I. Enzymatic activity of saliva. II. Synthesis and characterization of some amine derivatives of 2-chloro-1,4-naphtho quinone

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ACKNOWLEDGMENT Tli© author wishes to express his appreciation to Dr, J. C. Calandra for his encouragement and guidance throughout the course of these investigations.

Thanks are also due to

Miss leannette Mier for carrying out the studies of the inhibition of acid formation in saliva and for some of the nitrogen analysis; to Parke, Davis and Co* for the results against Entameba histolytica.




2 Eactors involved in dental caries .................. Role of ammonia as a possible factor in caries* .. . immunity.............................. . . o ♦« • 4 Sources of salivary ammonia ......................... 6 Review of the literature on amino acid oxidases .. • 8 Oxidation of glucose degradation products as a possible factor in caries immunity................14 Review of literature on oxidation of small molecular weight acids by enzyme s y s t e m s ...................... 14 Review of literature on fatty acid oxidases............16 Statement of Problem

. . . . . . . . . . . .




Principle of the Warburg Respirometer .............. 19 Calculation of flask constants................... 20 Calibration of flasks and manometers . . 23 Reading of the Warburg Manometer....... ............. 25 Temperature control .................................. 27 Use of L- and D- amino acid oxidase inhibitors. . . . 28 Experimental procedure.............. . ............... 32 Method for expressing the data......... ........... .. 34 Results. . . o • • .

............ ** ................



Graphs.......................................... 36-62 Table of Ac ti v i t i e s ............................. 63 Discussion

....................................... 64

Comparisons of rates of oxidations ofthe amino acids 64 Controlling factors in the rate ofammonia production 66 Possible importance of the oxidation of lactate, pyruvate, acetate, and propionate to caries immunity. .............. . . . . . . . . . . 70 Sources of the enzymes. ................... Conclusions.


. . . . . .


Summary. . . .

|influencing dental caries. ;under two headings:

These factors may be grouped

The rate of acid formation in the mouth;

|and the rate of acid destruction or neutralization in the mouth.

The factors under the two headings are listed in

ijthe following outline:1

j i ■ i l | 'il. Fosdick, L.S. "Dental Caries". Pamphlet, Chemistry ' Department, Northwestern University Dental School, Chicago, Illinois.

2 Rate of Acid Formation A.


Substrate 1.



Fermentable sugars

Enzyme Systems 1.

Bacterial flora


Tissue enzymes



Rate of Acid Destruction A.

Rate of Acid Neutralization 1.

Buffering Capacity a.



Flow of Saliva


Calcium and Phosphorus Content

Other Pathways of Degradation 1.


Diet alkalinity

Oxidation of pyruvic and lactic acids

Physical Features 1.


Anatomical a.

Diet and nutrition during formation


Pits, fissures, occlusions

Mechanical a.





Oral hygiene

3 This paper will deal mainly with the rate of acid for­ mation.

The role of the substrate has been adequately dis*1 cussed in the past and it has been decided that the control

of the substrate would be difficult except by severe restric­ tion of the diet or by removing the substrate from the teeth by immediate brushing after meals.

This leaves the enzyme

systems responsible for the degradation of carbohydrates as the major point of attack on the dental caries problem. Lactobacillus acidophilus has been assigned the major role in the acid production but this may be an overemphasis of this organism.

L. acidophilus has a high concentration

of the enzymes necessary to convert pyruvic acid to lactic i acid but a low concentration of the enzymes necessary to initiate the carbohydrate degradation.

L. acidophilus alone

1then would result in a slow production of lactic acid; how­ ever, it may act in a symbiotic relationship with other i ! Ibacteria which have a high concentration of the enzymes j|

necessary for the initiation of the degradation.


with such characteristics that have been isolated from the Ioral cavity are staphylococcus a l b u s , ^ yeast,® aerobacter l i » jjl. Fosdick, L.S. ”Dental Caries” . Pamphlet, Chemistry | j Department, Northwestern University Dental School, I Chicago, Illinois. i2. Fosdick, L.S. and G.W. Rapp. Arch. Biochem. 1, 379 ' (1943). |3. Fosdick, L.S. and G.W. Wessinger. 1. Amer. Dent. Assoc. 28, 234 (1941).

4 aerogenes, ^ Candida albicans, ^ and sarcina lutea. ® An inhibitor of acid production may act by interfering with some metabolic system of an organism that plays a role in acid production.

This is probably a biological antagonism

due to a structural analogy between the inhibitor and some necessary metabolite for growth of the organism.

A second

mechanism of inhibition may be the tying up of some coenzyme that is necessary for a degradation reaction in a precipitate or a complex.

The tying up of magnesium appears to be the

mechanism for fluoride inhibition.

A third mechanism acts

by altering some condition such as pH so that the organisms are operating under less optimum conditions.

An inhibition

of any one of these types might be responsible for the natural resistance of some individuals to dental caries. In 1934 Grove and Grove ^ emphasized the possible impor|tance of ammonia in checking dental caries.

They did not

'attribute its action to any antibacterial effect, but rather | jto its buffering qualities and its ability to dissolve mucin, I jthereby removing or interfering with the development of Jplaques on the caries-susceptible surfaces of the teeth. i I |They found definitely higher levels of ammonia in the salivas |of caries-free individuals than in subjects with active caries. 1. 2. 3. 4.

Fosdick, L.S. and A.F. Dodds, Arch. Bjochem. 6, 1 (1945). Fosdick, L.S. and T. Blohm, Unpublished work. Fosdick, L.S. and J.C. Calandra, Arch. Bjochem. 6. 9 (1945). Grove, C.T. and C.J. Grove, Dental Cosmos 76, 1029 (1934).

5 Neither White and Bunting ^ nor Youngburg ^ were able to find any statistically significant difference in the ammonia content of the saliva of caries-immune and caries-active individuals as the Groves had reported.

Grove and Grove

countered this criticism by stating that the aeration method used by their critics determined both protein and non-protein nitrogen, while the Folin-Bell permutit method used by them determined only non-protein nitrogen and that only ammonia !had an influence on dental caries.

They also reported that

ammonia had an inhibiting effect on the activity of L. aci1dophilus in concentrations as low as 1.4 x 10“4 moles per I !liter. Cary ^ was able to show that ammonia is continuously |evolving from saliva both in vitro and in the mouth, but I

iwas unable to correlate the amount of ammonia with caries |activity. Kesel et al. confirmed the inhibition ! ] iof the growth of L. acidophilus, but also were unable to ] find any significant difference in the concentration of J! :i j j l . White, J. and R.W. Bunting. 1. Amer. Dent. Assoc. 22, 468 (1955). 2. Youngburg, G.E.Dent. R e s. 1 5 , 246 (1936). !l3. Grove, C.T. and C.J. Grove. J. Amer. Dent. Assoc. 29, i j 1921 (1942). I)4. Cary, I.E. Australian J . Dent. 50 , 4, (1936). j5. Kesel, R.G., J.F. C^Donnell, and E.R. Kirch. Science ! 101, 230-231 (1945). |6. Kesel, R.G. ,I.E. O'Donnell, E.R. Kirch and E.C. V/ach. i iLi. Amer. Dent. Assoc. 55, 695-714 (1946) 7. Kesel, R.G.,J.F. O'Donnell, E.R. Kirch and E.C. Wach. Amer. J . Orthodontics Oral Surg. 33, 68-79 (1947). 18. Kesel, R.G., J.F. O'Donnell, E.R. Kirch and E.C. Wach. Amer. J ♦ Orthodontics Oral Surg. 33, 80-101 (1947). i

6 ammonium ion in the saliva of caries-immune and caries-active individuals; however, they postulated that there might be !a difference in the rate of ammonia production at certain I i tistrategic points. They discarded the alkalinity as the suggested inhibitor, as sodium acetate buffers of the same pH did not effect the growth of L. acidophilus. |

This would indicate that the possible sources of salivary

IIammonia should be investigated. Cary 1 collected saliva !i ; i |from the parotid gland at the submaxillary ducts so that jthe saliva was free of bacterial contamination. He found i‘ |that ammonia was not secreted by the salivary glands and |that normal saliva owes its ammonia content to an overwhelming, i l ;if not complete, extent to the presence of bacteria. The j i jpossible sources for salivary ammonia are the urea, protein, land amino acids of the saliva and any nitrogenous food resiijdues that might be in the mouth.

Stephan ^ emphasized the

jimportance of urea by calling attention to its presence in 'i Isaliva and to its ready conversion into ammonium carbonate by the enzyme urease, which i s ,invariably present in the oral cavity. Cary ■** showed that urea was not present in ' i ^sufficient amount to account for all the ammonia produced . 3 4 in saliva. Kesel et al. ’ also found that urea could |not be the sole source of salivary ammonia by innoculating jl. Cary, J.E. Australian J. Dent. 50, 4 (1936). i2. Stephan, R.N. Science 9 2, 578 (1940). 3. Kesel, R.G., J.F. O'Donnell and E.R. Kirch. Science 101, 230-31 (1945). |4. Kesel, R.G., J.F. O'Donnell, E.R. Kirch and E.C. Waeh, j Amer. Dent.Assoc. 5 5, 695-714 (1946) .

7 saliva into media that did not contain urea and observing that ammonia was still produced*

Also the organism Bacterium

lactis aerogenes, which they found most effective in the pro­ duction of ammonia in vitro, does not convert urea into ammonia.

This leaves the amino acids and proteins to be

considered as possible sources of ammonia.

Kesel 1 found

that ammonia was invariably produced from alanine and aspartic acid by immune saliva while ammonia was not produced by the majority of caries-active saliva.

The ammonia producing

ability of the immune saliva remained fairly consistent |while that of caries-active saliva fluctuated widely even

J in different specimens collected from the same individual. !Calandra and Fosdick ^ found that norleucine, valine, threojnine, and phenylalanine inhibited acid formation in saliva, |but that these amino acids did not occur in saliva in inhibi­ tory concentrations. Kirch et al. 3 found tryptophane, [ i i! arginine, valine, glutamic acid, phenylalanine, threonine, i Ilysine, glycine, tyrosine, proline, leucine, serine, iso!j

jleucine, cysteine, histidine, and methionine in saliva, but !neither he nor Kesel ^ were able to correlate the concentra­ tions of these amino acids with caries activity.


}suggests that the variable may be the activity of the amino |l. * 12. | 13. |

Kesel, R.G., J.F. O ’Donnell, E.R. Kirch, and E.C. Wach. American J . Orthodontics Oral Surg. 30, 68-79 (1947). Calandra, J.C. and L.S. Fosdick. J. Dent. Research 26. 303-8 (1947). Kirch, E.R., R.G. Kesel, J.F. O ’Donnell, and E.C. Wach. J. Dent. Research. 26, 297-301 (1947).

acid oxidases responsible for the liberation of ammonia by !

Ioxidative deamination of amino acids. Keselfs preliminary j |experiments ^ indicated that caries-immune individuals have enzyme systems capable of converting at least six amino acids i ! into ammonia; the six are arginine, alanine, aspartic acid, asparagine, glutamic acid, isoleucine, and serine.


suggested that caries immunity is based on the production of minute but continuous amounts of ammonia in the bacterial plaque resident on the tooth surface; the substrate is a I | ijsmall group of amino acids present in the mouth as a result j!of diet and body metabolism. i


While there have been no studies of possible amino acid


!;oxidases in saliva, much is known about such oxidases as they jlhave been found in mamaalian kidney and liver, in molds and i * ■| p r* |lbacteria, in snake venom, and in plants. Krebs was ijprobably the first to investigate the amino acid oxidases |found in liver and kidney.

He reported that slices of liver

iand kidney deaminated by oxidation both natural amino acids !and their optical isomerides. !acids are not deaminated

He found that the L-amino

in the presence of octyl alcohol,

jO.QIM HCN or in extracts of ground or dried tissue, while |the D-forms were deaminated under the same conditions. ijThese differences 'led Krebs to decide that there were two 'l. j 2. |3.

Kesel, R.G., J.F. Q ’Donnell and E.R. Kirch. Science 101. 230-231 (1945). Krebs, H.A. Z. Physiol. Chem. 217, 191 (1933). Krebs, H.A. Biochem. J. 29. 1620-43 (1935).

!different enzyme systems, but that the two systems could have components in common or even that the D-oxidase is a fragment of the L.

Krebs originally named the two enzyme systems D-

!amino acid deaminase and L-amino acid deaminase, ^ but later, |on observing that proline was not deaminated but was oxidized to oC-ketoaminovaleric acid, he changed the nomenclature to ' ! 2 amino acid oxidase. While there is general agreement that ■i

1there are separate D and L-oxidases, it is still disputed [whether there is more than one D and L-oxidase or whether Ithere is only one acting at different rates on the different i |amino acids. Adler ® reported the isolation of a glutamic jdehydrase which was different from Krebsf oxidase in that jit required a coenzyme and was specific for glutamic acid. A Bernheim and Bernheim concluded that tyrosine and phenyl­ alanine were oxidized in the presence of enzymes different i

'from the one that caused the oxidation of proline and alanine by observing the difference in rates of oxidation of these tamino acids by whole and broken cell liver and kidney suspen­ sions.

Braunstein and Bychkov ® considered the oxidation

i of L-amino acids to be actually the result of a combination |of trans-amination carried out by transaminase and 1. Krebs, H.A., Biochem. J. 29, 1680-43 (1935). 2. Krebs, H.A., Bnzymologia. 7. 53-57 (1939). 3. Adler, E . , Arkiv. Kemi. Mineral. Geol. 12B. oart 5. no. 42. (1938). 4. Bernheim, E. and M.L.C. Bernheim, J*. Biol. Chem. 107. 275-281 (1934). ;5. Braunshtein, A.E. and S.M. Bychkov, Biokhimiya. 5. 24-70 (1940); P.A. 35. 4788 (1941). ~

! Cr\ M m o l e s

~ o n ? 'r T ?4 in

tic ro litc rn






g iwt'


Ttme ca

id 40 60 80 l°° io 40 6o Tim e, ia





Ttrnc. tn M t n o t e s


20 T tpxe. to Mt/ivtCi

CH*C00H _____»

j OH ;Lactic Acid

0 Pyruvic Acid


o Acetic Acid

2 GOg + 2 HgO

An organism, Miorococcus lactilyticus, capable of causing the (oxidation of lactate and only lactate has been isolated from the oral cavity*

The products of the oxidation are propionate,

jaeetate, carbon dioxide, and water* 1 i

Lewkowicz 2 was the

(first to discover this organism. Hall and Howitt 3 and Foubert ' 4 iand Douglas further investigated this organism. Douglas 1 j pound the organism to occur in saliva in numbers ranging from | l % x 106 to 340 x 10^ per ml. In some cases fifty percent of the oral flora was made up of Micrococcus lactilyticus*


brganism in the presence of lactate can cause a marked rise jin pH when the medium is initially acidic due to the conversion j>f the strong lactic acid to the weaker propionic and acetic acids. Such oxidations then would quickly remove the condil Itions favorable for decalcification. This organism might well I jturn out to be the most important factor in the difference i

between caries immune and caries active individuals.


|vas very little oxidation of propionate and no oxidation of (citrate, succinate, malate, or acetoacetate. 1. Douglas, H.C., £. Dental Research. 29, 304-306 (1950). fe. Lewkowicz, X . , Arch, de Med., Fxper._ et d'anat. Path. 13, L £??760T ln9°«nri Howitt. B.. I. Infect. Dis. 37, 112-25 (1925) I* and Douglas! H7C m 35-36 (19481:?.

72 The oxidation of the fatty acids was slow compared to the oxidation of the amino acids.

Palmitate and stearate

were oxidized at about the same rate, indicating that the same system is involved.

Propionate was only very slowly

oxidized, while butyrate was not oxidized at all®

The oxi­

dation of the fatty acids required that a hydrogen carrier, ;jmethylene blue, be added to the reaction to obtain the faster ■I ijrate of oxidation.

Thus it is doubtful that such oxidation


joccurs in the mouth to any appreciable extent. The oxidation j (probably proceeds by the classical beta oxidation:

Thiamine phosphate CH3(CH2)l4CH2CH2COOH-t- H3PO4 ----------------» CH3 ( CH2 )l4CH2CH2 -C -0P03H 2 ATP 0 -Eg

H 20

CH3 ( C H g)1 4 CH = CH-C-OPOgHg ------- » CHg( CHg) ^CHCH g-CO PG -jH g DPN 0 OH 0



H 2°

^ GHg ( CHg) -^^C -C Eg-C -O PO gH g 0 0

> CHg-C-OPOgHg 0


! Thiamine W ' C H 2 )14C-0H phOSpha-^-e> CHgCOOH + CH3 (CH2 )14G-0P03H g ^ | ^ :!.1



H s0 :CH3 (CHg)j_gCH = CH-C-OPOgHg --- > etc. 0 I

i ! iin ; i 1 | i !'l* J '2.

It will be observed tbat a phosphorylation is involved the oxidation.

Marenzi and Cardini, 1 Lehninger, 2

Marenzi, A.D. and C.K. Cardini, J . Biol. Chem. 147, 355—63 r1943). Lehninger, A., J • Biol. Chem. 154, 309-10 (1944).

73 i

Champou, ^ Kennedy and Lehninger, ^ and Lehninger and Kennedy ^ | stressed the need of phosphorylation in the oxidation of fatty | acids.

It was found that the system required inorganic phos-

j phate, cytochrome c, ATP, neutral salts, magnesium or manga: nese ions, and catalytic amounts of malate, oxalacetate, or 2 3 4 5 j fumarate. * * * Thus the rate of oxidation of fatty acids , 1 j could be limited by the concentration of any one of the above 1 j g substances. Lehninger found the rate of fatty acid oxida■

! tion by liver slices to be limited by the ATP concentration. i ! Since this is the limiting factor, it would be expected that ! all the fatty acids would be oxidized at about the same rate t j


as has been found to be true for palmitate and stearate.


i system apparently is unable to break the acids down beyond the i I four carbon stage, as butyrate is not oxidized under the coni j ditions of the experiment. Another system catalyzes the oxi! dation of propionate and acetate as was discussed earlier. i The source of the various enzymes discussed in the pre- ceding pages is probably the bacterial flora of the oral cavity as was pointed out in the introduction.

The coenzymes

i 1. Champou, I. and E. Le Breton, Compt. rend.Soc. biol. 141, 450-53 (1947). 2. Kennedy, E.P. and A.L. Lehninger, J . Biol. Chem. 172, 847-8, (1948). ■ 3. Lehninger, A.L. and E.P. Kennedy, Biol. Chem. 173, 7531 * 71, (1948). i 4 . Marenzi, A.D. and C.E. Cardini, 1. Stumpf, P.K. and D.E. Green, J. Biol. Chem. 155, 389-399 (1944). I g. Horowitz, N.H., J » Biol. Chem. 154, 141-149 (1944). i 3 # Knight, S.G., J . Bact. 55, 401-47 (1948). : 4. Kesel, R.G. , J.F. O ’Donnell, B.R. Kirch and E.C. Wach, i Amer. J. Orthodontics Oral Surg. 23, 68-79 (1947).

75 I i

that other amino acids were not deaminated by either aeroge— ues alone or by the combination of the two organisms unless Iaspartic acid was added.

Deamination of other amino acids,

jhowever, occurred in the presence of saliva.

This could mean

|that the other amino acid oxidases are produced by other j |organisms yet unisolated from the oral cavity; or that the |i

|oxidation of the other amino acids in the presence of saliva |l

J proceeds by way of the transamination reaction mentioned h earlier and that the two organisms do not contain transaminase jwhich must be supplied by other organisms or by secretion |by the salivary glands.

It has been reported that the D-

amino acid oxidase is extractable while the L-ami no acid ioxidase is intracellular in both animal tissue and bacteria.1 *This was found to be true also for the oral amino acid oxi­ dases by passing saliva through a bacterial filter and testing the filtrate for its ability to cause oxidative deamination of amino acids.

There was no appreciable change in the oxi­

dation rate for the D-amino acids, but the oxidation of the L-amino acids was almost completely abolished. The source of the other oxidative enzymes is also without doubt tne bacterial flora, although there nas been little |investigation as to their presence in bacteria.

A pyruvate

j i !, Birlcofer, L. and R. Wetzel, Z_;_ Ptarslol. Cnem. 264. 31-33 1 * (1940).

76 oxidase Has been reported in certain bacteria* 1>2 >3 ,4 j :

The conclusions to be drawn from tnis work are tnat

i it is possible Tor ammonia to be produced from amino acids | at a sufficient rate to explain natural immunity to dental j caries and tnat such oxidative deamination may also be eon' cerned in periodontal diseases*

The oxidation of pyruvate,

lactate, and acetate is fast enough to be partially respon­ sible for this immunity.

The immunity may actually be due


to a balance between the three types of systems:

acid pro­

ducing, oxidative deamination, and pyruvate, lactate, and acetate oxidation.

In addition to the lines of investiga­

tion indicated earlier in the discussion, the organisms

j producing

these enzymes should be isolated and identified.

iThe effect of each organism alone and in combination with !

the others should be followed. Also the effect of varying i | the concentrations of the necessary coenzymes should be i


| investigated. This paper has served to lay the ground work I | for such future investigations. SUMMARY




The oxidative deamination activity for amino acids

I in the presence of pooled saliva has been determined by using i !

i 1. 1z ! * 3. I4I

Lipmann, , Enzymologia. 4. 65 (1937). Lipmann, F . , Cold Spring Harbor Symposia Quant. Biol. _7, 248 (1939). Lipmann, F., Advances Enzymology. 1, 99 (1941). Still, Biochem. J.~35. 580 (1941).

the Warburg respirometer* 2.

The rate of oxidation of various carbohydrate

degradation intermediates have been determined. 3.

The rate of oxidation of fatty acids have been

determined* 4*

The possible relation of such enzyme systems to


Idental caries activity and immunity and to periodontal I iisease has been discussed. 5. |nade.

Suggestions for further investigations have been


1. Adler, E. , Arkiv. Kemi. Mineral. Geol. 12B, part 5, #42 (1938). 2. Axelrod, A.E., Sober, H.A. and Elvehjem, C.A., Nature. 144, 6 70-1 (1939). 3. Axelrod, A.E., Sober, H.A. and Elvehjem, C.A., J. Biol. Chem. 134, 749-59 (1940). 4. Barcroft, 1. and Haldane, J.S., Physiol. 28, 232 (1902). |5. Barnes, H.F., Ph.D. Dissertation, Northwestern University (1948). :6 . Barron, E.S.G. and Lyman, C.H., J. Biol. Chem. 127, 143 (1939). i|7. Barron, E.S.G. and Singer, T.P., Biol. Chem. 157, 221-40 ! (1943). 18. Bartlett, G.R. , Amer. Chem. Soc. _70, 1010-11 (1948) . 9. Berg, M. and Fosdick, L.S., Dent. Research 25, 73-81 (1946). |10. Berg, M . , Burrill, D.Y. and Fosdick, L.S. , J. Dent. Research. i( 25, 231-46 (1946). |11. Berg, M. et al., J. Dent. Research. 26, 291-5 (1947). jl2. Bernheim, F. and Bernheim, M.L.C., cN Biol. Chem. 107. 275281 (1934). 113. Bernheim, F., J. Biol. Chem. 111. 217-224 (1935). ill4. Birkofer, L. and Wetzel, R. , Z. Physiol. Chem. 264. 31-33 j1 (1940). |l5. Blackwell, R.Q. , Ph.D. Dissertation, Northwestern University I (1949). 116• Blanchard, M . , Green, D.E., Nocito, V. and Ratner, S., J . Biol. Chem. 155. 421-40 (1944). 17. Blanchard, M . , Green, D.E., Nocito, V. and Ratner, S., J . Biol. Chem. 161. 583-597 (1945). 18. Braushtein, A.E.,Biokhimiya. 4. 667 (1939). 19. Braushtein, A.E., Enzymologia. 7. 25 (1939). 20. Braushtein, A.E., Nature. 143. 609 (1939). 21. Braunshtein, A.E. and Bychkov, S.M., Biokhimiya 5. 261-70 (1940); C.A. 35. 4788^ (1941). 22. Braushtein, A.E., Advances in Protein Chemistry. 3, l-52b (1947). 23. Brodie, T.G., 1. Physiol. 37. 391 (1910). 24. Burk, D. and Milner, R.T., Ind. Eng. Chem.. Anal. Ed. 4, 3 (1932). 25. Calandra,




| jit m i g h t b e m e n t i o n e d her e t h a t in all cases the p r o d u c t g i v e s ja b l o o d r e d color w i t h c o n c e n t r a t e d


sulfu r i c acid w h i l e the

!s t a r t i n g c o m p o u n d s w i l l not. Thus it w o u l d appear t h a t s u c h i la test i n d i c a t e s that the r e a c t i o n ha s t a k e n place.



|l. Sudborough, Jour. Ohem. Soc. 70, 534 (1901).



TABLE I Derivative

Amount Added Calcium Mg/100 cc. Miiimoies/Liter Mg/100 cc< 0 4 8 12 16 20 24 50

0 0.09 0,18 0, 24 0.36 0.45 0.54 1.14

19.8 11.0 5.1 3.1 2.4 2.9 4.4 2.0


0 50

0 1.38

19.8 10.3


0 4 8 12 16 20 24 50

0 0.09 0.18 0.27 0.36 0.45 0.54 1.14

21.8 5.4 3.9 3.2 3.1 2.1 3.7 2.4


0 12 16 20 24 50

0 0.27 0.36 0.45 0.54 1.12

21.8 5.7 2.6 3.4 2.9 2.0

'Sulf amerazine

0 50

0 1.14

25.4 2.2.

0 12 16 20 24 50

0 0.21 0.28 0.35 0.42 0.88

19.8 15.3 14.4 15.6 2.6 10.0

0 16 20 24 50

0 0.31 0.38 0.46 0.96

19.8 20.0 11.2 10.0 3.4





Sulf aguanidine I

i i

i !

Guanidine carbonate ■

i ' i

100 Table I, continued Derivative

Amount Mg/100 cc.

Added Calcium Milimoles/Liter Mg/100 cc


0 50

0 1.44

24. E 18.7


0 IE 16 E0 E4 50

0 0.44 0.59 0.73 0.88 1.83

24.4 14 .1 1E.E 10*4 8.2 4.0


0 IE 16 EO E4 50

0 0.13 0 .%B 0.$S“ O.^SL

24.4 22.4 19.9 15.6 14.3 4.5

2-Amino - 5-me tJaylpy r idine

0 8 IE E4 50

0 0.16 0.E5 0.49 1.02

17.8 2.5 3.3 3.8 4.0

Aminocaproic acid

0 E4 50

0 0 *82 1.70

23.8 17.0 11.3

4-Amino-iso-phtbaiic acid

0 5 10 25 50

0 0.13 0.27 0.67 1.34

17.8 10.4 6.0 4.1 3.5


0 0.1 0 o5 1.0 5.0 10.0 25.0 50.0

0 0.0034 0.017 0.034 0.17 0.34 0.85 1.7

28.8 12.0 8.8 3.8 3.3 3.0 2.5 1.8

101 Table I, continued Derivative

«ount Added Calcium 100 cc. Milimoles/liter Mg/100 cc,

0-Hydroxy etdylamine

0 1 5 10 85 50

0 0 #04 0.80 0.40 1.00 8.00

85.7 10.0 4.6 3.7 3.5 8.8


0 5 10 85 50

0 0.18 0.56 0.90 1.80

83.8 9.4 8.1 7.8 4.3


Derivative 1:500 2-Amino -3-me thylpyridine 2-Amino-5-methylpyridine n-Decylamine







103 DISCUSSION The results shown In Table I indicate that all the 2chloro-1,4-naphthoquinone derivatives are effective to some extent in inhibiting acid production from glucose in the presence of saliva.

The most effective inhibitor is 3-(2-

chloro-1,4-naphthoquinonyl) -N-2-amino-4-methylpyridine which |reduces the calcium dissolved from enamel extremely in con­ centrations as low as 1 miligram per 100 ml. and has moderate i .inhibition in concentrations even as low as 0.1 miligrams per i 1100 ml. The sulfonamides appear to be the next most effec­ tive; probably here the sulfonamide structure as well as the Inaphthoquinone is concerned in the inhibition.

The pyridine

derivatives with the exception of 2-amino-4-methylpyr Idine, Iwhile very effective at the maximum concentrations used, do jnot inhibit in as low concentrations as the sulfonamides. |The two amino acid derivatives are the least effective. ' The search for effective agents against Entamebae histoi jlytica, the causative agent of amebiasis, has become very i

Iimportant in the past few years. Although originally coni isidered a tropical disease, the infection has now spread i

ithroughout the temperate zone.

There was a great outbreak

of the disease at the Chicago Fair in 1934.

Today it is

estimated that at least ten percent of the population of the i lUnited States, and as high as thirty percent of the population I !of Chicago are infected with the organism; either as acute

104 and chronic infections or as carriers.

The organisms estab­

lish themselves in the lumen and tissues of the intestinal tract, and may spread to the liver and produce hepatic absces­ ses.

As with other protozoan diseases, recent infections

jyield fairly readily and quickly to active chemo-therapy, jbut may relapse after a time; and chronic infections are easily j improved, but difficult to eradicate.

Uncomplicated chronic

’intestinal amebiasis is the most frequent type in the United I jjStates. Carbarsone has been very effective against both the ;acute and chronic intestinal amebiasis; but it cannot be used jin hepatic abscesses, since the liver then is unable to detoxify i the arsenic compound. In the past emetine has been used for |the treatment of such abscesses.

Before the development of

|the arsenials, emetine was used for treatment of the other forms i jof the infection. Emetine, however, has the disadvantage of i

|being extremely toxic, especially to the heart and kidney. i ^The effective dose of the drug seriously overlaps the toxic !range; **■ toxic phenomenon was found to occur from effective i 2 'treatment in eighty percent of eaperimental animals. The heart muscle shows early functional and histological damage, jcloudy swelling, shrinking and atrophy of the muscle fibers, |which may be followed by scarring.

The liver and kidneys

lare affected later, chiefly with congestion and fat

1. Leake, C.D. , J. Amer. Med. Assoc. 98, 195 (1932). 2. Dobell, C. and Bishop, Parasitology. 21, 446 (1929).

105 infiltration.

IP 9

This toxicity to the heart makes emetine

extremely dangerous to use in the treatment of amebiasis; but in the case of hepatic abscesses, it is safer than carbarsone. |Thus the effective agent in the treatment of amebic abscesses Iof the liver must be a non-arsenial and must be less toxic ithan emetine to the heart.

It can even be less effective

iagainst the causative organism than emetine, if it is appre­ ciably less toxic.

The in vitro tests of these naphthoquinone

Iderivatives against E. histolytica appear promising.


2-amino-3-me thy lpyridine derivative is amebicidal at a 1; 50000 |dilution.

The in vitro tests, however, do not mean the drug

iwill be effective in vivo, as often effective agents in vitro i are inactivated by the plasma proteins in some manner. The in vitro results are promising enough for the compounds to ibe tried in rat amebiasis.

If they prove effective, these

jcompounds may be the long sought answer for an effective non­ toxic agent against amebiasis after liver damage has already developed to prevent the use of arsenials. There has been much speculation as to the mechanism of action of the naphthoquinones against microorganisms.


berg 3 found that the compounds interfered with the respiration of M. tuberculosis and that cysteine was antagonistic to the 1. Chopra, R.N. , Ghosh and De, Indian Med. Gaz. 59, 338 (1924). \zl Rinehart, I.E. and Anderson, Arch. Path. 1 1 . 546 (1931). !3. Zetterberg, B . , Acta Pathologies et Microbiologica Scandinavica Supplement urn EXXXXI (1949).

106 quinone inhibition.

Previous investigators ^->2,3,4 iia