Homozygosity mapping of the gene for alkaptonuria to chromosome 3q2.

Alkaptonuria, the first human disorder recognized by Garrod as an inborn error of metabolism, is a rare recessive condition that darkens urine and causes a debilitating arthritis termed ochronosis. We have studied two families with consanguineous parents and four affected children in order to map the gene responsible for alkaptonuria. Coinheritance of either neonatal severe hyperparathyroidism or sucrase-isomaltase deficiency and alkaptonuria provided a candidate location for the mutated genes on chromosome 3. Homozygosity mapping with polymorphic loci identified a 16 centiMorgan region on chromosome 3q2 that contains the alkaptonuria gene. Analysis of two additional nonconsanguineous families supports linkage of alkaptonuria to this single locus (combined lod score = 4.3, theta = 0).

Simulations of non-neutral slab systems with long-range electrostatic interactions in two-dimensional periodic boundary conditions.

We introduce a regularization procedure to define electrostatic energies and forces in a slab system of thickness h that is periodic in two dimensions and carries a net charge. The regularization corresponds to a neutralization of the system by two charged walls and can be viewed as the extension to the two-dimensional (2D)+h geometry of the neutralization by a homogeneous background in the standard three-dimensional Ewald method. The energies and forces can be computed efficiently by using advanced methods for systems with 2D periodicity, such as MMM2D or P3M/ELC, or by introducing a simple background-charge correction to the Yeh-Berkowitz approach of slab systems. The results are checked against direct lattice sum calculations on simple systems. We show, in particular, that the Madelung energy of a 2D square charge lattice in a uniform compensating background is correctly reproduced to high accuracy. A molecular dynamics simulation of a sodium ion close to an air/water interface is performed to demonstrate that the method does indeed provide consistent long-range electrostatics. The mean force on the ion reduces at large distances to the image-charge interaction predicted by macroscopic electrostatics. This result is used to determine precisely the position of the macroscopic dielectric interface with respect to the true molecular surface.

Hypertension in Latin America: Current perspectives on trends and characteristics.

The region of Latin America, which includes Central America, the Caribbean and South America, is one that is rapidly developing. Signified by socio-economic growth, transition and development over the last few decades, living standards in countries like Brazil and Mexico have improved dramatically, including improvements in education and health care. An important marker of socio-economic change has been the epidemiological shift in disease burden. Cardiovascular disease is now the leading cause of death in Latin America, and the drop in prevalence of infectious diseases has been accompanied by a rise in non-communicable diseases. Hypertension is the major risk factor driving the cardiovascular disease continuum. In this article we aim to discuss the epidemiological and management trends and patterns in hypertension that may be specific or more common to Latin-American populations - what we term 'Latin American characteristics' of hypertension - via a review of the recent literature. Recognizing that there may be a specific profile of hypertension for Latin-American patients may help to improve their treatment, with the ultimate goal to reduce their cardiovascular risk. We focus somewhat on the countries of Brazil, Mexico and Venezuela, the experience of which may reflect other Latin American countries that currently have less published data regarding epidemiology and management practices.

Pectin: its interaction with serum lipoproteins.

In vitro studies of interaction between grapefruit (Citrus paradisi) pectin and various human serum lipoproteins indicated that pectin interacts specifically with low-density lipoprotein. Examination of observed interaction between the pectin and low-density lipoprotein under variable experimental conditions revealed the electrostatic nature of this interaction. The results obtained from these studies suggest a possible biochemical basis by which dietary pectin may cause lowering of serum and/or tissue cholesterol levels.

Urinary 4-pyridoxic acid excretion in 24-hour versus random urine samples as a measurement of vitamin B6 status in humans.

Urinary excretion of 4-pyridoxic acid (4PA) in 19 men (n = 5) and women (n = 14) was measured to evaluate the validity of determining the 4PA/creatinine ratio in random urine samples as an alternative to total 24-h 4PA excretion in assessing vitamin B6 nutritional status. The relationships among dietary vitamin B6 intake, 4PA excretion, plasma pyridoxal 5'-phosphate levels, and erythrocyte aspartate aminotransferase activity and in vitro stimulation by added plasma pyridoxal 5'-phosphate were examined. The subjects consumed all meals for 3 days in a metabolic unit, and protein intake was kept constant. Plasma pyridoxal 5'-phosphate concentration was positively correlated with vitamin B6 intake of the previous day (r = 0.61, p less than 0.01). There was a positive correlation (r = 0.59, p less than 0.01) between total 4PA and 4PA/creatinine in the 24-h urine samples. No difference (p greater than 0.05) in 4PA/creatinine between the 24-h samples and either morning or afternoon random samples taken the next day was found. These findings support the use of the 4PA/creatinine ratio in random urine samples as an alternative to 24-h urinary 4PA excretion.

Comparative bioavailability of folate and vitamin C from a synthetic and a natural source.

Intraluminal perfusion of the human small intestine has not been used extensively to study comparative bioavailability of vitamins. In this study a triple lumen tube with a 30-cm study segment was used to measure absorption of water-soluble vitamins from the human proximal jejunum. Fifteen normal subjects served as their own controls to quantitate absorption of folic acid and vitamin C from an orange juice solution and from a solution of synthetic vitamins. Despite a predictably greater water absorption from the glucose containing orange juice solution, the absorption of the two water-soluble vitamins did not differ significantly from the two solutions. Natural and synthetic ascorbate and folate were avidly absorbed in the first 30 cm of jejunum and with the exception of synthetic folate correlated positively with water absorption. This method, previously applied to the absorption of sugars, amino acids, and electrolytes, can be reliably applied to the study of comparative bioavailability of nutrients from food sources. The advantages of triple lumen perfusion over previous methods are: 1) it overcomes the necessity for urine collections in metabolic studies, 2) it can be used to study sites and mechanism of absorption, and 3) it is a direct measurement of absorption capacity.

Methanol production from the degradation of pectin by human colonic bacteria.

When ingested, pectin can lower serum cholesterol levels in humans. Pectin is degraded by fecal bacteria in the colon. We examined the release of methanol (MeOH) by this degradation. A 0.2% glucose (2 g/L) mixture was used as the control medium. A pure culture of pectinolytic Erwinia carotovora was the control bacterium. The chief substrates were, in set 1, 0.2% pectin (2 g/L) and, in set 2, 0.1% glucose (1 g/L) and 0.1% pectin (1 g/L). Cultures of fecal bacteria and E carotovora grew for 72 h in each of the solutions. By 72 h the fecal flora culture in set 1 cleaved 30% of the possible methoxyl groups on pectin. The fecal flora in set 2 cleaved 90.7% of all possible methoxyl groups. Balance studies suggest that all of the free MeOH comes from methoxyl groups on pectin. This study demonstrates that fecal bacteria are capable of degrading pectin to release MeOH.

Genomic DNA methylation decreases in response to moderate folate depletion in elderly women.

BACKGROUND: Methylation of genomic DNA is dependent on an adequate supply of folate coenzymes. Previous data support the hypothesis that abnormal DNA methylation plays an integral role in carcinogenesis. To date, no studies assessing the effect of inadequate folate status on DNA methylation in older women (aged >63 y) have been reported. OBJECTIVE: The effect of moderate folate depletion followed by folate repletion on leukocyte genomic DNA methylation was investigated in elderly women (aged 60-85 y) to evaluate whether DNA methylation could be used as a functional indicator of folate status. DESIGN: Healthy, postmenopausal women (n = 33) consumed a moderately folate-depleted diet (118 microg folate/d) for 7 wk, followed by 7 wk of folate repletion with 200 or 415 microg/d, each provided as 2 different dietary treatments for a total of 4 treatment groups (n = 30). Leukocyte DNA methylation was determined on the basis of the ability of DNA to incorporate [(3)H]methyl groups from labeled S:-adenosylmethionine in an in vitro assay. RESULTS: Incorporation of [(3)H]methyl groups increased significantly (P: = 0.0025) in response to folate depletion, suggesting undermethylation of DNA. No significant changes were detected in [(3)H]methyl incorporation in any group over the 7-wk repletion period compared with postdepletion values. CONCLUSIONS: DNA methylation status may be used as a functional indicator of moderately depleted folate status. The slow response to the repletion diets observed suggests that normalization of DNA methylation after moderate folate depletion may be delayed in older women.

Stable-isotope methods for assessment of folate bioavailability.

Research was conducted to determine whether stable-isotope-labeled folates could be employed for studies of folate absorption and metabolism in human subjects. Two deuterium-labeled forms of folic acid were evaluated for simultaneous in vivo use, with quantification of relative bioavailability by measurement of urinary excretion of labeled folates. Adult male subjects (n = 11) were given saturation doses of 2 mg unlabeled folic acid/d for 7 d before the study. After an overnight fast each subject consumed 677 nmol each of 3',5'-labeled bideuterofolic acid and glutamate-labeled tetradeuterofolic acid. The 48-h urinary excretion of deuterated folates represented 5-6% of the ingested dose. The molar ratio of labeled folates in urine was not significantly different from the molar ratio in the ingested dose, which indicated equivalent absorption and metabolism of these labeled forms of the vitamin. These results support the validity of this protocol for in vivo studies of folate bioavailability.

Effect of physical activity on human potassium metabolism in a hot and humid environment.

Previous investigations have suggested that there are high potassium losses during heavy physical activities in hot climates. In order to determine if high levels of potassium losses could be offset by potassium loading, this study was conducted with five long-distance track runners who had trained in hot and humid environments. The liquid supplements containing 4.3, 98.0, and 0.0 mEq/liter of potassium were given 1 to 2 hr before physical activity. The daily diet contained 2.6 g of potassium. A sodium and potassium balance study was conducted in which stool, urine, and dermal losses were measured. In order to determine if there was a change in the distribution of body potassium during physical exercise, seven subjects total body potassium was estimated before and after exercise. This measurement was performed by counting 40K in a whole body counter. Although the subjects with potassium supplementation and higher urinary sodium and potassium losses, the 98 mEq/liter of potassium supplement resulted in a positive potassium balance. The subjects' potassium requirements exceeded the National Research Council suggested dietary intake. The total body potassium measurements indicated that the counting efficiency of 40K increases significantly immediately after the period of vigorous exercise.

Effect of diphenylhydantoin on the bioavailability of citrus folate.

Long term use of the drug diphenylhydantoin (DPH) has been associated with biochemical evidence of folic acid deficiency and rarely with megaloblastic anemia. The mechanism of this nutritional deficiency is uncertain but is thought to result from DPH-induced alteration in the intestinal absorption of conjugated and/or free dietary folate. The effect of DPH on the intestinal absorption of free folates from a food source has heretofore not been reported. In this study triple lumen tube perfusion of the human jejunum was used to quantitate folate absorption from a control solution of orange juice and from an identical solution containing DPH. The results in eleven volunteers serving as their own controls indicate no effectof DPH at a concentration of 20 microgram/ml on folate absorption from this food source. The predominant form of folate in orange juice as determined by differential microbiologic assay is N-5-methyltetrahydrofolate. DPH does not appear to interfere with the absorption of free food folate which is both methylated and reduced.

Red blood cell uptake of supplemental folate in patients on anticonvulsant drug therapy.

A group of epileptics (n = 18) and a control group (n = 10) of subjects aged 21-42 y were given 1-mg supplements of folate daily for 1 mo. Anticonvulsant therapy involved phenytoin alone or in combination with phenobarbital. Serum and red blood cell (RBC) folate levels were determined on days 1, 14, and 28. Mean serum and RBC folate levels were greater (p less than 0.05) for the control subjects compared with the epileptic subjects throughout the study. The percent increase in either serum or RBC folate was not different (p greater than 0.05) between the groups. The percent increase in serum folate when expressed as a percent of RBC folate was greater (p less than 0.05) for those epileptics who initially had deficient blood folate levels (serum folate less than 7 nmol/L; RBC folate less than 317 nmol/L) than those who did not. Deficient epileptics may have had an impaired RBC incorporation of circulating (serum) folate compared with nondeficient epileptics.

Relative bioavailability of deuterium-labeled monoglutamyl tetrahydrofolates and folic acid in human subjects.

The bioavailability of orally administered monoglutamyl folic acid and various (6S)-tetrahydrofolates was examined in humans with stable-isotope methods. Folic acid (PteGlu), tetrahydrofolate (H4folate), 5-formyl-H4folate, 10-formyl-H4folate, and 5-methyl-H4folate were prepared for oral administration in 3',5'-2H2 labeled (d2) form, and [glu-2H4]folic acid (d4-PteGlu) was prepared for intravenous injection. In each of five trials, fasting adult males (n = 7) on a folate saturation regimen (2 mg/d) were given a single oral dose of one of the d2-folates in apple juice, as well as an intravenous injection of d4-PteGlu as a control. Urine was collected for 48 h and the isotope labeling of urinary folates determined by mass spectrometry. Isotope excretion ratios of urinary folates were used as criteria of bioavailability (pooled SE = 0.10): PteGlu (1.53, least squares mean), 10-formyl-H4folate (1.02), 5-methyl-H4folate (0.99), 5-formyl-H4folate (0.1.13), and H4folate (0.71). These results indicate that differences exist in the bioavailability of monoglutamyl folates under these experimental conditions. This variation, whether due to differences in absorption or postabsorptive events, must be considered in quantitative studies of folate utilization with this type of protocol.

Relative bioavailability of deuterium-labeled monoglutamyl and hexaglutamyl folates in human subjects.

The bioavailability of orally administered mono- and polyglutamyl folates was examined in humans by using stable-isotope methods. [3',5'-2H2]Folic acid (d2-FA) and [3',5'-2H2]pteroylhexaglutamate (d2-PteGlu6) were prepared for oral administration and (glu-2H4)folic acid (d4-FA) was prepared for intravenous (iv) injection. In two trials, adult males (n = 7) on a folate saturation regimen (2 mg/d) were given a single 677-nmol oral dose of either d2-FA or d2-PteGlu6 in apple juice along with an iv injection of 502 nmol d4-FA as a control. Urine was collected for 48 h and the isotope labeling of urinary folates determined by mass spectrometry. The excretion ratio of urinary folates (% of d2-folate dose/% of d4-folate dose) resulting from oral d2-FA and iv d4-FA was 1.45 +/- 0.10 (mean +/- SEM) whereas the ratio for oral d2-PteGlu6 and iv d4-FA was 0.67 +/- 0.04. These results indicate that the d2-PteGlu6 is available to humans as a source of folate although its bioavailability is substantially less than that of d2-FA under these conditions.

Use of nutritional supplements in an ambulatory elderly population.

The use of nutritional supplements by 3,192 ambulatory elderly participants in a health screening program is described. The 2,009 women used vitamin (45.5 per cent) and mineral (22.4 per cent) products with significantly greater frequencies than did the 1,183 men (34.0 per cent and 15.0 per cent, respectively); chi-square, P less than 0.01. The most commonly used vitamin products were multiple vitamins, multiple vitamins with minerals, vitamin E, and vitamin C; for minerals, the ranking was potassium chloride, calcium salts, and ferrous sulfate. Eighty-two participants (2.5 per cent) reported the use of four or more supplements. Many older Americans are spending a great deal of money for nutritional supplements, whereas it would seem that the money could be better spent to improve the quality of their diet.

Methylenetetrahydrofolate reductase mutation (677C-->T) negatively influences plasma homocysteine response to marginal folate intake in elderly women.

Individuals who are homozygous for the methylenetetrahydrofolate reductase (MTHFR) 677C --> T mutation have depressed serum folate (SF) and elevated plasma total homocysteine (tHcy) concentrations, which may affect folate requirements and increase the risk for coronary artery disease. A controlled metabolic study (14 weeks) using a depletion/repletion protocol was performed in women (aged 60 to 85 years, N = 33) to provide age-specific data on the effects of the MTHFR mutation on SF and tHcy status. Subjects consumed a moderately folate-deplete diet (118 microg/d) for 7 weeks, followed by 7 weeks of folate repletion with 200 or 415 microg/d provided as two different treatments. Following folate depletion, the mean SF concentration was lower for homozygous (P = .017) versus heterozygous subjects. Homozygotes for the 677C --> T mutation showed a higher (P = .015) percent increase in plasma tHcy (44%) than heterozygous (20%) or normal (15%) subjects. At week 7, the mean plasma tHcy concentration was higher in homozygous subjects (12.5 +/- 5.3 micromol/L, mean +/- SD) versus the heterozygous (10.8 +/- 3.8 micromol/L, P = .008) or normal (11.3 +/- 2.7 micromol/L, P = .001) genotype groups. Following folate repletion, plasma tHcy concentrations were not different between genotype groups, despite a higher (P < .016) SF concentration in subjects with the homozygous genotype. These data suggest that older women who are homozygous for the MTHFR 677C --> T mutation may be at risk for greater elevations in plasma tHcy in response to moderately low folate intake as compared with individuals with the normal or heterozygous genotypes.

Diet and gastrointestinal disease.

Diet therapy in the treatment of gastrointestinal disease has become better defined. Because maldigestion or malabsorption frequently occurs, malnutrition is a common complication. Careful assessment of nutritional status by the health care providers is mandatory. Diet therapy, by eliminating offending foods such as lactose or gluten or by addition of specialized enteral formulas containing MCT, or elemental diets can be instituted with marked relief in symptoms. The role of dietary fiber is now better defined in the treatment and prevention of many diseases. Further refinement and rational uses of diet therapy can be expected in future years.

Remedial education: can this doctor be saved?

In the first two years of the program 30 physicians have completed the program. A list of the distribution of specialties/practice areas [table: see text] served is provided in Table 1. The data reveal that the distribution of practice areas corresponds approximately to the distribution of physicians practicing in the state. The UF C.A.R.E.S Program provides a great benefit to physicians and their patients. It provides an atmosphere of professional collaboration and encouragement to address specific educational needs and underscores a commitment to providing continuing medical education, meaningful doctor-to-doctor collaboration, better patient care, and reflects a medical model of diagnosis and treatment of specific problems.

Total parenteral nutrition in the mouse: body composition and plasma chemistries.

Mice that were maintained in energy and nitrogen (N) balance by total parenteral nutrition (TPN) for 12 days were analyzed for changes in organ weight, carcass and liver N and fat, and plasma glucose, urea N, and total protein. The results are compared with two other groups: (1) PO, which consisted of mice that were given the TPN solution per os in amounts equivalent to the TPN group, and (2) AL, which consisted of mice allowed to consume a stock diet ad libitum. In comparison with group AL, the TPN-fed mice had normal liver, kidney, and lung weights but heavier spleens and hearts. Group PO had an increase in liver weight only. Hepatic lipid content declined in group TPN but increased markedly in PO-fed mice. The latter group also demonstrated a 35% increase in carcass fat whereas it was unchanged in the TPN group. No differences were found in plasma urea N and total protein among the groups but plasma glucose increased 2-fold in group PO. It appears that our technique of parenteral feeding in mice maintains fairly normal body composition and plasma chemistries. However, mice drinking the TPN solution (group PO) exhibited the greatest number of alterations. These results are discussed in relation to differences in route of feeding, diet composition, feeding pattern, and the possible influence of circadian rhythms. The dilemma of choosing appropriate control groups in TPN studies is also discussed.

Total parenteral nutrition in the mouse: development of a technique.

A method for total parenteral nutrition in the mouse was developed using commercially available supplies and equipment. The mouse's inferior vena cava was catheterized and the catheter was exteriorized from the tail. Mice (average body weight 22.5 g) were not tethered but instead were partially restrained by immobilizing the tail to protect the infusion tubing. A solution was formulated to contain 40% dextrose and 4.3% amino acids plus vitamins, electrolytes, and trace elements. It was administered via pump in graded fashion for the first 3 days to allow the mice to adapt, and then at a rate of 8 ml/day thereafter. This volume provided approximately 12 kcal and 54 mg nitrogen per day and was calculated to meet the needs of the mouse fed per os according to guidelines of the National Research Council. During the adaptation period mice lost weight but they were in positive N balance thereafter. At the end of 12 days body weight was not different than at the start, indicating the adequacy of nutrient input. Further, there were no differences in nitrogen balance or body weight in total parenteral nutrition fed mice in comparison with tail-restrained mice given the same solution per os. All mice appeared to tolerate the partial restraint without incidence and showed no untoward side effects. The rationale and validity of this technique is discussed in detail.