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The Rapid Induction of Human Riboflavin Deficiency with Galactoflavin

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The Rapid Induction of Human Riboflavin Deficiency with Galactoflavin The Rapid Induction of Human Riboflavin Deficiency with Galactoflavin Montague Lane, … , Maureen A. Doherty, Jean Doherty J Clin Invest. 1964;43(3):357-373. https://doi.org/10.1172/JCI104921. Research Article Find the latest version: https://jci.me/104921/pdf Journal of Clinical Investigation Vol. 43, No. 3, 1964 The Rapid Induction of Human Riboflavin Deficiency with Galactoflavin * MONTAGUE LANE, CLARENCE P. ALFREY, CHARLES E. MENGEL,t MAUREEN A. DOHERTY, AND JEAN DOHERTY (From the Departments of Pharmacology and Medicine, Baylor University College of Medicine, and the Veterane Administration Hospital, Houston, Texas; and the National Cancer Institute, Bethesda, Md.) Signs of riboflavin deficiency require many an analog reported to produce regression of months to develop in patients placed on riboflavin- lymphosarcoma in mice (10). Riboflavin defi- restricted diets (1-6). The demonstrations that ciency was rapidly induced in six consecutive pa- riboflavin deficiency induced by dietary restriction tients by means of galactoflavin and a semisyn- or riboflavin antagonists resulted in regression of thetic, riboflavin-deficient, protein-rich diet. The rodent tumors (7-13) prompted several efforts in clinical and laboratory manifestations of this de- this laboratory to produce riboflavin deficiency in ficiency state are reported. An anemia responsive patients with neoplastic disease (14, 15). Ribo- to the administration of riboflavin occurred in flavin antagonists were used in these studies with each patient. This is believed to be the first re- the objective of producing the deficiency state producible evidence of riboflavin-deficiency ane- rapidly, since a regimen requiring many months of mia in man. dietary restriction would not lend itself to thera- Methods peutic evaluation in patients with metastatic can- Selection of patients. Six male patients with inop- cer. These earlier investigations did not result erable neoplastic diseases participated in these studies. in the production of clinical evidences of riboflavin Five of the men weighed within 10% of their usual deficiency or in objective demonstrations of tu- body weight when the study was begun; patient V.T. mor regression. The administered riboflavin an- had lost 25% of his body weight. None of the patients tagonists, selected on the basis of their effective- had impairment of renal function as judged by routine urinalysis and blood urea nitrogen concentration. Two ness in rats, were shown to be metabolized differ- patients (C.R. and R.C.) had slightly elevated Brom- ently in man. The most active of these agents sulphalein retention, and one (R.C.) had a slightly ele- against rat tumors, 6,7-dimethyl-9- (2'-acetoxy- vated serum alkaline phosphatase. All patients were ethyl) -isoalloxazine, was rapidly and almost com- informed of the nature and purposes of the study and pletely hydrolyzed in man, although a significant were strongly motivated volunteers. Plan of study. Patients were given a regular diet for amount of the absorbed drug was excreted un- several days during which caloric intake was estimated. changed by rats. With the objective of producing Base-line clinical and laboratory observations were made. human riboflavin deficiency, an analog was sought Four patients were given the following semisynthetic that would be an effective riboflavin antagonist in diet formula: dextrose, 280 g; safflower oil emulsion, rodents and would be metabolized similarly in 100 g; calcium caseinate,2 120 g; 2% methyl cellulose, 100 ml; sodium chloride, 6 g; potassium chloride, 6 g; rodents and in man. and water to make 1,500 ml total volume. The 2% The present paper describes studies with 1O-D- methyl cellulose was prepared by wetting 80 g methyl cel- dulcityl -7,8- dimethyl- isoalloxazine, galactoflavin, lulose with 1,000 ml water at 820 C. The mixture was poured over 2,000 ml ice chips and stirred constantly. Ice * Submitted for publication June 3, 1963; accepted No- was added until the total volume was 4,000 ml. Each vember 5, 1963. 1,000 ml of formula provided 1,400 calories and 300 pg This investigation was supported in part by U. S. riboflavin. A different protein source, to be described Public Health Service research grant CA 0-5630 from below, was used in the diet of patients B.P. and G.H. the National Cancer Institute and by the Anna Fuller Subjects were encouraged to consume sufficient formula Fund. to equal their prestudy caloric intake. The formula, t Markle Scholar in Academic Medicine. Present ad- dress: Department of Medicine, Duke University, Dur- 'Abbott Laboratories, North Chicago, Ill. ham, N. C. 2Mead Johnson Laboratories, Evansville, Ind. 357 358 M. LANE, C. P. ALFREY, C. E. MENGEL, M. A. DOHERTY, AND J. DOHERTY given in 3 to 6 equal feedings, was prepared each morning, minutes before each feeding. Oral administration was refrigerated, and mixed in a blendor before serving. employed because the limited solubility of galactoflavin L-cystine, 0.75 g per 1,400 calories of diet, was ad- precluded adequate parenteral therapy. Doses greater ministered in gelatin capsules, 0.25 mg each, to com- than 3 g a day had been associated with nausea and did pensate for the L-cystine deficiency of casein. The not result in larger urine flavin recovery in previous formula was supplemented with a vitamin and mineral clinical pharmacology studies (17). Galactoflavin ther- mixture.8 Vitamin B,,, 15 lAg, was injected intramuscu- apy was continued through the entire study, including larly every 14 days. The diet given patients B.P. and the period of riboflavin supplementation (reversal of de- G.H. was modified to limit the riboflavin content even ficiency). Patients V.T., C.R., and R.C. were given further. The protein source was "Vitamin Free" Casein.4 kanamycin sulfate or neomycin sulfate based on specula- One hundred g was added to the formula in place of the tion that deficiency might develop more rapidly if syn- calcium caseinate. "Vitamin Free" Casein contained thesis of riboflavin by intestinal bacteria could be sup- 14.5 g nitrogen (dry weight basis) and only 50 ,ug ribo- pressed. These drugs were given orally, 0.25 g four flavin per 100 g casein. Sodium bicarbonate, 1N, 71 ml times daily. Patients S.H., B.P., and G.H. were not per 1,500 ml of formula, was added to dissolve the ca- given antibiotics. Patients were examined daily with sein, and the sodium chloride addition was reduced to 2 emphasis on the condition of the eyes, skin, lips, tongue, g per 1,500 ml. Three cups of coffee, prepared from oral mucous membranes, and nervous system. Slit-lamp individually packaged 2.2-g servings of powdered cof- examinations were performed once a week. Photographs fee (15 ,ug of riboflavin per package), were permitted were taken before study, at the height of deficiency, and daily when requested by patients. Small servings of raw after administration of riboflavin. carrots and celery were given to maintain the integrity of Laboratory studies. Routine hematologic and chemical dental structures. The patients were allowed supplements determinations were performed by the clinical labora- of stewed fruits for variety when they tired of the liquid tories. The technique of Brecher and Schneiderman was diet alone. The riboflavin content of these servings was employed for reticulocyte enumeration (18). Serum determined from tables of standard food values (16). electrophoresis 6 was performed, and vitamin A and beta- Galactoflavin5 administration was begun shortly after carotene in serum T and serum B12 levels 8 were deter- the patients had adjusted to the liquid diet. Four pa- mined. Riboflavin (19), folic acid (20), and pantothe- tients received 1.5 to 3.0 g a day divided into 3 doses nate (21) were determined by microbiological methods every 8 hours. Patients B.P. and G.H. received 1.5 and after acid hydrolysis of urine and growth were estimated 3.0 g a day, respectively, as 6 equal doses administered 30 turbidometrically.9 Urinary galactoflavin was meas- ured fluorometrically (14, 15). The radioactivity of 3 The daily vitamin and mineral supplement contained plasma was determined at frequent intervals for 8 hours the following: vitamin A, 5,000 U; vitamin D, 1,000 U; after iv injection of 10 Ac of Fe' globulin. The labeled thiamin 3 mg; mg; hydrochloride, nicotinamide, 15 pyri- globulin was prepared by addition of ferrous5' citrate to doxine hydrochloride, 2 mg; folic acid, 5 mg; cyanoco- AB negative plasma from normal donors. The amount balamin, 1 ,ug; calcium pantothenate, 5 mg; ascorbic acid, of iron added to the plasma did not exceed the iron bind- 75 mg; d-a-tocopheryl acid succinate, 5 mg; cobaltous ing capacity. One-ml samples of whole blood and plasma chloride-6 H20, 2 mg; ferrous sulfate, 22 mg; cupric were counted in a single channel pulse-height analyzer 10 sulfate 5 H2O, 4 mg; potassium iodide, 0.2 mg; manga- to a maximal counting error of 3.5%o. All counts were nous sulfate-5 H20, 1.5 mg; sodium molybdate-2 H2O, corrected for background radioactivity and physical 0.5 mg; zinc sulfate-7 H2O, 5.3 mg; magnesium sul- decay. The counts per minute per milliliter of plasma fate-7 H20, 61 mg. 4 Nutritional Biochemicals Corp., Cleveland, Ohio. 6 Performed by Dr. John L. Fahey. 5 Galactoflavin (10-D-dulcityl-7,8-dimethyl-isoalloxazine) 7Determined by Dr. John Sieri. used in these studies was generously provided by Dr. 8 Determined by Dr. Donald Watkin. Augustus Gibson, Merck & Co., Inc., Rahway, N. J., 9 Dehydrated bioassay media were obtained from through the Cancer Chemotherapy National Service Difco Laboratories, Detroit, Mich. Center. 10 Nuclear-Chicago Corp. Des Plaines, Ill. TABLE I Data on patients Patient Age Sex Usual weight Admission weight Diagnosis years kg kg V.T. 35 Male 72 54 Carcinoma of lung S.H.
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