- Email: [email protected]
SEPARATION OF RUBELLA IgM, IgA, AND IgG ANTIBODIES BY GEL FILTRATION ON AGAROSE
1278 We thank Prof. W. A. Gillespie, Dr. D. C. E. Speller, and Mr. H. J. Espiner for their interest and advice.
Requests for reprints should be addressed to J. S., Departof Pathology, Royal Infirmary, Bristol BS2 8HW.
ment
Since these methods do not achieve tion of rubella IgM, IgA, and IgG we have used a new gel material.
complete separaantibody activity
Methods
REFERENCES
1. Medical Research Council. Report of Subcommittee on Aseptic Methods in Operating Theatres. Lancet, 1968, i, 705, 763, 831. 2. Stuart, R. D. Publ. Hlth Rep., Wash. 1959, 74, 431. 3. Lowbury, E. J. L., Lilly, H. A., Bull, J. P. Br. med. J. 1960, ii, 1039. 4. Davies, G. E., Francis, J., Martin, A. R., Rose, F. L., Swain, G. Br. J. Pharmac. Chemother. 1954, 9, 192. 5. Davidson, A. I. G., Smith, G., Smylie, H. G. Br. J. Surg. 1971, 58, 326. 6. Gillespie, W. A., Simpson, K., Tozer, R. C. Lancet, 1958, ii, 1075. 7. Lowbury, E. J. L., Lilly, H. A., Bull, J. P. Br. med. J. 1964, ii, 531. 8. Lidwell, O. M. in Infection in Hospitals: Epidemiology and Control (edited by R. E. O. Williams and R. A. Shooter); p. 43. Oxford, 1963.
Removal of Non-specific Inhibitor A major amount of H.I. p-lipoprotein is eluted from the agarose column together with IgM, and has, therefore, to be removed before fractionation. 1 ml. of serum is mixed with 3 ml. of 25% kaolin in dextrose-gelatin-barbitonebuffer pH 7-2, incubated for 20 minutes, and centrifuged. The supernatant is concentrated to 0-8 ml. by centrifugation in ultrafilter cones (’ Centriflo’ Amicon; cut-off molecular
weight 50,000). The removal of
P-lipoprotein is complete, as judged by immunoelectrophoresis and micro-Ouchterlony tests. In four test sera the loss of immunoglobulin was estimated quantitatively by radial immunodiffusion (’Partigen’, Behringwerke). After kaolin adsorption the following amounts of immunoglobulin were still present: IgM 32-62%, IgA 100%, IgG 73-76%. "
SEPARATION OF RUBELLA IgM, IgA, AND IgG ANTIBODIES BY GEL FILTRATION ON AGAROSE ANNEMARIE BÜRGIN-WOLFF ROSEMARIE HERNANDEZ MAX JUST University Children’s Hospital, Basle, Switzerland
Identification of the
immunoglobulin
Summary classes of rubella antibodies is the
Gel Filtration
on
Agarose
Fractionation was done on agarose’Biogel A5-m’ 200-400 mesh (Calbiochem) equilibrated with O-lM " tris" buffer pH 7-5 with 0-9% saline in a column 85 x 2-4 cm. Two fractions of 2-5 ml. were collected per hour. After 20 hours the column can be charged with the next sample. The same column filling is used continuously for months with sodium azide added to the buffer.
only reliable tool for the diagnosis of a recent primary infection with rubella within 6-8 weeks of infection. Gel filtration permits the separation of IgM and IgA antibodies, both characteristic for a recent infection, from persistent rubella IgG antibody (which indicates pre-existing immunity). Gel filtration on agarose offers advantages over the other methods for separating immunoglobulin classes. Fractionation of specific rubella antibodies is important not only in all cases where a serum sample was taken too late and a rise of antibody titre missed, but also in cases with significant antibody rise to distinguish a secondary immune response after rubella reinfection. Introduction
IN
single determination of neutralising hsemagglutination-inhibiting (H.I.) antibodies in serum cannot be used for the diagnosis of a recent virus infection because specific antibodies are always present in acquired immunity. A 4-fold rise of antibody titre in two samples of sera taken at different times is regarded as a proof of recent infection. However, the first blood-sample is often collected too late, and a rise in antibody titre may be missed. In this situation, a recent infection can only be proved by identification of specific antibodies in the IgM globulin fraction. In rubella the serological diagnosis of a recent infection is very important in pregnancy. Congenital infections are also identified by the demonstration of specific antibodies in the IgM globulins which do not pass the placenta and so must general,
a
or
by the fetus. 1,2 Immunoglobulins are usually
have been formed
separated into fractions of different molecular weight by ultracentrifugation 3-5 or gel filtration on ’‘ Sephadex ’.1
Fig. I-Immunoglobulin fractionation on agarose gel of serum samples from a patient with rubella. (a) Ig levels in mg. per 100 ml. 4 days after exanthem.
(b) Distribution of H.I. antibody activity on day 4. (c) Distribution of H.I. antibody activity at 7 weeks.
1279
of the IgM and IgA classes which disappeared in a third serum sample taken a few weeks later. In one case only IgG antibody was found. The distribution of antibody after the onset of exanthem is summarised in fig. 2. In all three immunoglobulin classes the antibodies rise shortly after appearance of the clinical symptoms. After a few weeks both IgM and IgA antibody titres decline simultaneously, and 2 months later a primary infection can no longer be diagnosed. IgG antibodies remain in a constantly high titre and are responsible for the
lifelong immunity. Discussion
Since the clinical
diagnosis of rubella is unreliable, exposed to or who develop rubellalike illnesses in early pregnancy have to be tested for rubella antibodies. 80-90% of women of childbearing age have high rubella-antibody titres, so differentiation between recent infection and pre-existing immunity is vital. A 4-fold rise of antibody titre in two serum samples is generally accepted as a proof for a recent infection. However, high H.I. titres are attained quite rapidly, so a rise in titre is often missed. Complementfixing antibodies develop later, and rises in titre can be detected a long time after infection. But in rubella infection complement fixation is of less diagnostic
patients who
range of H.I. antibody titres in patients to three times after appearance of exanthem.
Fig. 2-Median and tested
one
Testing for Rubella Antibody For the development of the method and for special investigations fractions 19-33 containing immunoglobulin were concentrated to 0-2 ml. by ultrafiltration and the 1-1.1. test carried out as published by Stewart et aI.,6 but using pigeon erythrocytes.’ When a column has been calibrated once for the distribution of immunoglobulin classes for routine purposes two fractions from each of the IgM, IgA, and IgG peaks can be pooled, concentrated to 0.2 ml., and tested for H.I.
antibody. In an emergency the result can be obtained within 24 hours of receipt of the blood-sample.
Results 1 shows the fractionation on agarose gel of samples from the same patient taken 4 and 7 weeks after onset of exanthem. The days distribution of rubella-antibody activity in (b) over three different peaks is characteristic for a recent infection. The antibodies of the first peak belong to the IgM globulin class, the second peak is IgA, and the
Fig.
two serum
third peak is IgG. The
antibody activity disappeared precipitation with the corresponding antiglobulin antisera or incubation with specific immunosorbents. In serum (c) the IgM and IgA rubella antibodies have disappeared. Only the IgG fraction contains rubella antibody activity. Fig. 1 (a) shows the corresponding distribution of the total immunoglobulin of the 4-day serum. The variable molecular size of IgA from 7 to 11 S results in a partial overlapping with IgG. We have used this method in sixty-six patients for whom only one serum was available; recent infection was diagnosed if rubella antibody activity was distributed over three peaks, and earlier acquired immunity was diagnosed if activity was present in only one peak. In another thirty-seven cases two different serum samples were titrated for rubella antibody. A recent infection seemed already to be proved by a 4-fold rise of the antibody titre in the second serum. All but one patient in fact developed rubella antibodies
after
are
value than the H.I. test: Banatvalafound considerable variation in the c.F.-antibody response among laboratory-confirmed cases of rubella, probably due to the varying potency of different batches of antigen. Even a significant rise of titre between the first and the second serum sample may not always be a reliable test for recent primary infection. In one of our cases there was a pronounced increase of IgG antibody, but no IgM or IgA rubella antibody could be found. The identification of the immunoglobulin class of specific rubella antibody is therefore indispensable for the diagnosis of a primary infection. Rubella IgM, IgA, and IgG antibodies can be detected by two different ways. Rubella-virusinfected tissue is overlaid with the patient’s serum, and specific antibody is differentiated with fluorescent heavy-chain-specific anti-human Ig antiserum. Until commercial tissue slides become more generally available, the immunofluorescent technique is restricted to specialised laboratories with facilities for preparing virus-infected tissue-culture slides. Alternatively, the serum is separated into the different immunoglobulin classes before testing for specific antibody. Up to now serum separation has been done by ultracentrifugation or by gel filtration on sephadex G-200. Specialised equipment is needed for ultracentrifugation, and neither method differentiates IgA antibody. Gel filtration on agarose offers new possibilities for the study of the role of IgA in primary and secondary rubella infections. Since IgA is not absorbed to kaolin it offers another safety factor for not missing a primary infection. Increases of rubella antibody in the IgG class only may occur as a result of a reinfection and a secondary immune response.1o It is not likely that reinfection causes any rubella-induced embryopathies, so the decision to terminate a pregnancy should not be made
1280
without fractionation of the serum and identification of rubella IgM and IgA antibodies. Requests for reprints should be addressed
to
M. J.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9.
10.
Bellanti, J. A., Artenstein, M. S., Olson, L. C., Buescher, E. L., Luhrs, C. E., Milstead, K. L. Am. J. Dis. Child. 1965, 110, 464. Alford, C. A. ibid. p. 455. Vesikari, T., Vaheri, A. Br. med. J. 1968, i, 221. Desmyter, J., South, M. A., Rawis, W. E. J. med. Microbiol. 1971, 4, 107. Best, J. M., Banatvala, J. E. Lancet, 1969, ii, 65. Stewart, G. L., Parkman, P. D., Hopps, H. E., Douglas, R. D., Hamilton, J. P., Meyer, H. M. New Engl. J. Med. 1967, 276, 554. Peetermans, J., Huygelen, C. Presse méd. 1967, 75, 2177. Banatvala, J. E., Best, J. M., Bertrand, J., Bowern, N. A., Hudson, S. M. Br. méd. J. 1970, iii, 247. Brown, G. C., Maassab, H. F., Veronelli, J. A., Francis, T. Science, 1964, 145, 943. Boué, A., Nicolas, A., Montagnon, B. Lancet, 1971, i, 1251.
Introduction
discovery that I could
THE chance
vary my
own
serum-cholesterol between 140 and 230 mg. per 100 ml. simply by varying my vitamin-C intake, and regardless of cholesterol consumption, led me to investigate this in other people. Volunteers and Methods
Fifty-eight healthy volunteers, consisting of hospital staff and their relatives, were used. Serum-cholesterol levels were estimated every week for 6 weeks. The volunteers were then given vitamin C 1 g. daily, and cholesterol TABLE I-MEAN AND S.D. OF SERUM-CHOLESTEROL LEVELS IN THREE GROUPS OF VOLUNTEERS AND IN PATIENTS WITH ATHEROSCLEROSIS BEFORE AND AFTER VITAMIN-C SUPPLEMENTS
ATHEROSCLEROSIS AND VITAMIN C CONSTANCE R. SPITTLE
Pathology Department, Pinderfields Hospital, Wakefield, Yorkshire
Following the chance observation that the author’s serum-cholesterol could be varied between 140 and 230 mg. per 100 ml. by increasing the vitamin-C intake or by lowering it, a study was undertaken in healthy individuals and in patients with atherosclerosis. In healthy people under the age of 25, cholesterol levels tended to fall when 1 g. of vitamin C per day was added to an otherwise normal diet. In older people, no consistent pattern of serumcholesterol change was seen, but in patients with a history of atherosclerosis, most of whom were on clofibrate and/or anticoagulants, the serum-cholesterol increased in the weeks when vitamin-C supplements It is suggested that this rise in serumwere given. cholesterol is caused by mobilisation of the arterial Summary
*
0.05
>
p >
0.02.
t
P > 0-1.
:j:
P <
0.001.
levels were estimated for a further 6 weeks. No dietary restrictions were imposed. Serum-cholesterol levels were measured by the method and materials supplied by Stayne Laboratories. Since the response to vitamin C differed from my own in some cases, and seemed to vary with age, I divided the results into three groups depending on age. The vitamin-C experiment was repeated in twenty-five patients with atherosclerosis. All had electrocardiographic evidence of coronary thrombosis, and all had had the thrombosis at least three months previously. No alterations were made in their diet or treatment (table n).
cholesterol.
in volunteers under the age of 25, for choleto fall when extra vitamin C is administered. are shown as deviations from the per 100 0 which is the mean for weeks 1-6.
Fig. I-Tendency, sterol levels
Results baseline
("
(mg.
")
ml.)
Fig. 2-Tendency, in patients with atherosclerosis, for cholesterol levels
to
Plotted
1.
as
in
fig.
serum-
increase when vitamin C is administered
Requests for reprints should be addressed to J. S., Departof Pathology, Royal Infirmary, Bristol BS2 8HW.
ment
Since these methods do not achieve tion of rubella IgM, IgA, and IgG we have used a new gel material.
complete separaantibody activity
Methods
REFERENCES
1. Medical Research Council. Report of Subcommittee on Aseptic Methods in Operating Theatres. Lancet, 1968, i, 705, 763, 831. 2. Stuart, R. D. Publ. Hlth Rep., Wash. 1959, 74, 431. 3. Lowbury, E. J. L., Lilly, H. A., Bull, J. P. Br. med. J. 1960, ii, 1039. 4. Davies, G. E., Francis, J., Martin, A. R., Rose, F. L., Swain, G. Br. J. Pharmac. Chemother. 1954, 9, 192. 5. Davidson, A. I. G., Smith, G., Smylie, H. G. Br. J. Surg. 1971, 58, 326. 6. Gillespie, W. A., Simpson, K., Tozer, R. C. Lancet, 1958, ii, 1075. 7. Lowbury, E. J. L., Lilly, H. A., Bull, J. P. Br. med. J. 1964, ii, 531. 8. Lidwell, O. M. in Infection in Hospitals: Epidemiology and Control (edited by R. E. O. Williams and R. A. Shooter); p. 43. Oxford, 1963.
Removal of Non-specific Inhibitor A major amount of H.I. p-lipoprotein is eluted from the agarose column together with IgM, and has, therefore, to be removed before fractionation. 1 ml. of serum is mixed with 3 ml. of 25% kaolin in dextrose-gelatin-barbitonebuffer pH 7-2, incubated for 20 minutes, and centrifuged. The supernatant is concentrated to 0-8 ml. by centrifugation in ultrafilter cones (’ Centriflo’ Amicon; cut-off molecular
weight 50,000). The removal of
P-lipoprotein is complete, as judged by immunoelectrophoresis and micro-Ouchterlony tests. In four test sera the loss of immunoglobulin was estimated quantitatively by radial immunodiffusion (’Partigen’, Behringwerke). After kaolin adsorption the following amounts of immunoglobulin were still present: IgM 32-62%, IgA 100%, IgG 73-76%. "
SEPARATION OF RUBELLA IgM, IgA, AND IgG ANTIBODIES BY GEL FILTRATION ON AGAROSE ANNEMARIE BÜRGIN-WOLFF ROSEMARIE HERNANDEZ MAX JUST University Children’s Hospital, Basle, Switzerland
Identification of the
immunoglobulin
Summary classes of rubella antibodies is the
Gel Filtration
on
Agarose
Fractionation was done on agarose’Biogel A5-m’ 200-400 mesh (Calbiochem) equilibrated with O-lM " tris" buffer pH 7-5 with 0-9% saline in a column 85 x 2-4 cm. Two fractions of 2-5 ml. were collected per hour. After 20 hours the column can be charged with the next sample. The same column filling is used continuously for months with sodium azide added to the buffer.
only reliable tool for the diagnosis of a recent primary infection with rubella within 6-8 weeks of infection. Gel filtration permits the separation of IgM and IgA antibodies, both characteristic for a recent infection, from persistent rubella IgG antibody (which indicates pre-existing immunity). Gel filtration on agarose offers advantages over the other methods for separating immunoglobulin classes. Fractionation of specific rubella antibodies is important not only in all cases where a serum sample was taken too late and a rise of antibody titre missed, but also in cases with significant antibody rise to distinguish a secondary immune response after rubella reinfection. Introduction
IN
single determination of neutralising hsemagglutination-inhibiting (H.I.) antibodies in serum cannot be used for the diagnosis of a recent virus infection because specific antibodies are always present in acquired immunity. A 4-fold rise of antibody titre in two samples of sera taken at different times is regarded as a proof of recent infection. However, the first blood-sample is often collected too late, and a rise in antibody titre may be missed. In this situation, a recent infection can only be proved by identification of specific antibodies in the IgM globulin fraction. In rubella the serological diagnosis of a recent infection is very important in pregnancy. Congenital infections are also identified by the demonstration of specific antibodies in the IgM globulins which do not pass the placenta and so must general,
a
or
by the fetus. 1,2 Immunoglobulins are usually
have been formed
separated into fractions of different molecular weight by ultracentrifugation 3-5 or gel filtration on ’‘ Sephadex ’.1
Fig. I-Immunoglobulin fractionation on agarose gel of serum samples from a patient with rubella. (a) Ig levels in mg. per 100 ml. 4 days after exanthem.
(b) Distribution of H.I. antibody activity on day 4. (c) Distribution of H.I. antibody activity at 7 weeks.
1279
of the IgM and IgA classes which disappeared in a third serum sample taken a few weeks later. In one case only IgG antibody was found. The distribution of antibody after the onset of exanthem is summarised in fig. 2. In all three immunoglobulin classes the antibodies rise shortly after appearance of the clinical symptoms. After a few weeks both IgM and IgA antibody titres decline simultaneously, and 2 months later a primary infection can no longer be diagnosed. IgG antibodies remain in a constantly high titre and are responsible for the
lifelong immunity. Discussion
Since the clinical
diagnosis of rubella is unreliable, exposed to or who develop rubellalike illnesses in early pregnancy have to be tested for rubella antibodies. 80-90% of women of childbearing age have high rubella-antibody titres, so differentiation between recent infection and pre-existing immunity is vital. A 4-fold rise of antibody titre in two serum samples is generally accepted as a proof for a recent infection. However, high H.I. titres are attained quite rapidly, so a rise in titre is often missed. Complementfixing antibodies develop later, and rises in titre can be detected a long time after infection. But in rubella infection complement fixation is of less diagnostic
patients who
range of H.I. antibody titres in patients to three times after appearance of exanthem.
Fig. 2-Median and tested
one
Testing for Rubella Antibody For the development of the method and for special investigations fractions 19-33 containing immunoglobulin were concentrated to 0-2 ml. by ultrafiltration and the 1-1.1. test carried out as published by Stewart et aI.,6 but using pigeon erythrocytes.’ When a column has been calibrated once for the distribution of immunoglobulin classes for routine purposes two fractions from each of the IgM, IgA, and IgG peaks can be pooled, concentrated to 0.2 ml., and tested for H.I.
antibody. In an emergency the result can be obtained within 24 hours of receipt of the blood-sample.
Results 1 shows the fractionation on agarose gel of samples from the same patient taken 4 and 7 weeks after onset of exanthem. The days distribution of rubella-antibody activity in (b) over three different peaks is characteristic for a recent infection. The antibodies of the first peak belong to the IgM globulin class, the second peak is IgA, and the
Fig.
two serum
third peak is IgG. The
antibody activity disappeared precipitation with the corresponding antiglobulin antisera or incubation with specific immunosorbents. In serum (c) the IgM and IgA rubella antibodies have disappeared. Only the IgG fraction contains rubella antibody activity. Fig. 1 (a) shows the corresponding distribution of the total immunoglobulin of the 4-day serum. The variable molecular size of IgA from 7 to 11 S results in a partial overlapping with IgG. We have used this method in sixty-six patients for whom only one serum was available; recent infection was diagnosed if rubella antibody activity was distributed over three peaks, and earlier acquired immunity was diagnosed if activity was present in only one peak. In another thirty-seven cases two different serum samples were titrated for rubella antibody. A recent infection seemed already to be proved by a 4-fold rise of the antibody titre in the second serum. All but one patient in fact developed rubella antibodies
after
are
value than the H.I. test: Banatvalafound considerable variation in the c.F.-antibody response among laboratory-confirmed cases of rubella, probably due to the varying potency of different batches of antigen. Even a significant rise of titre between the first and the second serum sample may not always be a reliable test for recent primary infection. In one of our cases there was a pronounced increase of IgG antibody, but no IgM or IgA rubella antibody could be found. The identification of the immunoglobulin class of specific rubella antibody is therefore indispensable for the diagnosis of a primary infection. Rubella IgM, IgA, and IgG antibodies can be detected by two different ways. Rubella-virusinfected tissue is overlaid with the patient’s serum, and specific antibody is differentiated with fluorescent heavy-chain-specific anti-human Ig antiserum. Until commercial tissue slides become more generally available, the immunofluorescent technique is restricted to specialised laboratories with facilities for preparing virus-infected tissue-culture slides. Alternatively, the serum is separated into the different immunoglobulin classes before testing for specific antibody. Up to now serum separation has been done by ultracentrifugation or by gel filtration on sephadex G-200. Specialised equipment is needed for ultracentrifugation, and neither method differentiates IgA antibody. Gel filtration on agarose offers new possibilities for the study of the role of IgA in primary and secondary rubella infections. Since IgA is not absorbed to kaolin it offers another safety factor for not missing a primary infection. Increases of rubella antibody in the IgG class only may occur as a result of a reinfection and a secondary immune response.1o It is not likely that reinfection causes any rubella-induced embryopathies, so the decision to terminate a pregnancy should not be made
1280
without fractionation of the serum and identification of rubella IgM and IgA antibodies. Requests for reprints should be addressed
to
M. J.
REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9.
10.
Bellanti, J. A., Artenstein, M. S., Olson, L. C., Buescher, E. L., Luhrs, C. E., Milstead, K. L. Am. J. Dis. Child. 1965, 110, 464. Alford, C. A. ibid. p. 455. Vesikari, T., Vaheri, A. Br. med. J. 1968, i, 221. Desmyter, J., South, M. A., Rawis, W. E. J. med. Microbiol. 1971, 4, 107. Best, J. M., Banatvala, J. E. Lancet, 1969, ii, 65. Stewart, G. L., Parkman, P. D., Hopps, H. E., Douglas, R. D., Hamilton, J. P., Meyer, H. M. New Engl. J. Med. 1967, 276, 554. Peetermans, J., Huygelen, C. Presse méd. 1967, 75, 2177. Banatvala, J. E., Best, J. M., Bertrand, J., Bowern, N. A., Hudson, S. M. Br. méd. J. 1970, iii, 247. Brown, G. C., Maassab, H. F., Veronelli, J. A., Francis, T. Science, 1964, 145, 943. Boué, A., Nicolas, A., Montagnon, B. Lancet, 1971, i, 1251.
Introduction
discovery that I could
THE chance
vary my
own
serum-cholesterol between 140 and 230 mg. per 100 ml. simply by varying my vitamin-C intake, and regardless of cholesterol consumption, led me to investigate this in other people. Volunteers and Methods
Fifty-eight healthy volunteers, consisting of hospital staff and their relatives, were used. Serum-cholesterol levels were estimated every week for 6 weeks. The volunteers were then given vitamin C 1 g. daily, and cholesterol TABLE I-MEAN AND S.D. OF SERUM-CHOLESTEROL LEVELS IN THREE GROUPS OF VOLUNTEERS AND IN PATIENTS WITH ATHEROSCLEROSIS BEFORE AND AFTER VITAMIN-C SUPPLEMENTS
ATHEROSCLEROSIS AND VITAMIN C CONSTANCE R. SPITTLE
Pathology Department, Pinderfields Hospital, Wakefield, Yorkshire
Following the chance observation that the author’s serum-cholesterol could be varied between 140 and 230 mg. per 100 ml. by increasing the vitamin-C intake or by lowering it, a study was undertaken in healthy individuals and in patients with atherosclerosis. In healthy people under the age of 25, cholesterol levels tended to fall when 1 g. of vitamin C per day was added to an otherwise normal diet. In older people, no consistent pattern of serumcholesterol change was seen, but in patients with a history of atherosclerosis, most of whom were on clofibrate and/or anticoagulants, the serum-cholesterol increased in the weeks when vitamin-C supplements It is suggested that this rise in serumwere given. cholesterol is caused by mobilisation of the arterial Summary
*
0.05
>
p >
0.02.
t
P > 0-1.
:j:
P <
0.001.
levels were estimated for a further 6 weeks. No dietary restrictions were imposed. Serum-cholesterol levels were measured by the method and materials supplied by Stayne Laboratories. Since the response to vitamin C differed from my own in some cases, and seemed to vary with age, I divided the results into three groups depending on age. The vitamin-C experiment was repeated in twenty-five patients with atherosclerosis. All had electrocardiographic evidence of coronary thrombosis, and all had had the thrombosis at least three months previously. No alterations were made in their diet or treatment (table n).
cholesterol.
in volunteers under the age of 25, for choleto fall when extra vitamin C is administered. are shown as deviations from the per 100 0 which is the mean for weeks 1-6.
Fig. I-Tendency, sterol levels
Results baseline
("
(mg.
")
ml.)
Fig. 2-Tendency, in patients with atherosclerosis, for cholesterol levels
to
Plotted
1.
as
in
fig.
serum-
increase when vitamin C is administered