Herman
Baker,2
K. S. Rajan,5
Ph.D.,
M.D.,
Oscar Willem
tn
ABSTRACT natural liver
Frank,3
Ph.D.,
Hove,6
Absorption food
source-
disease
riboflavin
due and
yeast.
(the
predominant
absorbed folates
synthetic
folylmonoglutamate
common
in
foods.
alcoholic
Am.
J. C/in.
had
in yeast)
liver
disease
ensue
28:
1377-1380,
Nutr.
Folate deficit is common in chronic alcoholics with or without hematological abnormalities (1 -4). Poor diet, defective folate storage (5-7), toxicity of alcohol, especially for the jejunum (8, 9) have been suggested as causes for the depressed serum folate. Defective absorption of food folates might induce folate deficits in chronic alcoholics. We therefore determined whether chronic alcoholics with liver disease could absorb naturally occurring folylpolyglutamates and other vitamins from yeast-a food rich in folylpolyglutamates and other B-vitamins (10). We found that chronic alcoholics with liver disease indeed had a limited capacity to absorb folates, thiamin, and vitamin B6 from yeast but absorbed pantothenate and riboflavin. Details are described here. Materials
and
serve
from
that
was subjects.
absorption not thiamin. of
vitamin
absorb
All
folate
The alcoholics group consisted of 37 patients, 4 females and 33 males aged 26-52 years, admitted to the liver service. A percutaneous liver biopsy served to confirm clinical and histological diagnosis: 61% of the biopsies had fat involving 10 70% of the total biopsy; 38% had bile stasis, and 23% were classified as alcoholic hepatitis (I I); cirrhosis was a common feature in all biopsies studied. Liver function was evaluated by determination of serum bilirubin, alkaline phosphatase. transaminases, proteins, and prothrombin time. None of the patients admitted to the use of toxic agents or drugs other than commercial alcoholic beverages: they were taking no drugs, vitamins, or alcohol for 5 8 days before this
28:
DECEMBER
and
a
with
absorbed vitamin
B6
folylpolyglutamates for
the
alcoholics,
B6, and thiamin these
from
alcoholics
Ingested
the folate, to
riboflavin in 37 patients
of thiamin
as a source
inability
and studied
specific
but
deficits
vitamins
so from
1975.
study and were being fed the regular hospital diet. All were chronic alcoholics whose alcohol consumption consisted of whiskey, wine and/or beer; all had a history of heavy alcohol intake for at least 4 years. Almost all appeared malnourished: 14 alcoholics had macrocytosis due to folate deficiency . No patient had had gastrointestinal disease or surgery. Four alcoholics had had peroral biopsies of the jejunum: none had had alterations in the jejunal mucosa on light microscopy. volunteers (6 females and 6 males, without history of alcoholism, liver,
Twelve healthy aged 24 50 years) or gastrointestinal
disease, chosen from laboratory personnel, served as controls. Absorption of selected vitamins common in yeast. vide infra, was estimated for all subjects after they ingested 35 g of brewer’s-type yeast (Saccharomvces cerevisiae) suspended in water: the yeast was ingested within 5 mm. The differential responses to folates of Lactobaci//us casei (ATCC 7469), Streptococcus /aecium (ATCC 8043), and Pediococcus cerevisiae (ATCC 8081) differentiated the folates (12) in yeast before and after deconjugation (deglutamylation) to the monoglutamate with hog kidney conjugase (13.14). Lactobaci/lus casei responds to oxidized and reduced mates and N’-methyltetrahydrofolate:
methods
TheAmericanfournalofClinica/Nutrition
not
We suggest
forms
lowered B6, but
M.D.
B6, pantothenate
nonalcoholic
vitamin
M.D.,
Leevy,7
synthetic
12 healthy.
could
served.
M.
vitamin
a significantly
synthetic
Zen’erman,4
Carroll
respective
and but
K.
thiamin,
their
alcoholism,
Alcoholics
and
of folates,
pantothenate
from
M.D.
yeast-and
to
Rowen
of
1
From
the
Departments
folylmono-to-triglutaP. cerevisiae
of Medicine
and
only
Preventive
Medicine and Community Health, New Jersey Medical School, 88 Ross Street, East Orange. New Jersey 07018 and Newark, New Jersey 07103. 2 Professor of Medicine and Preventive Medicine and Community Health. 3Associate Professor of Medicine. Research Fellow in Medicine, Present address: Walter Reed Hospital, Washington. D.C. ‘Assistant Professor of Medicine. Present address: Henry Ford Hospital, Detroit, Michigan. 6Associate Professor of Medicine. Professor of Medicine.
1975,
pp.
1377-1380.
Printed
in U.S.A.
1377
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1377/4716604 by Open University of Hong Kong user on 13 January 2019
Inability of chronic alcoholics with liver disease to use food as a source folates, thiamin and vitamin B61
1378
BAKER
14Lg/g was due to reduced folates (P. cerevisiae activity). Approximately 99% of the available folate in this yeast was present as the polyglutamate, which could be deglutamylated to the monoglutamate stage by the enzymes in hog kidney extract. One gram of yeast also contained 0.6 ± 0.1 mg of thiamin, 0.4 ± 0.1 mg of vitamin B6, 0.2 ± 0.04 mg riboflavin, and 6.0 ± l.2 mg pantothenate. All of the riboflavin and pantothenate existed as the free forms since enzymatic hydrolysis did not significantly increase vitamin activity. Approximately 50 of the thiamin and B6 was in a bound form: enzymatic hydrolysis ( 12) was needed to increase vitamin recoveries from the sample. Thirty-five grams of yeast therefore contained 1.9 mg of available folates (as polyglutamates). 21 mg ofthiamin, 14 mg ofvitamin B6, 7 mg of riboflavin, and 210 mg of pantothenate. Blood was drawn from all subjects before. then 2 hours and 5 hours after ingestion of yeast and analyzed for folate, thiamin, vitamin B6, riboflavin and pantothenate by described methods (12). One week later the absorption test was repeated using the same concentrations of synthetic vitamins present as natural vitamins in yeast, i.e., .9 mg folylmonoglutamate, 21 mg thiamin HCI, 14 mg pyridoxine HCI (vitamin B.), 7 mg riboflavin, and 210 mg calcium pantothenate. Blood was drawn during the same intervals (vide supra) and saved for folate analysis with L. casei; thiamin, vitamin B6, riboflavin, and pantothenate were analyzed with protozoa (12). Peak increment (i), i.e., the difference in value between heights of plasma and whole blood vitamin activity before and after yeast or synthetic vitamins, were calculated as means ± standard error (SE) and their statistical significance (P) calculated by the Student’s i-distribution test. Activity of folate conjugase (pteroylpolyglutamate hydrolase) in plasma was evaluated by adding 1.0 ml of plasma to 7.0 ml of yeast suspension (2mg/mI which had been autoclaved for 10 mm in a solution of pH 6.0 containing 1.25% ascorbic acid and 0.5% NaCI and then adding 6.0 ml of 0.1 si acetate buffer at pH 4.7. The plasma yeast suspension was incubated overnight at 37 C and then autoclaved for 10 mm. One-milliliter of saline, instead of plasma, was used as control. After incubation, folate activity was measured with the aforementioned folate-requirers, before and after incubation with plasma to determine increase in folate activity due to deglutamylation of yeast folylpolyglutamates to the monoglutamate stage by plasma folate conjugase.
AL.
Results From the 37 alcoholics in this study 14 had a circulating folate level below 4 ng/ml, 16 had a B6 level below 25 ng/ml, 12 had a thiamin level below 20 ng/ml and I had a riboflavin level below 80 ng/ml. These alcoholics were classified as having a hypovitaminemia by published criteria (12). Multiple vitamin deficits were usually noted in the alcoholics. None of the alcoholics had a pantothenate deficit. Results listed in Table I show that patients with alcoholic liver disease have a similar for pantothenate and riboflavin as normal subjects had after ingesting yeast. In contrast, the for thiamin and vitamin B6 is significantly depressed. Except for thiamin, all alcoholics had values similar to those of the healthy subjects when the synthetic form of the vitamin was ingested. Alcoholics with liver disease did not significantly increase the folate (P 0.005) after yeast ingestion as compared to the increase by healthy subjects (Table 1), but did show increased circulating folate similar to the controls after ingesting synthetic folylmonoglutamate. The plasma of patients with alcoholic liver disease contained (per ml) as much folate
0.05 0.05 0.05 >0.05
Thiamin
18±5 71 ±
Riboflayin Pantothenate
a Values significance
5.4
P
219 given
±
31
are means
P is compared
5.1±2.6
251 ±
standard
to healthy
±
0.05 statistical
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1377/4716604 by Open University of Hong Kong user on 13 January 2019
to reduced folylmono-to-triglutamates; and S.faecium to oxidized and reduced folylmonoglutamates. This established analytical scheme, combined with protozoal analyses for vitamin B6, thiamin, riboflavin, and pantothenate (12) indicated that: yeast had almost no folylmonoglutamate: the differential assays with folate-requirers revealed 0.5 ± 0.1, 0.2 ± 0.04, and 0.1 ± 0.02 g folylmonoglutamate/g of yeast as assayed, respectively. with L. casei, P. cerevisiae, and S. faecium. More than 40% of the total folylmonoglutamate existed in the reduced state (P. cerevisiae activity) while 5 times more folyltriglutamates and/or N’-methyltetrahydrofolate was present (L. casei compared to S. faecium activity). Deglutamylation with hog kidney extract yielded 55 ± 2.8 .tg/g L. casei and S.faecium folate activity: 19 ± 1.1
ET
USE
OF
FOOD
BY
CHRONIC
±
Discussion A degree of folate deficiency is almost universal among alcoholics despite adequate dietary intake of folates (15-17). Our results (Table I) indicate that alcoholics with liver disease have an impaired ability to process folylpolyglutamates in foods for absorption. This would eventually lead to a folate deficit even though the folate requirement can be satisfied by the folylpolyglutamates in foodstuffs (18) which usually serve as well as the synthetic folylmonoglutamate as a folate source (19). Since folate conjugase levels in alcoholics were similar to those of healthy subjects (see “Results”) it may not play a central role in preparing folylpolyglutamates for absorption in the alcoholic. Others have observed this in cases of tropical sprue (20, 21); malabsorption of folate polyglutamates occurred despite normal serum and jejunal folate conjugase. Despite inability to utilize folylpolyglutamates as a folate source, these alcoholics absorbed folylmonoglutamate (Table 1). Since folate deficits are s’ prevalent in alcoholics perhaps this pharmacologic dose of folylmonoglutam ate (> 2 mg) should be used to treat folate deficits in such patients rather than relying on a nutritionally adequate diet to supply folates: our results show that the latter may not suffice as a folate supply for alcoholics with liver disease. Treatment with pharmacologic doses of the monoglutamate should be considered since smaller doses, e.g., 25-50 zg may not be absorbed by alcoholics (17) consuming alcohol. This may
1379
be due to interference of alcohol with the active, rather than the passive transport mechanisms during folate absorption. Such results have been shown with vitamin B12 (22). Alcoholics cannot absorb synthetic thiamm as the HCI (Table I): previously, we and others have made this observation (23, 24). Thiamin and vitamin B6 in foodstuff are also poorly available to the alcoholic with liver disease (Table I As with folate, the alcoholic seems impaired in processing these two vitamins into an absorbable form from foods. To make thiamin absorbable there is a need to dephosphorylate thiamin prior to absorption. Absorption may be hindered by an inability of the alcoholic to dephosphorylate the natural forms or else the electric charge may hinder penetration or attachment to a transfer system within the intestinal cell (25): B6 absorption may follow a similar pattern (26). Since pantothenate and riboflavin are absorbed from yeast by the alcoholic it is possible that these two vitamins may be less tightly bound to carrier-protein and so require minimal processing by the alcoholic. Since results (Table I) indicate that a malabsorpt ion of only specific vitamins, i.e., folate, B6, and thiamin from food exists in the alcoholic with liver disease it may explain why these vitamin deficits are so common in ).
blood and tissues of such alcoholics (5, 7, 12, 27, 28). Because of their inability to use food as a source of some essential nutrients (Table I). nutritional deficiency becomes inevitable in this type of alcoholic. This then leads to a vicious circle: alcohol leads to liver injury-which leads to less vitamin absorption from food-which leads to more intensified liver injury due to unavailable nutrients necessary to repair liver cells, e.g., folate and B6 (5, 28). Some workers believe that alcohol per se is responsible for vitamin malabsorption (4), however in this study alcohol was not responsible for the vitamin malabsorption since these patients were not consuming any alcoholic beverages for at least 5- 10 days prior to these studies. Even a prolonged hospital stay (> 3 weeks), without alcohol consumption, did not improve their ability to absorb food vitamins. The common feature seen in the alcoholics studied here is the
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1377/4716604 by Open University of Hong Kong user on 13 January 2019
conjugase activity as plasma from subjects without liver disease. One milliliter of plasma from alcoholics or controls increased the folate activity present in I .0 mg of yeast from 0.5 0.1 ng (untreated with plasma) to 64 ± 7 ng (treated with plasma as a source of folate conjugase). The plasma from healthy subjects and subjects with alcoholic liver disease therefore contained comparable folate conjugase activity. All results shown for the alcoholics (Table I) were not influenced by the amount of fat, bile, severity of the fibrotic process in the liver, abnormality of liver function tests, or by the degree of hypovitaminemia.
ALCOHOLICS
1380
BAKER
ET
AL.
alcohol-induced liver disease. The mechanisms responsible for the malabsorption of only selected vitamins from yeast by these alcoholics are still obscure. We are now investigating whether unconjugated dihydroxy bile acids are possibly inhibiting folate conjugase in cirrhotics. Others (30) have observed that the dihydroxy bile acids (deoxycholic and chenodeoxycholic acids) can inhibit guinea pig intestinal mucosa folate conjugase activity.
14.
M. F., 0. FRANK, H. AQUINO, A. D. THOMSON AND H. BAKER. In vivo and in vitro penetration of vitamins into human red blood cells. Am. J. Clin. Nutr. 24: 924, 1971. LEEVY, C. M., ANt) H. BAKER. Nutritional deficiencies in liver disease. Med. Clin. N. Am. 54: 467. 1970. KLIPSTEIN, F. A. AND J. LiNDENBAUSI. Folate deficiency in chronic liverdisease. Blood 25: 443, 1965. HALSTED, C. H., E. A. ROBLES ANt) E. MEZES. Decreased jejunal uptake of labeled folic acid (3HPGA) in alcoholic patients: roles of alcohol and nutrition. New EngI. J. Med. 285: 70l, 1971. BUTTERWORTH, C. E., JR. The availability of food folates. Brit. J. Haematol. 14: 339, 1968. NELSON, E. W., R. R. STREIFI AND J. J. CERDA. Absorption of vitamin C and folic acid from natural and synthetic sources. Clin. Res. 22: 366, 1974. SANT1NI, R., E. PEREZ-SANTIAGO. L. WALKER ANt) C. E. BUTTERWORTH, JR. Folic acid conjugase in normal human plasma and in the plasma of patients with tropical sprue. Am. J. Clin. Nutr. 19: 342, 1966.
16. 17.
U
References I.
2.
3.
4. 5.
6.
19.
BAKER, H., V. HERBERT, 0. FRANK, I. PAStIER, S. H. HUTNER, L. R. WASSERMAN ANI) H. SOBOTKA. A microbiologic method for detecting folic acid deficiency in man. Clin. Chem. 5: 275, 1959. LEEVY, C. M., W. S. GEORGE, H. ZIEFER ANt) H. BAKER. Pantothenic acid, fatty liver and alcoholism. J. Clin. Invest. 39: 1005, 1960. HERBERT, V., R. ZAIUSK’i ANI) C. S. DAVIDSON. Correlation of folate deficiency with alcoholism and associated macrocytosis, anemia and liver disease. Ann. Internal Med. 58: 977, l963. EICIINER. E. R. The hematologic disorders of alcoholism. Am. J. Med. 54: 621, 1973. BAKER, H., ANI) 0. FRANK. World Review of Nutrition, edited by G. H. Bourne. New York: Karger, 1968, vol. 9, p. 124. CHERRiCK, 0. R., H. BAKER, 0. FRANK ANt) C. M. LEES. Observations of hepatic avidity for folate in Laennec’s 1965.
7.
cirrhosis. H.,
BAKER,
0.
FRANK,
J.
Lab. H.
Clin.
Med.
66:
S. GOLDFARB,
ZIFFER,
intestinal produced
damage and by ethanol
changes ingestion
C.
in cell populain the rat.
Gastroenterology 66: 226, 1974. 10. SCHERTEI., M. E., W. J. BOEHSIE ANt) D. A. Ltans. Folic acid derivatives in yeast. J. Biol. Chem. 240: 3154, 1965. II. LEEvY, C. M. Clinical diagnosis, evaluation and treatment of liver disease in alcoholics. Federation Proc. 26: 1474, 1967. 12. BAKER, H.. AND 0. FRANK. Clinical Vitaminology:
13.
Methods and Interpretation. ence, 1968. BIRD, 0. D., V. M. MCGLOHN Naturally occurring folates the
rat.
Anal.
Biochem.
New
York:
20.
21.
22.
23.
24.
446,
M. LEESY AND H. SOBOTKA. Effect of hepatic disease on liver B-complex vitamin titers. Am. J. Clin. Nutr. 14: 1, 1964. 8. JARROID, T., J. J. WILL, R. A. DAVIES, P. H. DUFFY ANI) J. L. BRASISCIIREIBER. Bone marrow-erythroid morphology in alcoholic patients. Am. J. Clin. Nutr. 20: 716, 1967. 9. BARAONA, E., R. C. PIROLA AND C. S. LIEBER. Small tion
18.
25.
26.
27.
28.
29.
Intersci30.
J. W. VA1TKLS. in the blood and liver of
12: l8,
AND
1965.
A. V., ANI T. F. NECIIELES. Malabsorption of folate polyglutamates in tropical sprue. Brit. Med. J. 2: 543, 1969. CARRERA, G., S. MirJAS-ii.A ANt) R. DERACHE. Studies on active absorption of vitamin B,, by isolated rat enterocytes. Biol. Gastroenterol., Paris 7: 43, 1974. TOMASULO, P. A., R. M. H. KATER AND F. L. IBER. Impairment of thiamine absorption in alcoholism. Am. J. Clin. Nutr. 21: 1340, 1968. THOMSON, A. D., H. BAKER AND C. M. LEEVY. Patterns of “S-thiamine hydrochloride absorption in the malnourished alcoholic patient. J. Lab. Clin. Med. 76: 34, 1970. BAKER, H., A. D. THOMSON, 0. FRANK AND C. M. LEEV’I. Absorption and passage of fat- and watersoluble thiamin derivatives into erythrocytes and cerebrospinal fluid of man. Am. J. Clin. Nutr. 27: 676, 1974. SORRELL., M. F., 0. FRANK, H. AQUiNO, A. D. THOMSON, M. M. HOWARD ANt) H. BAKER. Vitamin interchange between ascitic fluid and blood in man. Am. J. Clin. Nutr. 25: 125, 1972. FENNEILY, J., 0. FRANK, H. BAKER AND C. M. LEEVY. Peripheral neuropathy of the alcoholic. I. Aetiological role of aneurin and other B-complex vitamins. Brit. Med. J. 2: 1290, 1964. LEEVY, C. M., H. BAKER. W. TEN HovE, 0. FRANK AND 0. R. CHERRICK. B-complex vitamins in liver disease of the alcoholic. Am. J. Clin. Nutr. 16: 339, 1965. HALSTED, C. H., E. A. ROBLES AND E. MEZES. Intestinal malabsorption in folate-deficient alcoholics. Gastroenterology 64: 526, 1973. BERNSTEIN, L. H., S. GUTSTEIN AND S. WEINER. Folic acid conjugase: inhibition by unconjugated dihydroxy bile acids. Proc. Soc. Exptl. Biol. Med. 132: 1167, 1969. HOFFBRAND,
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1377/4716604 by Open University of Hong Kong user on 13 January 2019
15.
SORRELL,
Baker,2
K. S. Rajan,5
Ph.D.,
M.D.,
Oscar Willem
tn
ABSTRACT natural liver
Frank,3
Ph.D.,
Hove,6
Absorption food
source-
disease
riboflavin
due and
yeast.
(the
predominant
absorbed folates
synthetic
folylmonoglutamate
common
in
foods.
alcoholic
Am.
J. C/in.
had
in yeast)
liver
disease
ensue
28:
1377-1380,
Nutr.
Folate deficit is common in chronic alcoholics with or without hematological abnormalities (1 -4). Poor diet, defective folate storage (5-7), toxicity of alcohol, especially for the jejunum (8, 9) have been suggested as causes for the depressed serum folate. Defective absorption of food folates might induce folate deficits in chronic alcoholics. We therefore determined whether chronic alcoholics with liver disease could absorb naturally occurring folylpolyglutamates and other vitamins from yeast-a food rich in folylpolyglutamates and other B-vitamins (10). We found that chronic alcoholics with liver disease indeed had a limited capacity to absorb folates, thiamin, and vitamin B6 from yeast but absorbed pantothenate and riboflavin. Details are described here. Materials
and
serve
from
that
was subjects.
absorption not thiamin. of
vitamin
absorb
All
folate
The alcoholics group consisted of 37 patients, 4 females and 33 males aged 26-52 years, admitted to the liver service. A percutaneous liver biopsy served to confirm clinical and histological diagnosis: 61% of the biopsies had fat involving 10 70% of the total biopsy; 38% had bile stasis, and 23% were classified as alcoholic hepatitis (I I); cirrhosis was a common feature in all biopsies studied. Liver function was evaluated by determination of serum bilirubin, alkaline phosphatase. transaminases, proteins, and prothrombin time. None of the patients admitted to the use of toxic agents or drugs other than commercial alcoholic beverages: they were taking no drugs, vitamins, or alcohol for 5 8 days before this
28:
DECEMBER
and
a
with
absorbed vitamin
B6
folylpolyglutamates for
the
alcoholics,
B6, and thiamin these
from
alcoholics
Ingested
the folate, to
riboflavin in 37 patients
of thiamin
as a source
inability
and studied
specific
but
deficits
vitamins
so from
1975.
study and were being fed the regular hospital diet. All were chronic alcoholics whose alcohol consumption consisted of whiskey, wine and/or beer; all had a history of heavy alcohol intake for at least 4 years. Almost all appeared malnourished: 14 alcoholics had macrocytosis due to folate deficiency . No patient had had gastrointestinal disease or surgery. Four alcoholics had had peroral biopsies of the jejunum: none had had alterations in the jejunal mucosa on light microscopy. volunteers (6 females and 6 males, without history of alcoholism, liver,
Twelve healthy aged 24 50 years) or gastrointestinal
disease, chosen from laboratory personnel, served as controls. Absorption of selected vitamins common in yeast. vide infra, was estimated for all subjects after they ingested 35 g of brewer’s-type yeast (Saccharomvces cerevisiae) suspended in water: the yeast was ingested within 5 mm. The differential responses to folates of Lactobaci//us casei (ATCC 7469), Streptococcus /aecium (ATCC 8043), and Pediococcus cerevisiae (ATCC 8081) differentiated the folates (12) in yeast before and after deconjugation (deglutamylation) to the monoglutamate with hog kidney conjugase (13.14). Lactobaci/lus casei responds to oxidized and reduced mates and N’-methyltetrahydrofolate:
methods
TheAmericanfournalofClinica/Nutrition
not
We suggest
forms
lowered B6, but
M.D.
B6, pantothenate
nonalcoholic
vitamin
M.D.,
Leevy,7
synthetic
12 healthy.
could
served.
M.
vitamin
a significantly
synthetic
Zen’erman,4
Carroll
respective
and but
K.
thiamin,
their
alcoholism,
Alcoholics
and
of folates,
pantothenate
from
M.D.
yeast-and
to
Rowen
of
1
From
the
Departments
folylmono-to-triglutaP. cerevisiae
of Medicine
and
only
Preventive
Medicine and Community Health, New Jersey Medical School, 88 Ross Street, East Orange. New Jersey 07018 and Newark, New Jersey 07103. 2 Professor of Medicine and Preventive Medicine and Community Health. 3Associate Professor of Medicine. Research Fellow in Medicine, Present address: Walter Reed Hospital, Washington. D.C. ‘Assistant Professor of Medicine. Present address: Henry Ford Hospital, Detroit, Michigan. 6Associate Professor of Medicine. Professor of Medicine.
1975,
pp.
1377-1380.
Printed
in U.S.A.
1377
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1377/4716604 by Open University of Hong Kong user on 13 January 2019
Inability of chronic alcoholics with liver disease to use food as a source folates, thiamin and vitamin B61
1378
BAKER
14Lg/g was due to reduced folates (P. cerevisiae activity). Approximately 99% of the available folate in this yeast was present as the polyglutamate, which could be deglutamylated to the monoglutamate stage by the enzymes in hog kidney extract. One gram of yeast also contained 0.6 ± 0.1 mg of thiamin, 0.4 ± 0.1 mg of vitamin B6, 0.2 ± 0.04 mg riboflavin, and 6.0 ± l.2 mg pantothenate. All of the riboflavin and pantothenate existed as the free forms since enzymatic hydrolysis did not significantly increase vitamin activity. Approximately 50 of the thiamin and B6 was in a bound form: enzymatic hydrolysis ( 12) was needed to increase vitamin recoveries from the sample. Thirty-five grams of yeast therefore contained 1.9 mg of available folates (as polyglutamates). 21 mg ofthiamin, 14 mg ofvitamin B6, 7 mg of riboflavin, and 210 mg of pantothenate. Blood was drawn from all subjects before. then 2 hours and 5 hours after ingestion of yeast and analyzed for folate, thiamin, vitamin B6, riboflavin and pantothenate by described methods (12). One week later the absorption test was repeated using the same concentrations of synthetic vitamins present as natural vitamins in yeast, i.e., .9 mg folylmonoglutamate, 21 mg thiamin HCI, 14 mg pyridoxine HCI (vitamin B.), 7 mg riboflavin, and 210 mg calcium pantothenate. Blood was drawn during the same intervals (vide supra) and saved for folate analysis with L. casei; thiamin, vitamin B6, riboflavin, and pantothenate were analyzed with protozoa (12). Peak increment (i), i.e., the difference in value between heights of plasma and whole blood vitamin activity before and after yeast or synthetic vitamins, were calculated as means ± standard error (SE) and their statistical significance (P) calculated by the Student’s i-distribution test. Activity of folate conjugase (pteroylpolyglutamate hydrolase) in plasma was evaluated by adding 1.0 ml of plasma to 7.0 ml of yeast suspension (2mg/mI which had been autoclaved for 10 mm in a solution of pH 6.0 containing 1.25% ascorbic acid and 0.5% NaCI and then adding 6.0 ml of 0.1 si acetate buffer at pH 4.7. The plasma yeast suspension was incubated overnight at 37 C and then autoclaved for 10 mm. One-milliliter of saline, instead of plasma, was used as control. After incubation, folate activity was measured with the aforementioned folate-requirers, before and after incubation with plasma to determine increase in folate activity due to deglutamylation of yeast folylpolyglutamates to the monoglutamate stage by plasma folate conjugase.
AL.
Results From the 37 alcoholics in this study 14 had a circulating folate level below 4 ng/ml, 16 had a B6 level below 25 ng/ml, 12 had a thiamin level below 20 ng/ml and I had a riboflavin level below 80 ng/ml. These alcoholics were classified as having a hypovitaminemia by published criteria (12). Multiple vitamin deficits were usually noted in the alcoholics. None of the alcoholics had a pantothenate deficit. Results listed in Table I show that patients with alcoholic liver disease have a similar for pantothenate and riboflavin as normal subjects had after ingesting yeast. In contrast, the for thiamin and vitamin B6 is significantly depressed. Except for thiamin, all alcoholics had values similar to those of the healthy subjects when the synthetic form of the vitamin was ingested. Alcoholics with liver disease did not significantly increase the folate (P 0.005) after yeast ingestion as compared to the increase by healthy subjects (Table 1), but did show increased circulating folate similar to the controls after ingesting synthetic folylmonoglutamate. The plasma of patients with alcoholic liver disease contained (per ml) as much folate
0.05 0.05 0.05 >0.05
Thiamin
18±5 71 ±
Riboflayin Pantothenate
a Values significance
5.4
P
219 given
±
31
are means
P is compared
5.1±2.6
251 ±
standard
to healthy
±
0.05 statistical
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to reduced folylmono-to-triglutamates; and S.faecium to oxidized and reduced folylmonoglutamates. This established analytical scheme, combined with protozoal analyses for vitamin B6, thiamin, riboflavin, and pantothenate (12) indicated that: yeast had almost no folylmonoglutamate: the differential assays with folate-requirers revealed 0.5 ± 0.1, 0.2 ± 0.04, and 0.1 ± 0.02 g folylmonoglutamate/g of yeast as assayed, respectively. with L. casei, P. cerevisiae, and S. faecium. More than 40% of the total folylmonoglutamate existed in the reduced state (P. cerevisiae activity) while 5 times more folyltriglutamates and/or N’-methyltetrahydrofolate was present (L. casei compared to S. faecium activity). Deglutamylation with hog kidney extract yielded 55 ± 2.8 .tg/g L. casei and S.faecium folate activity: 19 ± 1.1
ET
USE
OF
FOOD
BY
CHRONIC
±
Discussion A degree of folate deficiency is almost universal among alcoholics despite adequate dietary intake of folates (15-17). Our results (Table I) indicate that alcoholics with liver disease have an impaired ability to process folylpolyglutamates in foods for absorption. This would eventually lead to a folate deficit even though the folate requirement can be satisfied by the folylpolyglutamates in foodstuffs (18) which usually serve as well as the synthetic folylmonoglutamate as a folate source (19). Since folate conjugase levels in alcoholics were similar to those of healthy subjects (see “Results”) it may not play a central role in preparing folylpolyglutamates for absorption in the alcoholic. Others have observed this in cases of tropical sprue (20, 21); malabsorption of folate polyglutamates occurred despite normal serum and jejunal folate conjugase. Despite inability to utilize folylpolyglutamates as a folate source, these alcoholics absorbed folylmonoglutamate (Table 1). Since folate deficits are s’ prevalent in alcoholics perhaps this pharmacologic dose of folylmonoglutam ate (> 2 mg) should be used to treat folate deficits in such patients rather than relying on a nutritionally adequate diet to supply folates: our results show that the latter may not suffice as a folate supply for alcoholics with liver disease. Treatment with pharmacologic doses of the monoglutamate should be considered since smaller doses, e.g., 25-50 zg may not be absorbed by alcoholics (17) consuming alcohol. This may
1379
be due to interference of alcohol with the active, rather than the passive transport mechanisms during folate absorption. Such results have been shown with vitamin B12 (22). Alcoholics cannot absorb synthetic thiamm as the HCI (Table I): previously, we and others have made this observation (23, 24). Thiamin and vitamin B6 in foodstuff are also poorly available to the alcoholic with liver disease (Table I As with folate, the alcoholic seems impaired in processing these two vitamins into an absorbable form from foods. To make thiamin absorbable there is a need to dephosphorylate thiamin prior to absorption. Absorption may be hindered by an inability of the alcoholic to dephosphorylate the natural forms or else the electric charge may hinder penetration or attachment to a transfer system within the intestinal cell (25): B6 absorption may follow a similar pattern (26). Since pantothenate and riboflavin are absorbed from yeast by the alcoholic it is possible that these two vitamins may be less tightly bound to carrier-protein and so require minimal processing by the alcoholic. Since results (Table I) indicate that a malabsorpt ion of only specific vitamins, i.e., folate, B6, and thiamin from food exists in the alcoholic with liver disease it may explain why these vitamin deficits are so common in ).
blood and tissues of such alcoholics (5, 7, 12, 27, 28). Because of their inability to use food as a source of some essential nutrients (Table I). nutritional deficiency becomes inevitable in this type of alcoholic. This then leads to a vicious circle: alcohol leads to liver injury-which leads to less vitamin absorption from food-which leads to more intensified liver injury due to unavailable nutrients necessary to repair liver cells, e.g., folate and B6 (5, 28). Some workers believe that alcohol per se is responsible for vitamin malabsorption (4), however in this study alcohol was not responsible for the vitamin malabsorption since these patients were not consuming any alcoholic beverages for at least 5- 10 days prior to these studies. Even a prolonged hospital stay (> 3 weeks), without alcohol consumption, did not improve their ability to absorb food vitamins. The common feature seen in the alcoholics studied here is the
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conjugase activity as plasma from subjects without liver disease. One milliliter of plasma from alcoholics or controls increased the folate activity present in I .0 mg of yeast from 0.5 0.1 ng (untreated with plasma) to 64 ± 7 ng (treated with plasma as a source of folate conjugase). The plasma from healthy subjects and subjects with alcoholic liver disease therefore contained comparable folate conjugase activity. All results shown for the alcoholics (Table I) were not influenced by the amount of fat, bile, severity of the fibrotic process in the liver, abnormality of liver function tests, or by the degree of hypovitaminemia.
ALCOHOLICS
1380
BAKER
ET
AL.
alcohol-induced liver disease. The mechanisms responsible for the malabsorption of only selected vitamins from yeast by these alcoholics are still obscure. We are now investigating whether unconjugated dihydroxy bile acids are possibly inhibiting folate conjugase in cirrhotics. Others (30) have observed that the dihydroxy bile acids (deoxycholic and chenodeoxycholic acids) can inhibit guinea pig intestinal mucosa folate conjugase activity.
14.
M. F., 0. FRANK, H. AQUINO, A. D. THOMSON AND H. BAKER. In vivo and in vitro penetration of vitamins into human red blood cells. Am. J. Clin. Nutr. 24: 924, 1971. LEEVY, C. M., ANt) H. BAKER. Nutritional deficiencies in liver disease. Med. Clin. N. Am. 54: 467. 1970. KLIPSTEIN, F. A. AND J. LiNDENBAUSI. Folate deficiency in chronic liverdisease. Blood 25: 443, 1965. HALSTED, C. H., E. A. ROBLES ANt) E. MEZES. Decreased jejunal uptake of labeled folic acid (3HPGA) in alcoholic patients: roles of alcohol and nutrition. New EngI. J. Med. 285: 70l, 1971. BUTTERWORTH, C. E., JR. The availability of food folates. Brit. J. Haematol. 14: 339, 1968. NELSON, E. W., R. R. STREIFI AND J. J. CERDA. Absorption of vitamin C and folic acid from natural and synthetic sources. Clin. Res. 22: 366, 1974. SANT1NI, R., E. PEREZ-SANTIAGO. L. WALKER ANt) C. E. BUTTERWORTH, JR. Folic acid conjugase in normal human plasma and in the plasma of patients with tropical sprue. Am. J. Clin. Nutr. 19: 342, 1966.
16. 17.
U
References I.
2.
3.
4. 5.
6.
19.
BAKER, H., V. HERBERT, 0. FRANK, I. PAStIER, S. H. HUTNER, L. R. WASSERMAN ANI) H. SOBOTKA. A microbiologic method for detecting folic acid deficiency in man. Clin. Chem. 5: 275, 1959. LEEVY, C. M., W. S. GEORGE, H. ZIEFER ANt) H. BAKER. Pantothenic acid, fatty liver and alcoholism. J. Clin. Invest. 39: 1005, 1960. HERBERT, V., R. ZAIUSK’i ANI) C. S. DAVIDSON. Correlation of folate deficiency with alcoholism and associated macrocytosis, anemia and liver disease. Ann. Internal Med. 58: 977, l963. EICIINER. E. R. The hematologic disorders of alcoholism. Am. J. Med. 54: 621, 1973. BAKER, H., ANI) 0. FRANK. World Review of Nutrition, edited by G. H. Bourne. New York: Karger, 1968, vol. 9, p. 124. CHERRiCK, 0. R., H. BAKER, 0. FRANK ANt) C. M. LEES. Observations of hepatic avidity for folate in Laennec’s 1965.
7.
cirrhosis. H.,
BAKER,
0.
FRANK,
J.
Lab. H.
Clin.
Med.
66:
S. GOLDFARB,
ZIFFER,
intestinal produced
damage and by ethanol
changes ingestion
C.
in cell populain the rat.
Gastroenterology 66: 226, 1974. 10. SCHERTEI., M. E., W. J. BOEHSIE ANt) D. A. Ltans. Folic acid derivatives in yeast. J. Biol. Chem. 240: 3154, 1965. II. LEEvY, C. M. Clinical diagnosis, evaluation and treatment of liver disease in alcoholics. Federation Proc. 26: 1474, 1967. 12. BAKER, H.. AND 0. FRANK. Clinical Vitaminology:
13.
Methods and Interpretation. ence, 1968. BIRD, 0. D., V. M. MCGLOHN Naturally occurring folates the
rat.
Anal.
Biochem.
New
York:
20.
21.
22.
23.
24.
446,
M. LEESY AND H. SOBOTKA. Effect of hepatic disease on liver B-complex vitamin titers. Am. J. Clin. Nutr. 14: 1, 1964. 8. JARROID, T., J. J. WILL, R. A. DAVIES, P. H. DUFFY ANI) J. L. BRASISCIIREIBER. Bone marrow-erythroid morphology in alcoholic patients. Am. J. Clin. Nutr. 20: 716, 1967. 9. BARAONA, E., R. C. PIROLA AND C. S. LIEBER. Small tion
18.
25.
26.
27.
28.
29.
Intersci30.
J. W. VA1TKLS. in the blood and liver of
12: l8,
AND
1965.
A. V., ANI T. F. NECIIELES. Malabsorption of folate polyglutamates in tropical sprue. Brit. Med. J. 2: 543, 1969. CARRERA, G., S. MirJAS-ii.A ANt) R. DERACHE. Studies on active absorption of vitamin B,, by isolated rat enterocytes. Biol. Gastroenterol., Paris 7: 43, 1974. TOMASULO, P. A., R. M. H. KATER AND F. L. IBER. Impairment of thiamine absorption in alcoholism. Am. J. Clin. Nutr. 21: 1340, 1968. THOMSON, A. D., H. BAKER AND C. M. LEEVY. Patterns of “S-thiamine hydrochloride absorption in the malnourished alcoholic patient. J. Lab. Clin. Med. 76: 34, 1970. BAKER, H., A. D. THOMSON, 0. FRANK AND C. M. LEEV’I. Absorption and passage of fat- and watersoluble thiamin derivatives into erythrocytes and cerebrospinal fluid of man. Am. J. Clin. Nutr. 27: 676, 1974. SORRELL., M. F., 0. FRANK, H. AQUiNO, A. D. THOMSON, M. M. HOWARD ANt) H. BAKER. Vitamin interchange between ascitic fluid and blood in man. Am. J. Clin. Nutr. 25: 125, 1972. FENNEILY, J., 0. FRANK, H. BAKER AND C. M. LEEVY. Peripheral neuropathy of the alcoholic. I. Aetiological role of aneurin and other B-complex vitamins. Brit. Med. J. 2: 1290, 1964. LEEVY, C. M., H. BAKER. W. TEN HovE, 0. FRANK AND 0. R. CHERRICK. B-complex vitamins in liver disease of the alcoholic. Am. J. Clin. Nutr. 16: 339, 1965. HALSTED, C. H., E. A. ROBLES AND E. MEZES. Intestinal malabsorption in folate-deficient alcoholics. Gastroenterology 64: 526, 1973. BERNSTEIN, L. H., S. GUTSTEIN AND S. WEINER. Folic acid conjugase: inhibition by unconjugated dihydroxy bile acids. Proc. Soc. Exptl. Biol. Med. 132: 1167, 1969. HOFFBRAND,
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1377/4716604 by Open University of Hong Kong user on 13 January 2019
15.
SORRELL,
