the enzyme ismissing.

An example: One may determ ine vitam in B12 by its effect on thegrowth of Lactobacillus le ichmannii, Euglena gracilis, O chromonasmaihamensis and a vitam in B12-less mutant of Bacillus coli. L .ieichmannii requires vitam in B12 primarily for the synthesis ofdesoxyribosides, whereas (a) the form ation of folinic acid and (b)the methylation of uracil m ay be taken care of by alternative en-zymes. Hence, the organism w ill get along w ithout vitam in B12, ifprovided w ith sufficient nucleosides. In contrast, Euglena an d Ochro-monas, require vitam in B12 at two or more spots in their m etabolism ;thus, it becomes impossible to replace its enzymatic functions by ad-dition of an end product. H ence, the high, one may say multiple,specificity of vitam in B12 for these organism s.

One may discern in Fig. 2 the involvement of our enzyme systemin the methylation of uracil. The absence of B12 may be overcome byaddition of relatively high concentrations of m ethionine, a m ethyldonor. In this instance the subnorm al concentration of an enzyme ism ade up for by the excessive concentration of one of the reactants,follow ing the mass action law . A s a practical application of this ob


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From J. B . Ford (4).

100 SOBOTKA Cl in ic a l Chem is tr yservation the assay organism may be used for the quantitation ofmethionine.

W hen we discuss vitam in B12, we must consider the numerouscongeners of the true vitam in B12, in which nature or directed bio-synthesis in the laboratory have replaced the benzim idazole groupin the nucleotide portion of the molecule by other cyclic groups suchas adenine, for example, or have om itted the nucleotide portion alto-gether, as in factor B . These various factors are inferior substitutesfor the true vitam in, and the extent varies to which they may replaceits functions; their effectiveness as a vitam in B12-substitute in higheranimals is negligible, but assumes different values for differentm icroorganism s (Table 3). The presence of these congeners, whichone may call pseudovitam ins or paravitam ins, in body fluidsw ill produce discrepancies in the response of diverse assay organ-ism s. Among them , O chrom onas m aiham ensis is in our experiencethe most finicky and thus the most specific for the true vitam in. Byvirtue of its high specificity , the response of this phagotrophic or-ganism approaches the response-and that m eans the requirem ents-of the mammalian organism . It has been our preference in clinicalstudies.

There is one more hurdle to be taken before practical application-the varying amounts of the vitam in in the body fluids and in the tis-sues in bound form . The binding factor may be identical or relatedw ith the intrinsic factor, but we have discovered distinctly differentbinding factors in various m icroorganism s. S ince only the free vita-m in elicits response in the assay organism , the fluid must be hydro-lysed to deconjugate the complex and to destroy the binding fac-tor. This is accomplished by heating at 118#{176}or 30 m inutes at pH4.5, conditions which do not affect the vitam in itself; the presence ofcyanide or bisulfite is necessary to prevent oxidation.

Table 3. REL&rIvE vIvAMm B12 RESPONSE TO DIPFERENT COM POUNDSSubstance

Lb .Ieicbmannii

EuglenagranUle

Ochromonasmaihamensis

Cyanocobalam in 100 100 10 0Factor A 64 137 3.4Factor B 60.3 4.2 0Factor C 14 100 0Pseudovitamin B12 400 800 1. 2


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Vol. 4 , No. 2 M ICROBIOLOGIC ASSAY OF VITAM INS 101I have already described some features of the m icrobiologic assay

for vitam in B12, especially the specificity of various organism s andthe specificity for various pseudovitam ins. The norm al content ofvitam in B12 in human serum ranges from 300 to 1000 ,g./m l. or, toexpress this in more fam iliar dim ensions, 0.03 to 0.1 11g. in 100 m l. ofw hole blood.

CLIN ICA L S TUD IE SIn a study of the distribution of B12 between blood plasma and

erytlirocytes (RBC) in untreated cases of pernicious anem ia, wefound that the RBCs hold on to their B12 and that the extrem ely lowvalues of B12 in whole blood in pernicious anem ia are due to its signaldecrease in the plasm a (5). The RBCs contain about two-thirds ofthe norm al amount, but their contribution to the over-all titer is ofcourse reduced by the low hematocrit figures in anem ia (Table 4).

In a study of our obstetric material w e have compared the vitam inB12 content of mothers and their infants blood (6, 7). W e found, inagreement w ith other authors, that the newborn baby holds on toits vitam in B12 at the expense of the mother, whose B12 sinks in themajority of unmedicated cases to levels typical for megaloblasticanem ia. W e have supplem ented this investigation with determ ina-tions of folic acid (PGA).

W e have developed a thermophiic bacillus, a strain of B. coagu-tans, for the m icrobiologic assay of PGA and its congeners (8). Quan-titatively sim ilar responses are obtained by folio acid, folinic acid,presumably its active form , and by its polyglutam ic acid conjugates(w ith due allowance made for their labifity). A s these results areobtained without previous deconjugation, B . coagulans appears su-perior to Streptococcus fecalis which requires pretreatment w ith de-conjugase, an enzyme supplied from chicken pancreas and which is

Table 4. VITAMIN B12 CONTENT O P P LA SM A, RBC s AND BLOO DS/hO. VU . B1 2

Hematocr itlas,no RBOe BloodSubject (1 m l.) (1 m l.) (1 m l.) reading

Normal 1 62 0 17 0 42 5 44Norm al 2 60 0 14 0 410 42Normal 3 32 0 18 5 26 0 47Peru. anem ia 1 32 115 45 15Peru. anem ia 2T ota l g astre eto my

2350

9518 5

3575

1520


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102 SOBOTKA Clin ical C hem istryinvariably accompanied by folic acid, thus necessitating large correc-tions for this added extrinsic folio acid. But the most striking advan-tage of the thermophiic bacillus is the complete absence of anycontam inants viable at 55#{176}.his greatly simplifies operations, as itelim inates the need for the various precautionary measures which Ihave mentioned previously in the description of the general technic.

PGA in the blood during pregnancy was found high in contrast tovitam in B12. An important role ascribed to this vitam in in pregnancyis its protective action toward progesterone in overcom ing the in-hibitory effects exerted upon this hormone by PGA-antim etabolitessuch as am inopterin . Toward the tim e of parturition, progesteroneand folic acid fall simultaneously, and the fetus draws relentlessly onthe maternal stores of PGA as well as of vitam in B12, both vitam insbeing needed for fetal grow th, more specifically for nucleic acid syn-thesis. The differential thus established between maternal and in-fantile blood is even higher for PGA than for vitam in B12. Thesestudies indicate the importance of vitam in supplem entation duringpregnancy (Table 5).

The difficulties encountered in any attempt to disentangle the etiol-ogy of multiple sclerosis are well known. The vaccilating, unpredict-able course of this disease makes it doubtful whether one should ex-pect chem ical stigm ata to be perpetually evident or only during orbefore attacks and episodes of exacerbation. This question may beraised for any deviation from the norm al in the chem ical findings,whether one deals w ith proteins, lipoids, enzymes, or vitam ins. Thegreat variations in acuteness and degree of severity from patient topatient impel us to keep our sights and our hopes low in this battle.W e have, nonetheless, studied the PQA and B12 level in the cerebro-spinal fluid (CSF) of multiple sclerosis patients (9).

Table 5. B12 AN D PG A CONTENT OF MATEun1 AND INFANT BLOOD

QuartileB (g./,ni.) P GA (ng.Jm i.)

M asrnaZ InjatU Maternal Infant

let 25-120 35-250 1-4 1- 92n d 120-190 250-390 4-7.5 9-40Median 19 0 39 0 7. 5 44 )3r d 190-270 390-600 7.5-22.5 40-904th 270 600 22.5 90No. patients 10 4 70 67 38


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Vol. 4 , No. 2 M ICROBIOLOG IC ASSAY OF VITAM INS 103PGA in the CSF of norm al controls ranges from zero to 30 mLg./m l.,

about the same as in blood. Values of B12 in CSF are substantiallylower-0 to 30 g./m l.-as against 300 to 1000 ipg./m l. in blood. Inmultiple sclerosis the PGA values in the spinal fluid run as follows:

Quartile 11111g.F irst 10-60Second 60-200Third 200-500Fourth 500-4000

This means that more than three-fourths of the cases (to be exact 34out of 42) show elevations, m any of them to enormous amounts.

In the case of vitam in B12, 14 out of 41 cases showed elevated valuesin CSF, 9 of these to more than tw ice the upper lim it of the normalrange. L ikew ise, about one-third of the B12 values in serum were ele-vated. These findings point to a relationship w ith the neurologiceffects of PGA therapy in pernicious anem ia; first it seem s to mobilizeth e a va ila ble B12-stores with ephemeral clinical benefits but eventual-ly it produces combined lateral sclerosis. Possibly the deviationin the level of these vitam ins and of the pertaining enzymes influencesthe methyl transfer between lecithins and cephalins and thus playssome part in the demyelinating process. The tests m ay serve for thedifferential diagnosis of neurologic conditions brought on by disturb-ance of B12 metabolism , as in multiple sclerosis and combined lateralsclerosis and, on the other hand, other disorders in which B12 is notinvolved.

Peculiar deviations in the B12 situation have been claim ed in dia-betes and diabetic retinopathy (10). U sing a greater number of casesthan Becker e t at., we have shown that these claim s are untenable(11). In the course of this study we made however the follow ing ob-servation. If a norm al subject is injected intramuscularly w ith a loaddose of 60 g. of vitam in B12, h e excretes in the urine 10-40 g. (15-65per cent) of the load dose w ithin the next 8 hours (12, 13). In a seriesof 40 cases of cirrhosis, hepatitis, and other parenchymatous liverdisorders, w e find the excretion reduced below 10 g. w ith very fewexceptions. The diseased liver is known to have lost its binding powerand its function as a storehouse for vitam in B12, but this function hasbeen taken over by the circulating blood to prevent depletion of B12 inthe organism . The vitam in B12 load test has been compared w ith anumber of other liver function tests and was found to be well corre-


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104 SOBOTKA Clinical C hem istrylated w ith them , but more sensitive at the onset and during recoveryof the patient.

In another project, w e utilized the vitam in B12 load test in patientsw ith thyroid disorders. In thyrotoxic subjects we found the B12 levelsin the blood low and the 8-hour urinary excretion at an abnorm allylow level. By contrast, m yxedematous patients, starting out from anormal level, display a higher rise than norm als after 4 and 8 hoursand a higher excretion (Table 6). These findings suggest that B12turnover is accelerated in hyperthyroidism and slowed down in hypo-thyroidism (14).

FAT.SOLUBLE VITAMINSA few words must be devoted to vitam ins outside the B group.

Aside from ascorbic acid, these vitam ins comprise the fat-soluble fac-tors A , D , E , and K . They are involved in the metabolism of themore highly specialized tissue system s of vertebrates and otherhigher phyla w ith features such as a bony skeleton, a coagulable cir-culating fluid, a complex, strongly differentiated integument, and thelike. Thus, their mode of action may be expected to be more diversi-fled and more complex.

M icrobiologists and biochem ists alike evince a kind of abstem iouspreference for aqueous media; in the chem ists eyes the grow th ofm icroorganism s in a medium is a heterogenous reaction. Thus, theintroduction of such lipids as fat-soluble vitam ins would further com -plicate m edium and mechanism -an unpopular m easure, especiallyin the absence of efficient and at the same tim e nontoxic emulsifyingagents. Only in recent years have m icrobiologists turned to the studyof organism s among the ciliate and flagellate protozoa that are phago-trophic and, thus, capable of devouring fat droplets. Paramoebathrives on lipid media (15) and Paramecium is stim ulated by sterolsTable 6. BLOOD LsvziS AND ExcanTloN or B12 IN LoAD PasT (50 ng.) IN Tnmom ParIENrs

Thyrotozictlhbjacic

NormaZcontrols

Myaud.,natotzssubjects

No.cases 10pLO.

7VITAMIN B12 IN 1 ML .

4

B ef or e i nje ct io n 32 0 57 5 60 0After 4 hours 605 1210 1675A fter 8 hours 410 825 1315Urinary excretion in 8 hours(% of load dose) 8 17 37


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Vol. 4 . No. 2 M ICROBIOLOG IC ASSAY OF VITAMINS 10 5Table 7. M icroorganism s and Sensitivity of B ioassay

Vitamin Microorganism Sensitivity per m l.PoSe acid B . c oa gu ia ns (thermophil) 10 mig. i0Cyanocobalamin Euglena graciii8 1 jeng. 1012Cyanocobalamin Ochroinonas cnalhamensia 1 ng. 1012Thiamine Ochromonas malkamenRis 10 m,ag. 10.8Biotin O ch ro,n on as m aih arn ensia 1 1012B 6 group T etr cjiyinena pijr ifor mis 30 0 qog. 10 +Thioctic acid T etrah yrnen a p ijrifo rm ss 30 10b0+Pantothenic acid T etrahym ena pyrifornus 3 mpeg. 108 +Panto then ic acid Lactobaciiius arabinosu. 10 mng . 10 8

(16, 17) ; Lab yrint hula has absolute sterol requirem ents (18). M icro-organisms with unorthodox tastes of this type may eventually be de-veloped for the assay of fat-soluble vitam ins. A t present their deter-m ination must be achieved by conventional biologic or chem icalmethods.

SUMMARYThe examples which have been given above in the cyanocobalam in

and folic acid field illustrate the usefulness of the m icrobiologic assayof vitam ins in investigate work. The application of these methods toroutine determ inations w ill follow , once the indications for theirusefulness are established. Table 7 summarizes the vitam ins forwhich we have developed assay methods, the m icroorganism s used,an d th eir sensitiv ity .

The methods described are much simpler than they may appear tobe at first sight, and they require no expensive or special equipment.They can become in due course routine procedures and w ill contributeto one of the lesser known chapters of clinical chem istry.

REFERENCES1. Sobotka, H ., J. M t. Sinai H o8p. 24 , 1231 (1957).2. Sonno, S ., and Sobotka, H., A rch. B iochem . 14, 93 (1947).3. Baker, H ., Sobotka, H ., and Hutner, S . H ., J. G en. M iorobio i. 9, 485 (1953).4. Ford, J . E ., B r-it. J. Nutrition 7, 2 09 (1 95 3).5. Baker , H ., Pasher, I., Sobotka, H ., Hutner, S. H ., Aaronson , S., and Ziffer, H . Nolure

180, 1043 (1957).6. Baker , H., Erdberg, R ., Pasher, I., and Sobotka, H ., Proc. Soc. E xp . B toi. M ed. 94 ,

51 3 (19 57).7. Baker, H ., Z iffer, H ., Pasber, I., and Sobotka, H ., Brit . M ed. J ., in p re ss.8 . B aker, H ., Hutner, S . H ., and Sobotka, H ., Proc. Soc. Exp. Biol. M ed. 89, 210 (1955).9. Sobotka, H ., Christoff, N ., and Baker, H ., In ternati. Neurol. Congress, B russels (1957).


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106 SOBOTKA C linica l C hem istry10 . Becker , B ., I*ng, C . A ., and Chow , B . F ., 1 . C hn , N utr itio n 1, 417 (1953).11. Bookman, J. J. , Joelson , B . H ., Toll, W . 0., Baker, H ., and Dolger, H ., Ass. J . Cisn .Nutrition 5, 26 (1957).12. Baker, H ., Pastier, I., Dolger, H ., a nd S obo tka , H., C hn . C he ss . 2, 328 (1956).13. Baker, H ., Brill, 0., Packer, I., Sobotka, K ., Olin. Chess. 4, 27 (1958).14 . Ziffer, H ., G utm an, A., Packer, I., Sobotka, H., and Baker, H ., Proc. Soc. E zp. Bsoi .

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Micro Assay of Vit in Clinical Chemisty

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