S-adenosylhomocysteine hydrolase deficiency in a 26-year-old man.

This paper reports the third proven human case of deficient S-adenosylhomocysteine (AdoHcy) hydrolase activity. The patient is similar to the only two previously reported cases with this disorder in having severe myopathy, developmental delay, elevated serum creatine kinase (CK) concentrations, and hypermethioninaemia. Although he has been followed from infancy, the basic enzyme deficiency was established only at age 26 years. The diagnosis was based on markedly elevated plasma concentrations of both AdoHcy and S-adenosylmethionine, some 20% of the mean control activity of AdoHcy hydrolase activity in haemolysates of his red-blood cells, and two missense mutations in his gene encoding AdoHcy hydrolase. He had low values of erythrocyte phosphatidylcholine and plasma free choline and marginally elevated excretion of guanidinoacetate, suggesting that the elevated AdoHcy may have been inhibiting methylation of phosphatidylethanolamine and guanidinoacetate. His leukocyte DNA was globally more methylated than the DNA's of his parents or the mean extent of methylation measured in age-matched control subjects.

Increase in human exposure to methylamine precursors of N-nitrosamines after eating fish.

Consumption of fish has been encouraged recently because it may prevent mortality due to heart disease. Fish contains methylamines, which are precursors of N-nitrosamines. Nitrosamines can act as potent carcinogens in a wide variety of animal species, and there is no reason to assume that humans are resistant. Human subjects (n = 5) ingested a diet of known methylamine content for 2 days. On Day 3, they ate fish at the luncheon and dinner meals. On Day 4, they again ate the control diet. A single portion of fish contained as many methylamines as were normally excreted by the human in 2 days. Urinary excretion of monomethylamine remained constant (1.3 to 1.5 mumol/24 h/kg of body weight) throughout the study. Dimethylamine excretion increased more than 4-fold after fish was eaten (from 5.6 to 24.1 mumol/24 h/kg of body weight), while trimethylamine excretion increased more than 8-fold (from 0.2 to 1.6 mumol/24 h/kg of body weight). We conclude that the consumption of fish significantly increased exposure to methylamines, particularly to dimethylamine. Although there is potential for the in vivo conversion of dimethylamine to nitrosodimethylamine, a carcinogen, we know of no studies that have determined that the ingestion of fish increases the risk of cancer. This should be carefully investigated prior to recommending that humans change their eating habits.

Effects of choline deficiency and methotrexate treatment upon liver folate content and distribution.

We examined the effects of feeding rats a choline deficient diet, of treating rats with low doses of methotrexate (MTX, 0.1 mg/kg, daily), and of combined choline deficiency and MTX treatment upon the content and distribution of folates in liver. We used a newly devised technique for analysis of folates which utilized affinity chromatography followed by high pressure liquid chromatography. Compared to control rats, total hepatic folate content decreased by 31% in the choline deficient rats, by 48% in the MTX treated rats, and by 60% in rats which were both choline deficient and treated with MTX. In extracts of livers from control rats, folates were present predominantly as penta (35%) and hexaglutamyl (52%) derivatives. The pteridine ring structure distribution of these folates was as follows: 48% 5-methyltetrahydrofolate, 14% formylated tetrahydrofolate, and 39% tetrahydrofolate. In choline deficient animals, there was a decrease in the relative concentration of pentaglutamyl folates and an increase in the relative concentration of heptaglutamyl folates. In livers from MTX treated animals, MTX-polyglutamates with 2-5 glutamate residues accumulated. The consequences of MTX treatment were: a) an elongation of the glutamate chains of the folates as the proportion of hepta- and octaglutamyl derivatives was increased relative to penta- and hexaglutamyl folates; b) the occurrence of unreduced folic acid; c) a decrease in the relative concentration of 5-methyltetrahydrofolate and an increase in the relative concentration of formylated tetrahydrofolate, and d) no change in the relative concentrations of tetrahydrofolate. In livers from animals that were both choline deficient and treated with MTX, the tetrahydrofolate concentrations were 50% of control while formylated tetrahydrofolate concentrations increased 3-fold. These data are discussed from the standpoint of the current understanding of mechanisms that regulate the elongation of the glutamic acid chains of folates and those that regulate folate dependent synthesis and utilization of one carbon unit.

Formation of unesterified choline by rat brain.

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees C at pH 7.4-8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37 degrees C. Concentrations of phosphocholine rose (by 2.3 nmol/mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.

Characterization of choline metabolism and secretion by human placental trophoblasts in culture.

Choline is an essential nutrient for fetal development and may be utilized to form phospholipids such as phosphatidylcholine and sphingomyelin; to synthesize the neurotransmitter, acetylcholine; and to donate methyl groups after being oxidized to betaine. Since the majority of choline required for fetal growth must be transported by the placenta from the maternal circulation, we examined the ability of isolated human trophoblasts to metabolize choline and to release choline and its metabolites into culture medium. Cytotrophoblasts were isolated from normal, full-term human placentas and incubated with [14C]choline for 3 h; the cells were washed to remove extracellular radiolabel, and the changes in intracellular and medium choline pools were followed for an additional 24 h. During the incubation, choline rapidly reached steady state intracellularly and label was incorporated into betaine, phosphocholine, cytidylyldiphosphocholine, phosphatidylcholine, glycerophosphocholine, lysophosphatidylcholine, and sphingomyelin. All labeled choline metabolites in cells, except glycerophosphocholine, decreased at 6 and 27 h of incubation (3 and 24 h, respectively, after labeled choline was removed), and labeled metabolites appeared in media. By 24 h after labeled choline was removed, the major labeled metabolites in the media were choline (82%), betaine (11%), and glycerophosphocholine (5%). Small amounts of phosphatidylcholine (1%), and lysophosphatidylcholine (1%) were found. Acetylcholine was a very minor choline metabolite in these cells. When placental cells were incubated for 66 h after isolation, they formed syncytiotrophoblasts, which incorporated labeled choline into metabolites in a similar pattern to cytotrophoblasts. These data indicate that isolated trophoblast cells can metabolize choline to form all of its major metabolites and that several metabolites are released to the medium in significant amounts. Thus, our data suggest that the major metabolite supplied to the fetus may be choline, but that betaine and glycerophosphocholine may also be vehicles for transfer of choline equivalents from mother to fetus.

Hepatic protein kinase C is not activated despite high intracellular 1,2-sn-diacylglycerol in obese Zucker rats.

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein X min(-1) vs. 780 pmol/mg protein X min(-1) in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.

1,2-sn-diacylglycerol accumulates in choline-deficient liver. A possible mechanism of hepatic carcinogenesis via alteration in protein kinase C activity?

Choline deficiency is associated with triacylglycerol accumulation in the liver, and is the only nutritional state known to trigger hepatic cancer spontaneously. In two different experiments, rats were pair-fed for 6 weeks with control (0.2% choline), or choline-deficient (CD) (0.002% choline) diets. Hepatic choline and phosphocholine declined in CD animals to 54% and 16% of control levels, respectively. In control livers, 1,2-sn-diacylglycerol (1,2-sn-DAG) concentration was (in nmol/g wet wt) 144 (+/- 25; mean +/- SE); while in CD livers it was 792 (+/- 140) in the first experiment. In the second experiment the values were 375 (+/- 26) and 1147 (+/- 74), respectively. 1,2-sn-DAG, a precursor of triacylglycerol, is an endogenous activator of protein kinase C (PKC). PKC is the presumed site of action of the tumor-promoting phorbol esters. We suggest that the 1,2-sn-DAG accumulating in CD liver could bind PKC, altering its activity, and thus contribute to the carcinogenic effect of CD diets.

Is there a metabolic basis for dietary supplementation?

To be efficacious, dietary supplements must either provide a nutrient that is normally undersupplied to cells or exert a pharmacologic effect on cellular processes. In the first case, optimal function is achieved when a nutrient required by the organism reaches a specific concentration within the cell. A supplement has benefit only when the normal intake of a bioavailable form of a nutrient is lower than the amount that would provide maximum benefit as judged from all biological perspectives. Metabolic, environmental, and genetic factors can make individual nutrient requirements differ from the estimated needs calculated from population-based data. For example, under certain circumstances intracellular antioxidants may be depleted and a dietary supplement might restore optimal antioxidant protection. In the second case, the dietary supplement contains a constituent that is normally not required by the cell, but this substance is capable of altering normal cell function. For example, herbal preparations may contain ephedrine (a drug), which might alter heart rate so that the amount of blood pumped by the heart is enhanced. An understanding of how the variation in nutrient requirements comes about and of the pharmacologic actions of nutrient supplements can help to identify which individuals are most likely to benefit from dietary supplements.

Nutrients, neurotransmitters and infant behavior.

In recent years, short-term effects of the composition of each meal on the synthesis of brain neurotransmitters have been studied. This paper reviews studies of the influence of dietary precursors such as tryptophan and other competing amino acids on serotonin synthesis and metabolism and emphasizes the important influence of insulin. The paper then focuses on assessment of newborn state behavior, since evidence in adult humans has suggested a relationship between sleep behavior and brain serotonin levels. Several studies are then summarized. First, a study of healthy full-term newborns examining the relationship between diet and sleep behavior showed that infants fed tryptophan entered active and quiet sleep sooner than infants fed valine and low carbohydrate. Other studies designed to examine the influence of hyperinsulinemia on this system are then described. An observational study of newborns of diabetic mothers during the first weeks of life showed that they were quieter babies, with difficulties in visual orientation and motor performance. Plasma amino acid ratios studied during a glucagon-stimulation test in an infant with hyperinsulinemia showed a marked increase in parallel with changes in insulin levels. The results suggest that infant sleep behavior can be a sensitive dependent variable in studies of behavioral effects of diet and suggests that variations in serotonin levels may play a modulating role.

Getting nutrition education into medical schools: a computer-based approach.

Despite awareness of the importance of nutrition as part of medical student's education, numerous barriers exist to incorporating nutrition education into the medical school curriculum. Chief among such barriers is that most medical schools do not have faculty trained specifically in nutrition. A curriculum is needed that can deliver comprehensive nutrition information that is consistent across medical schools. One way to deliver this information is to use computer-assisted instruction (CAI). To meet the different needs of medical schools and provide a consistent base of nutrition information, we developed a series of interactive, multimedia educational programs (Nutrition in Medicine) that teach the basic principles of nutritional science and apply those principles in a case-oriented approach. Curriculum content is derived from the American Society for Clinical Nutrition consensus guidelines. These modules offer the advantages of accessibility, self-paced study, interactivity, immediate feedback, and tracking of student performance. Modules are distributed free to all US medical schools. Preliminary data from surveys gathered by our team at the University of North Carolina at Chapel Hill indicate that 73 US medical schools use, or are planning to use, these modules; more schools are currently evaluating the programs. Successful implementation of CAI requires easy program access, faculty training, adequate technical support, and faculty commitment to the programs as a valuable resource. CAI fails when the program is just placed in the library and students are told to use it when they can find the time.

Introducing cancer nutrition to medical students: effectiveness of computer-based instruction.

BACKGROUND: Computer-based instruction has been introduced at the University of North Carolina at Chapel Hill to augment its nutrition course for first-year medical students. Seven program modules have been completed; 2 more are planned. Each module explains the biochemistry and physiology of nutrition through interactive lessons, exercises, and a video case study. OBJECTIVE: The goal of this study was to evaluate the instructional efficacy and acceptability of the nutrition and cancer module when used by first-year medical students. DESIGN: The module was used by 163 first-year medical students at the university's medical school as an obligatory component of the nutrition course. Before and after using the module, students were asked to answer multiple-choice questions concerning their knowledge and attitudes; each question had 5 possible answers. RESULTS: On average, students spent approximately 3 h studying the lessons. The percentage of correct responses to 20 knowledge questions increased from 22% before the module was used to 86% immediately after its use. When a randomly selected subsample of 25% of the students took the same test 3 mo later, they answered 62% of the questions correctly. The increase in the percentage of students who felt prepared to provide advice regarding nutrition's role in cancer prevention (from 5.7% to 66.9%) suggested a successful subjective learning experience. Neither the students' initial level of interest in cancer nutrition nor their acceptance of computer-based instruction was related to learning outcome. CONCLUSIONS: The tested module is a useful and effective aid for teaching nutritional principles of cancer prevention. The evaluation strategy helped identify areas for instructional improvement.

Prolonged fasting in humans results in diminished plasma choline concentrations but does not cause liver dysfunction.

Choline is a major donor of methyl groups, a precursor for membrane synthesis, and a component of the neurotransmitter acetylcholine. Choline-deficient diets deplete humans of choline and cause hepatic dysfunction and steatosis. In this study we determined whether acute starvation also depletes choline, as indicated by changes in plasma choline or phosphatidylcholine. Healthy humans (n = 10) fasted for 7 d, ingesting only water and mineral-vitamin supplements. Their mean (+/- SEM) plasma choline concentration was 9.5 +/- 0.5 micromol/L at the start of the study and dropped to 7.8 +/- 0.3 micromol/L after 1 wk of fasting (P < 0.01). The plasma phosphatidylcholine concentration did not change significantly (2.2 +/- 0.1 mmol/L at the start of the study and 2.4 +/- 0.2 mmol/L after 1 wk of fasting). Capacity of the liver to secrete lipoproteins was not affected by prolonged fasting. The mean plasma concentration of low-density-lipoprotein cholesterol was 3.3 +/- 0.2 mmol/L (126 +/- 8 mg/dL) at the start of the study and 4.9 +/- 0.5 mmol/L (188 +/- 19 mg/dL) after 1 wk of fasting. Liver damage assessed by serum alanine aminotransferase activity occurred in only 1 of 10 subjects. We conclude that prolonged fasting in humans modestly diminished plasma choline but was not associated with signs of choline deficiency, such as perturbed lipoprotein secretion and liver damage.

Choline and choline esters in human and rat milk and in infant formulas.

Large amounts of choline are required in neonates for rapid organ growth and membrane biosynthesis. Human infants derive much of their choline from milk. In our study, mature human milk contained more phosphocholine and glycerophosphocholine than choline, phosphatidylcholine, or sphingomyelin (P < 0.01). Previous studies have not recognized that phosphocholine and glycerophosphocholine exist in human milk. Concentrations of choline compounds in mature milk of mothers giving birth to preterm or full-term infants were not significantly different. Infant formulas also contained choline and choline-containing compounds. In infant formulas derived from soy or bovine milk, unesterified choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin concentrations varied greatly. All infant formulas contained significantly less phosphocholine than did human milk. Soy-derived formulas contained significantly less glycerophosphocholine (P < 0.01) and phosphocholine (P < 0.01) and more phosphatidylcholine (P < 0.01) than did human or bovine milk or bovine milk-derived infant formulas. Rat milk contained greater amounts of glycerophosphocholine (almost 75% of the total choline moiety in milk) and phosphocholine than did human milk. When dams were provided with either a control, choline-deficient, or choline-supplemented diet, milk composition reflected the choline content of the diet. Because there are competing demands for choline in neonates, it is important to ensure adequate availability through proper infant nutrition. Although the free choline moiety is adequately provided by infant formulas and bovine milk, reevaluation of the concentrations of other choline esters, in particular glycerophosphocholine and phosphocholine, may be warranted.

Plasma choline concentration in humans fed parenterally.

Choline is an essential nutrient for some mammals; it is used for membrane and neurotransmitter synthesis. We analyzed plasma samples, obtained periodically during TPN therapy, for choline concentration. Malnourished patients referred to a nutrition support service were prospectively assigned to be treated with daily infusions of amino acids with, and without, supplemental daily infusions of lipid emulsion for a period of 1 wk. After the first week, all subjects received intravenous lipid, and most were offered enteral food supplements. Initial plasma choline concentrations in the 25 malnourished patients were significantly lower than those measured in plasma samples from 23 hospitalized patients known to be eating well (6.5 +/- 0.6 vs 9.7 +/- 0.7 nmol/ml; mean +/- SEM; p less than 0.001). During the first week of TPN therapy, plasma choline concentrations in the lipid-restricted group tended to decrease (from 7.3 +/- 1.0 to 4.7 +/- 0.5 nmol/ml; mean +/- SEM; p less than 0.05), while in the lipid-supplemented group plasma choline tended to increase (from 5.6 +/- 0.5 to 6.2 +/- 0.7 nmol/ml; mean +/- SEM; p less than 0.05). Plasma choline concentration increased during wk 2-4, when all patients were treated with lipid emulsions, and some were offered enteral foods. We conclude that malnourished humans who eat no choline have diminished stores of plasma (and possibly tissue) choline.

Short-term TPN containing n-3 fatty acids ameliorate lactic acidosis induced by endotoxin in guinea pigs.

We evaluated the effect of total parenteral nutrition (TPN) enriched with n-3 fatty acids on the physiologic response to endotoxin in guinea pigs. Animals were randomly assigned to receive TPN differing only in lipid source for 3.5 d. Group 1 received soybean fat emulsion (Intralipid) whereas group 2 received fish (menhaden) oil. During the last 7 h of TPN, animals were further randomized to have either saline or E coli endotoxin added to the infusate. Acid-base status and serum lactate concentrations were determined. Animals infused with soybean fat emulsions and endotoxin developed a significant metabolic acidosis, lactic acidemia, and decrease in mixed venous O2 compared with controls and fish-oil-treated animals (p less than 0.05). The significantly reduced serum lactate and higher mixed venous O2 in fish-oil-infused animals suggests that the underlying mechanism involves improvement in endotoxin-induced tissue hypoperfusion, presumably through alterations in prostaglandin metabolism.

Normal plasma choline responses to ingested lecithin.

We examined plasma choline changes after ingestion of diets composed of common foodstuffs, with choline contents bracketing the average daily intake in the American diet, and ingestion of diets supplemented with exogenous purified lecithin. A diet with low choline content did not increase plasma choline concentrations; a diet with high choline content doubled plasma choline levels. A lecithin-supplemented (25 gm; 80% phosphatidylcholine) low-choline diet increased plasma choline levels 400%. These findings indicate that normal diets cause only small elevations in plasma choline; purified lecithin supplements are likely to have greater effects in treating neurologic diseases.

Lecithin and choline in human health and disease.

Choline is involved in methyl group metabolism and lipid transport and is a component of a number of important biological compounds including the membrane phospholipids lecithin, sphingomyelin, and plasmalogen; the neurotransmitter acetylcholine; and platelet activating factor. Although a required nutrient for several animal species, choline is not currently designated as essential for humans. However, recent clinical studies show it to be essential for normal liver function. Additionally, a large body of evidence from the fields of molecular and cell biology shows that certain phospholipids play a critical role in generating second messengers for cell membrane signal transduction. This process involves a cascade of reactions that translate an external cell stimulus such as a hormone or growth factor into a change in cell transport, metabolism, growth, function, or gene expression. Disruptions in phospholipid metabolism can interfere with this process and may underlie certain disease states such as cancer and Alzheimer's disease. These recent findings may be appropriate in the consideration of choline as an essential nutrient for humans.

Assessing effects of serotonin precursors on newborn behavior.

While traditional studies of newborn diet have focused on the effects of malnutrition on the central nervous system, there is now interest in how qualitative differences in the composition of early newborn feeding might influence behavior. This paper reviews the available techniques for assessing newborn perception and cognition, as well as behavioral organization. The paper then focuses intensively on measures of newborn state behaviour in view of evidence in adult humans, as well as in non-human species, suggesting a relationship between sleep behavior (sleep onset, night waking) and brain serotonin levels. A study designed to examine the relationship between dietary precursors of brain serotonin (within the range of concentrations found in human milk) and newborn state behavior after feeding is described to illustrate the application of these techniques. Healthy, fullterm newborns were fed a modified formula, containing tryptophan or valine, on one day, a routine formula on another day, and observed continuously for 3 h after each feeding for the observation and recording of newborn state. Data from individual infants in the tryptophan and valine groups are presented to illustrate the findings that infants fed tryptophan entered quiet sleep and active sleep sooner than infants fed valine and spent more time in active sleep and less time alert. These results illustrate the value of newborn behavior as a sensitive dependent variable in studies of behavioral effects of diet and suggests that variations in serotonin levels in the newborn brain may modulate the newborn's sleep/wake behaviour.