Thiamin status of gravidas treated for gestational diabetes mellitus compared to their neonates at parturition.

Since thiamin plays a role in glucose metabolism we wanted to know if blood thiamin influx from gravida to neonate was influenced by treatment of gravidas having gestational diabetes mellitus (GDM). In this study we found thiamin hypovitaminemia in 19% of the 77 pregnancies despite vitamin supplementation and treatment for GDM; neonates born to mothers with hypovitaminemia were also thiamin hypovitaminemic. All neonatal blood had significantly higher thiamin concentration than gravidas. Indeed, cord blood from neonates born to mothers treated with insulin for GDM had significantly higher thiamin concentration than other neonates in the study. A significant weight depression was noted in neonates born to treated GDM mothers. Healthy gravidas giving birth to macrosomia neonates, had significant thiamin hypovitaminosis, but only macrosomic neonates of treated diabetic mothers had significantly depressed blood thiamin concentrations. We noted that subclinical thiamin hypovitaminemia is prominent during pregnancy despite vitamin supplementation. Perhaps increased thiamin supplementation during pregnancy seems warranted to avoid metabolic stress in mother and fetus due to thiamin hypovitaminemia.

A practical assay of lipoate in biologic fluids and liver in health and disease.

A procedure for assaying lipoic acid concentration in biologic fluids and tissues was devised using a eukaryotic protozoan Tetrahymena thermophila. T.thermophila has a specific and sensitive (30 pg/ml) requirement for lipoic acid. Unlike humans and other microorganisms, T.thermophila can not synthesize lipoic acid; hence, its requirement for exogenous lipoic acid is specific. The lipoic acid supplied to T. thermophila by the processing of biologic fluids and tissues during the assay procedure, permits the derivation of a practical assay for lipoate concentration as described here. Lipoate concentration in biologic fluids and tissue obtained from healthy humans, compared to those obtained from patients with renal and liver disease, indicate deviations from normal during disease. Absorption chartings of 200 mg of DL-alpha-lipoic acid in humans indicate a peak concentration of lipoate in plasma 2 h after ingestion and then a steady descent of lipoate to a baseline level after 24 h. With this practical assay, it is now possible to chart lipoate's antioxidant activity and therapeutic action during health and disease.

Cobalamin (vitamin B12) and holotranscobalamin changes in plasma and liver tissue in alcoholics with liver disease.

OBJECTIVE: We wanted to know if alterations in plasma cobalamin (B12) concentration and B12 carriers, e.g., holotranscobalamins (holo TC), occur in blood and liver tissue from patients with severe alcoholic liver disease. Our purpose was to test the hypothesis that liver disease may disrupt B12 distribution. METHOD: Total B12, as well as B12 bound to transcobalamin I, II, III (holo TC), were measured to determine their concentration in plasma and in liver tissue; Poteriochromonas malhamensis--a protozoan reagent served to measure only metabolically active (true) B12. Total B12 as distributed in holo TC in plasma and liver tissue of healthy subjects (controls) were compared to patients with severe alcoholic liver disease. RESULTS: Severe liver disease initiates highly elevated B12 levels in plasma and a lowered liver tissue total B12 concentration. The percent of B12 distributed to holo TC II is significantly depleted during liver disease. In contrast, holo TC I and III are elevated in plasma during liver disease and contain more B12 than controls. Total B12 and B12 distributed to TC are lower in diseased liver tissue. CONCLUSION: Severe alcoholic liver disease involves leakage of total B12 from liver tissue into the plasma. Holo TC I and III concentration increases in plasma; this preserves the high plasma B12 from being excreted. However, plasma holo TC II B12 distribution is decreased, indicating that there is a depression of exogenous B12 entering the plasma and tissues. In severe liver disease, liver tissue B12 binding and storage by TC is disrupted and causes B12 to leak out of the liver into the circulation. Eventually liver disease could produce enough severe tissue B12 deficits to cause metabolic dysfunction despite elevated plasma total B12. Elevation of plasma B12, accompanied by a lowering of holo TC II distribution, seemed to be a useful index of liver disease severity suggesting preventive treatment.

Antioxidant survey to assess antagonism to redox stress using a prokaryotic and an eukaryotic system.

Using a prokaryote (Escherichia coli) and a metazoa-resembling eukaryote (Ochromonas danica), we surveyed antioxidants which might overcome redox stress imposed by menadione sodium bisulphite (MD) and buthionine sulphoximine (BSO). BSO oxidant stress was evident only in O. danica; MD oxidant stress was evident in both organisms. Glutathione, its precursors, e.g. cysteine, homocysteine, and 2-oxo-4-thiazolidine carboxylic acid, and red blood cells, emerged as prime antioxidants for relieving BSO and MD oxidant stress. BSO and MD oxidant activity and antioxidant-annulling effect in O. danica were judged comparable to those found in animal cells whereas the results E. coli were not entirely equivalent. The O. danica system emerged as a practical, rapid, and useful system for pinpointing oxidant stressors and antioxidants, and shows promise for studies with mammalian systems.

Human plasma patterns during 14 days ingestion of vitamin E, beta-carotene, ascorbic acid, and their various combinations.

OBJECTIVE: We wanted to learn about plasma patterns of ascorbic acid (AA), beta carotene (BC), and vitamin E (vit E) when each or their various combinations were fed to humans. Conceivably, the combined absorption of these antioxidants could synergize maximum plasma redox potential. METHODS: Vit E (800 mg/day), BC (30 mg/day), and AA (1000 mg/day) were fed individually or in various combinations with each other to 91 volunteers divided into different feeding groups for 14 days. Plasma vit E, carotenes, and AA patterns were analyzed by standardized methods; values were compared with each group's baseline value. RESULTS: AA feeding did not significantly increase already saturated plasma AA concentrations above baseline. Intake of BC did not influence vitamin A (vit A) levels. Feeding of only vit E or only BC, with or without AA addition, or a combination of BC and vit E significantly increased plasma vit E and carotene levels after 2 days. A statistically (ANOVA) significant increase in plasma vit E above baseline was noted when vit E was ingested combined with AA or BC; this increase in plasma vit E was not significant when AA, BC and vit E were taken in combination. CONCLUSION: Our results show that BC or AA ingestion in combination with vit E significantly increases circulating vit E above that seen when vit E is individually ingested. Vit E in combination with BC or AA seems a practical means or increasing the circulating antioxidant potential afforded by vit E. Reasons why such synergism does not exist when an AA, BC, vit E combination is ingested is not yet obvious.

Biocidal action of chlorhexidine is annulled by nicotinic acid.

An analytical system comprising a bacterium and a protozoan was used to pinpoint the metabolic lesion whereby chlorhexidine (CLX) produced cell death. Nicotinic acid but not nicotinamide annulled the biocidal action of CLX. The results suggest that CLX may not permit bioconversion of nicotinamide to nicotinic acid to annul the growth inhibition induced by CLX.

Tissue concentrations of water-soluble vitamins in normal and diabetic rats.

Changes in circulating and tissue concentrations of several vitamins have been reported in diabetic animals and human subjects. In this study, the effect of short-term (2 weeks) streptozotocin diabetes on folate, B6, B12, thiamin, nicotinate, pantothenate, riboflavin and biotin in liver, kidney, pancreas, heart, brain and skeletal muscle of rats was investigated. The tissue distribution of vitamins varied widely in normal rats. Diabetes significantly lowered folate in kidney, heart, brain, and muscle; B6 in brain; B12 in heart; thiamin in liver and heart; nicotinate in liver, kidney, heart and brain; pantothenate in all tissues; riboflavin in liver, kidney, heart, and muscle. These results indicate that experimental diabetes causes a depression of several water-soluble vitamins in various tissues of rats.

Absorption and excretion of L-carnitine during single or multiple dosings in humans.

Changes of short-chain (free) and long-chain (acyl) carnitine activity (CA) in plasma, whole blood, red blood cells (rbc) and urine were induced (a) by ingestion of L-carnitine as a single dose of 500 mg, or 2500 mg, or (b) by ingestion of a daily dose of 2500 mg for 10 days. A single 500-mg dose induced insignificant increases of CA in blood constituents. However elevated free CA was noted in urine. Single or daily high doses--e.g. 2500 mg of carnitine--significantly increased free and acyl-CA in plasma, whole blood and urine, but these increases were low. Variations in dosage or frequency of carnitine intake led to no changes in rbc CA; presumably CA in plasma diffused only slowly into rbc. The apparent low absorption of carnitine suggests that oral therapy may not effect rapid repletion of body stores of CA.

Effect of interleukin-2 on some micronutrients during adoptive immunotherapy for various cancers.

In 20 patients, we investigated the effect of interleukin-2 (IL-2) treatment during adoptive immunotherapy for various cancers on circulating levels of: thiamin; biotin; folate; pantothenate; riboflavin; nicotinate; vitamins A, B6, B12 and E; carotenes; free and total cholines; inositol; and free and total carnitines. Of the above micronutrients, only vitamins A, B6, B12, inositol, carotenes and folate varied markedly from normal levels (pre IL-2 exposure) to abnormal levels (post IL-2). Following IL-2 exposure, every patient's B12 level was significantly elevated; 50% of the levels were abnormally increased above 1000 pg/ml. Extreme significant elevations of inositol were also seen in 90% of the patients. In contrast, IL-2 exposure depressed normal vitamins A, B6, carotene, and folate levels to subnormal; 90% of the patients became B6 hypovitaminemic; 60% for vitamin A, 80% for carotene, and 45% for folate. Other micronutrients tested showed no clear deviations from normal levels post IL-2 exposure. Some reasons for micronutrient variations are discussed.

Effect of cyclosporine treatment on carnitine and myo-inositol in diabetic rats.

1. The effect of long-term (20 wk) treatment of cyclosporine A (CyA) was studied in urine, blood, liver, kidney and pancreatic concentrations of acid-soluble carnitine and free myo-inositol in streptozotocin diabetic rats. 2. Diabetic rats excreted significantly higher concentrations of carnitine and myo-inositol; CyA prevented the urinary loss of carnitine but not myo-inositol. 3. Blood carnitine levels were not different between normal and diabetic rats, however, CyA significantly decreased these levels. Conversely, blood myo-inositol concentrations were higher in diabetic than in normal rats; CyA prevented this increase. 4. Hepatic concentrations of both carnitine and myo-inositol were increased in diabetic rats; CyA treatment caused even further increase. 5. Pancreas from diabetic rats contained less carnitine and myo-inositol compared to normal pancreas. CyA treatment did not affect pancreatic carnitine, but it normalized myo-inositol in diabetic rats. 6. The kidney carnitine or myo-inositol levels were not influenced either by diabetes or by CyA treatment. 7. These results suggest that CyA treatment causes changes in carnitine and myo-inositol concentrations in biologic fluids and certain tissues.

Vitamin and micronutrient concentrations in cyclosporine-induced renal tumor from diabetic rats.

Concentrations of vitamins, biopterin, free inositol and acid-soluble carnitine were determined in cyclosporine A induced renal adenocarcinoma and uninvaded renal tissue from streptozotocin diabetic rats. Vitamin B6, thiamin, riboflavin, nicotinate, free inositol and acid-soluble carnitine were significantly decreased in tumor than nontumor tissue. Concentrations of folic acid, B12, biotin, pantothenate and biopterin were similar in both tissues. These studies suggest that renal adenocarcinoma affects concentrations of only certain vitamins and micronutrients.

Role of 5'-deoxy-5'-methylthioadenosine in growth of several microbial B12 requirers.

5'-Deoxy-5'-Methylthioadenosine (MTA) figures in cellular methionine and polyamine syntheses. It replaces B12 for growth of the chrysomonad protozoan Poteriochromonas malhamensis at a ratio of MTA:B12 of approximatly 10,000,000:1 (by weight). MTA does not replace B12 for other B12-requirers, e.g.: Euglena gracilis, Lactobacillus leichmannii, and Escherichia coli 113-3. The methionine synergism for P. malhamensis growth is also negated when B12 activity is annulled by alkali treatment; MTA is not inactivated by such treatment. The growth promoting activity of various deoxynucloesides and deoxynucloetides for P. malhamensis and other B12-requirers is reported here due to contamination by cobalamins. Ethionine antagonizes the growth-enhancing effect of MTA, methionine, and B12, individually and collectively -evidence that MTA plays a role in supplying methionine for P. malhamensis growth. MTA concentrations in body fluids and mammalian tissues are too low to interfere with the use of P. malhamensis for estimating only metabolically active B12.

Branched-chain amino acids overcome cycloleucine growth inhibition in B12 and non-B12-requiring microorganisms.

Because cycloleucine (CL) inhibits methionine, and probably B12, we studied CL activity in some B12 or methionine dependent microorganisms to determine whether methionine or other amino acids are targeted by CL. We found that branched-chain amino acids, valine in particular, effectively annulled CL growth inhibition, whereas B12 was ineffective. alpha-Ketoisovalerate was the only intermediate in pathways of branched-chain amino acids catabolism that overcome CL toxicity; propionate, methylmalonate, succinate, alpha-ketoisocaproate and alpha-ketoglutarate were inactive by themselves or in combination. This study suggests that CL antagonizes the action of not only B12 and methionine but also branched-chain amino acids. Results seem comparable to those with B12-deficient fruit bats having neurologic involvement.

Effect of short- and long-term diabetes on carnitine and myo-inositol in rats.

1. The effect of short- (2 wk) and long-term (20 wk) streptozotocin diabetes was studied on urine, blood, liver, heart, brain, skeletal muscle, pancreas and kidney concentrations of acid-soluble carnitine and free myo-inositol. 2. Short-term diabetic rats excreted significantly higher concentrations of carnitine as well as myoinositol than normal rats. Blood carnitine and myo-inositol were not different between normal and diabetic rats. Diabetes caused a decrease in liver, brain and pancreatic carnitine, but not in heart, skeletal muscle and kidney. Myo-inositol concentration was decreased in liver, heart and kidney but not in brain, pancreas and skeletal muscle. 3. Long-term diabetic rats had higher urinary excretions of both carnitine and myo-inositol. Blood carnitine did not change; however, myo-inositol was higher in diabetic than in normal rats. Diabetes caused a significant increase in liver and a decrease in heart, brain, skeletal muscle and pancreatic content of carnitine; no difference in kidney carnitine was noted. Myo-inositol content was elevated only in liver of diabetic rats. 4. We suggest that carnitine and myo-inositol concentrations are influenced both by short- and long-term diabetes through changes in tissue metabolism.

Effect of genetic diabetes and alcohol on tissue carnitine and inositol concentrations in mice.

Concentrations of acid-soluble L-carnitine and inositol were determined in heart, kidney, muscle, pancreas, liver, brain and blood of genetically diabetic obese db/db and their nondiabetic control C57BL/6J (CBL) mice. Results were compared to a group of diabetic and CBL mice fed ethanol (ETOH) 4 g/kg daily for 58-64 days. In CBL and db/db mice, heart muscle was found to have the greatest and brain the least content of carnitine. Diabetes caused a significant decrease in hepatic concentration of carnitine but did not affect carnitine concentration of heart, kidney, skeletal muscle, brain and pancreas. ETOH intake had no effect on carnitine content of any of the tissues studied. Free inositol content was highest in brain and lowest in skeletal muscle of CBL and db/db mice; diabetes or ETOH intake did not affect tissue inositol content. Except for liver, neither diabetes nor ETOH intake affects tissue carnitine or inositol concentration.

Interleukin-2 enhances biopterins and catecholamines production during adoptive immunotherapy for various cancers.

Biopterins production during three different protocols for adoptive immunotherapy for human cancer was investigated. Adoptive immunotherapy treatment with interleukin-2 (IL-2) was carried out for 13 patients with malignant melanoma; eight with metastatic renal cell carcinoma; and three with metastatic colon cancer. The authors estimated total biopterins in plasma and lymphokine (IL-2)-activated killer cells (LAK) from these patients before and during various treatment phases to determine if increased biopterins production reflects leukocyte activation by IL-2 or antitumor activity. They noted an increased synthesis of total "biopterins," i.e., biopterin; 7,8-dehydrobiopterin; and L-neopterin in LAK cells and plasma which correlated with IL-2 exposure. Mean plasma biopterins were normal (1.2 +/- 0.5 ng/ml) before therapy; in contrast, biopterins increased significantly to 3.4 +/- 1.9 ng/ml and 3.9 +/- 1.9 ng/ml during IL-2 and IL-2 + LAK treatment each, respectively. Similar biopterin elevations were noted irrespective of the different adoptive immunotherapy protocols used. Elevated biopterins decreased to normal levels (1.2 +/- 0.7 ng/ml) when IL-2 treatment was omitted. Tumor regression with adoptive immunotherapy did not correlate with increased plasma biopterins. Increased biopterins production was also associated with increase in plasma catecholamine after IL-2 treatment during adoptive immunotherapy. Conceivably increased biopterins, induced by IL-2 activation of a leukocyte population, is a cell-mediated consequence not necessarily serving as a signal for the antitumor effect associated with adoptive immunotherapy.

Vitamins and other metabolites in various sera commonly used for cell culturing.

Many cell culture media use different sera to enhance growth. We assayed vitamins and some related metabolites in different sera and identified the concentration of: thiamin, biotin, folates, riboflavin, pantothenates, nicotinates, vitamins B6, B12, A, E, C, and carotenes and some related metabolites: biopterins, free inositol, free and total choline, total carnitines in chicken, horse, rabbit, goat, pig, calf, newborn calf, fetal calf and human sera. Results indicate that vitamin and metabolite content of different sera vary. Such variations could produce fluctuant effects on cell culturings if the metabolite content of the serum is not documented.