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β-Carotene (BC), β-cryptoxanthin (CX) and α-carotene (AC) are common carotenoids that form retinol. The amount of retinol (vitamin A) formed from carotenoid-rich foods should depend chiefly on the bioavailability (absorption and circulation time in the body) of carotenoids from their major food sources and the selectivity and reactivity of carotene cleavage enzymes towards them. The objective of the present study was to estimate the apparent bioavailability of the major sources of provitamin A (AC, BC and CX) from the diet by comparing the concentrations of these carotenoids in blood to their dietary intakes. Dietary intakes were estimated by FFQ (three studies in this laboratory, n 86; apparent bioavailability calculated for six other studies, n 5738) or by food record (two studies in our laboratory, n 59; apparent bioavailability calculated for two other studies, n 54). Carotenoid concentrations were measured by reversed-phase HPLC. Apparent bioavailability was calculated as the ratio of concentration in the blood to carotenoid intake. Then apparent bioavailabilities for AC and CX were compared to BC. Eating comparable amounts of AC-, CX- and BC-rich foods resulted in 53 % greater AC (99 % CI 23, 83) and 725 % greater CX (99 % CI 535, 915) concentrations in the blood. This suggests that the apparent bioavailability of CX from typical diets is greater than that of BC. Thus, CX-rich foods might be better sources of vitamin A than expected.

Vitamin A containing (holo) free and transthyretin-bound (TTR) retinol binding protein (RBP) concentrations in plasma from rats with variable vitamin A status were determined by high performance liquid chromatography. Two different methods were used: (1) molecular exclusion with a TSK 2000 column and (2) reverse phase using a Protesil Octyl 300 column. Holo TTR-RBP peak areas were positively correlated to liver vitamin A concentrations (r = 0.79 for molecular exclusion 0.81 for reverse phase) in rats with marginal and normal vitamin A status. This correlation was higher than the correlation of serum retinol to liver vitamin A in these rats (r = 0.58). The correlation of holo-TTR-RBP to liver vitamin A was also higher than its correlations to plasma vitamin A. Therefore, plasma concentrations of holo—TTR-RBP may be influenced by marginal vitamin A liver stores to a greater extent than plasma retinol is. This suggests that holo—TTR-RBP protein concentrations may be the better indicator of marginal vitamin A nutritional status in rats. The correlations of both holo—TTR-RBP and serum retinol decreased sharply at high liver vitamin A concentrations. Neither method is suitable for measuring sub-toxicity in rats.

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR-RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR-RBP concentrations determined by this method are highly correlated to holo-TTR-RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR-RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR-RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.

Vitamin A deficiency is a health problem in Southeast Asia that can be corrected by feeding orange fruits and vegetables such as mango. Pumpkin is a traditional Korean food that is easy to store and is already believed to have health benefits. We extracted carotenoids from pumpkin by liquid-liquid extraction and by supercritical fluid extraction. We measured carotenoids by reversed-phase chromatography with diode array detection. The major carotenoid in pumpkin (> 80%) is beta-carotene, with lesser amounts of lutein, lycopene, alpha-carotene and cis-beta-carotene. Pumpkin is a rich source of beta-carotene and might be useful for preventing Vitamin A deficiency.

The search for a reliable, convenient indicator of Zn status was the focus of research for several decades. Plasma Zn concentration is still the most widely used clinical measurement, despite the known problems of interpretation. More recently, researchers suggested that isotopically determined kinetic parameters, such as the exchangeable Zn pool (EZP), may more accurately and reliably reflect body Zn status. The objective of this study was to examine the relationship between net body Zn loss and gain during acute changes in dietary Zn intake with biochemical and kinetic indices of Zn status. Five men participated in an 85-d Zn depletion/repletion study. Net body Zn loss and gain were determined from the difference between dietary plus intravenously administered Zn and Zn excretion. Biochemical indicators of Zn status included plasma Zn, plasma alkaline phosphatase activity, and plasma retinol binding protein concentration. Following intravenous administration of (70)Zn or (67)Zn, a compartmental model was used to determine EZP mass, fractional Zn absorption, endogenous zinc excretion (EZE), and plasma Zn flux. The changes in total body zinc correlated best with changes in plasma Zn (r(2) = 0.826, P < 0.001), EZE (r(2) = 0.773, P < 0.001), and plasma Zn flux (r(2) = 0.766, P < 0.001). This study confirms that plasma Zn concentration is a valid indicator of whole-body Zn status in the absence of confounding factors; however, further research is needed to determine how kinetic parameters respond to conditions where plasma Zn concentration is known to be unreliable.

Dietary intakes of carotenoids are highly variable in human populations as are serum carotenoid concentrations. However, there are few controlled data relating carotenoid intake to concentration. Most of the data that are available are from measurements of the absorption and decay of large pharmacologic doses of carotenoids, and are therefore of unknown physiologic relevance. Our objective was to determine the half-life (t(1/2)) of the most abundant carotenoids in blood serum from healthy adult women living under controlled conditions. As part of two carotenoid isotopic studies, we measured serum concentrations of beta-carotene, alpha-carotene, lutein, zeaxanthin, beta-cryptoxanthin and lycopene in 19 healthy young adult women that were fed controlled low carotenoid diets for approximately 10 wk. All other nutrients (vitamins A, E and C) were provided at 100-150% of the 1989 U.S. recommended dietary allowance levels. Exercise and activities were controlled throughout the studies to simulate usual activity patterns. Carotenoid concentrations were measured by reversed-phase HPLC. Serum carotenoid concentration decreases during depletion followed first-order kinetics. The half-lives determined in decreasing order were as follows: lutein (76 d) > alpha-carotene (45 d) = beta-cryptoxanthin (39 d) = zeaxanthin (38 d) = beta-carotene (37 d) > lycopene (26 d). Half-lives were unrelated to physical or demographic characteristics such as body mass, body fat, racial background or age in these relatively homogeneous groups. Carotenoids decreased by similar first-order mechanisms, although the rates differed for individual carotenoids.

BACKGROUND Blood beta-carotene and vitamin A responses to oral beta-carotene are variable in humans. Some individuals are characterized as responders and others as low- or nonresponders. A better understanding of the conditions that produce the variability is important to help design public health programs that ensure vitamin A sufficiency. OBJECTIVE Our objective was to assess variability in absorption and conversion of beta-carotene to vitamin A in vivo in humans by using a novel double-tracer ?hexadeuterated (D(6)) beta-carotene and D(6) retinyl acetate approach. DESIGN Eleven healthy women were housed at the US Department of Agriculture Western Human Nutrition Research Center metabolic unit for 44 d, where they consumed diets adequate in vitamins and minerals except for carotenoids. After an adaptation period, the women were given 30 micromol D(6) retinyl acetate orally, followed 1 wk later with 37 micromol D(6) beta-carotene (approximately equimolar doses). Time-dependent plasma concentration curves were determined for D(6) retinol, D(6) beta-carotene, and trideuterated (D(3)) retinol (derived from D(6) beta-carotene). RESULTS Mean (+/-SE) absorption of D(6) beta-carotene was 3.3 +/- 1.3% for all subjects. The mean conversion ratio was 0.81 +/- 0.34 mol D(3) retinol to 1 mol D(6) beta-carotene for all subjects. However, only 6 of the 11 subjects had plasma D(6) beta-carotene and D(3) retinol concentrations that we could measure. The mean absorption of D(6) beta-carotene in these 6 subjects was 6.1 +/- 0.02% and their conversion ratio was 1.47 +/- 0.49 mol D(3) retinol to 1 mol D(6) beta-carotene. The remaining 5 subjects were low responders with

We measured immunologically active (apo + holo) retinol-binding protein (RBP), vitamin A-carrying (holo) free RBP, and transthyretin-bound (TTR) holo-RBP in serum from 34 retrospective cases of fluctuating acute renal failure. All subjects had high serum creatinine concentrations caused by renal failure. Apo + holo, holo-TTR-RBP, and (especially) holo-free RBP all correlated poorly but significantly with serum creatinine concentration. Therefore, the use of any form of RBP to measure vitamin A status may be of limited value in subjects with high creatinine concentrations in serum. However, molecular-exclusion HPLC may be able to distinguish increases in RBP concentration associated with renal failure from those caused by altered vitamin A status, because renal failure causes abnormalities in the number and retention times of chromatographic peaks as well as their areas.

We conducted a pilot, open-label study to assess the effect of short-term beta-carotene administration (180 mg/d with meals for 4 weeks) on the plasma human immunodeficiency virus (HIV) RNA levels and CD4+ lymphocyte counts in 21 HIV-infected patients. We found that plasma HIV RNA levels and CD4+ lymphocyte counts did not change following this short course of beta-carotene supplementation. Patients with lower serum concentrations of beta-carotene before supplementation were no more likely to have an increase in their CD4+ lymphocyte count or plasma HIV RNA copy number than were those with higher concentrations. No correlation was found between pre- or postsupplementation beta-carotene or vitamin A concentrations and pre- or postsupplementation CD4+ lymphocyte counts or plasma HIV RNA titers. This study provides no support for beta-carotene supplementation for HIV-infected subjects with normal baseline serum levels of beta-carotene and vitamin A.

Vitamin A has several essential functions, including roles in dark adaptation, cell growth and maintenance, and immunological functions (Underwood, 1978; Bauernfeind, 1986; National Research Council, 1989; Sommer and West, 1996; Gerster, 1997). Vitamin A deficiency is the leading cause of preventable blindness in the world (Sommer, 1982). Furthermore, milder vitamin A inadequacy has been implicated as a leading cause of death in poor children of Southern Asia and Africa (Sommer et al., 1983; Sommer and West, 1987). A majority of people in the world derive most of their vitamin A from carotenoids found in many fruits and vegetables (Narasinga, 1991; Solomons and Bulux, 1993; Solomons and Bulux, 1994; FAO/INMU/South and East Asia Nutrition Research-cum-Action Network, 1995; Seshadri, 1996).

Dietary intakes are usually estimated by either a food frequency test, or by food records. We hypothesized that dietary intake estimates for fat soluble vitamins might be more accurate if information from both food frequency tests and food records were used. We estimated dietary intakes in 10 healthy adults by 4 food frequency questionnaires and ten 3-day food records collected over a year. Serum antioxidant nutrient concentrations (vitamins A, E, and the carotenoids) were measured by HPLC throughout the year. Few changes in intake occurred over the year. Estimates of nutrient densities correlated more often than estimates of nutrient intakes to serum nutrient concentrations. Nutrient density estimates derived from the average of food frequency and food record estimates were significantly correlated with serum nutrient concentrations more often than dietary estimates derived from either food frequency or food records alone. We suggest that nutrient density estimates derived from a combination of food frequency and food records may be useful for studies of free-living individuals.

The formation of chylomicrons by the intestine is important for the absorption of dietary fats and fat-soluble vitamins (e.g., retinol, alpha-tocopherol). Apo B plays an essential structural role in the formation of chylomicrons in the intestine as well as the VLDL in the liver. We have developed genetically modified mice that express apo B in the liver but not in the intestine. By electron microscopy, the enterocytes of these mice lacked nascent chylomicrons in the endoplasmic reticulum and Golgi apparatus. Because these mice could not form chylomicrons, the intestinal villus enterocytes were massively engorged with fat, which was contained in cytosolic lipid droplets. These mice absorbed D-xylose normally, but there was virtually no absorption of retinol palmitate or cholesterol. The levels of alpha-tocopherol in the plasma were extremely low. Of note, the absence of chylomicron synthesis in the intestine did not appear to have a significant effect on the plasma levels of the apo B-containing lipoproteins produced by the liver. The mice lacking intestinal apo B expression represent the first genetic model of defective absorption of fats and fat-soluble vitamins and provide a useful animal model for studying nutrition and lipoprotein metabolism.

being different. We all know many people who stop and start smoking, drinking, and dieting regularly. Yet, we tend to think of diet modification as a long-term, holistic, “natural,” non-specific method of health maintenance. We all know people who eat herbs (such as St. John’s Wort and gingko biloba), nutrients (such as vitamin C and antioxidant complexes), and nutrition-based drugs for years in the hope that they will prevent disease. Yet, we think of chemoprevention as being harsh, effective, and narrowly focused on short-term treatments or prevention of cancer. This narrow focus may have been especially true for retinoid (vitamin A) research. Researchers made exciting and rapid progress in retinoid genetics, metabolism, and cell differentiation in the 1980s and 1990s.4 This progress occurred at the same time, but generally in different laboratories, as exciting and rapid developments in ascertaining the causes, consequences, and prevention of vitamin A deficiency. 5 The explosion in information was difficult to assimilate and use effectively. Retinoid research essentially split into two camps. “Retinoid” researchers, mostly working in the United States and Europe, investigated cellular mechanisms of differentiation, and cancer chemoprevention. “Vitamin A” researchers, mostly working in the developing countries, investigated the effects of dietary intervention and modification on vitamin A deficiency. There were Retinoid conferences and Vitamin A conferences (few scientists attended both). However, the lines between “diet” modification and “drug”-based chemoprevention are not so difficult to cross. The retinoid family of nutrients provides some of the most successful nutrient-based cancer chemopreventatives we have, and is used in some of the most successful diet modification programs. Research on diet modification programs using vitamin A have been summarized elsewhere.5 Retinoids as cancer chemopreventive agents are reviewed in this issue.6 Retinoids are powerful chemicals that regulate cell differentiation and proliferation. Cancer involves the disruption of normal cell differentiation, so it was obvious that retinoid status might influence a cell’s potential for malignant transformation. Early studies showed that vitamin A deficiency could lead to cancer in animals, and that retinoids could interfere with cancer initiation and progression in cell culture. 6,7 These exciting results were followed by many more studies, which are summarized in this interesting review.6

The prenatal diagnosis and prenatal treatment of a fetus with the neonatal-onset variant of biotin-responsive multiple carboxylase deficiency are described. Pre-amniocentesis maternal urinary organic-acid profiles were normal. Amniotic-fluid methylcitrate (17 weeks' gestation) was ten standard deviations above the control mean. Amniotic-fluid-cell propionyl CoA carboxylase, pyruvate carboxylase, and 3-methylcrotonyl CoA carboxylase activities were indistinguishable from activities in controls after cell growth in biotin-supplemented media. In contrast, growth in biotin-restricted media resulted in carboxylase activities significantly below control values. The fetus was considered to be affected, and the mother was begun on oral biotin at 231/2 weeks' gestation. The full-term baby girl exhibited no clinical or chemical aberrations during the first 4 days of life, before the start of postnatal biotin supplementation. Cord-blood biotin was 34 times the upper normal neonatal concentration. The diagnosis was confirmed by carboxylase assays on cultured skin fibroblasts obtained after birth; by slightly raised urinary organic-acid levels later in infancy; and by demonstration of· fibroblast holocarboxylase synthetase with an elevated K m(biotin) and a depressed V max . Growth and development of the baby have been normal to age 11/4 years on oral biotin. The prenatal diagnosis of neonatal-onset biotin-responsive multiple carboxylase deficiency can be made by means of enzymatic studies of amniotic-fluid cells. Amniotic-fluid methylcitrate should be a useful adjunct to enzymatic studies. Prenatal treatment was effective in our patient, and there was no apparent toxicity to mother or baby from biotin supplementation during the last 16 weeks of pregnancy.

Erythrocyte superoxide dismutase activities and erythrocyte and plasma glutathione peroxidase activities have been determined in Duchenne muscular dystrophy patients, genetic carriers, and normal controls. A 19% decrease in red cell superoxide dismutase activity was observed in patients with Duchenne muscular dystrophy. Genetic carriers showed a level between that of the dystrophy patients and the normal controls. No abnormality was seen in the red cell or plasma activities of glutathione peroxidase.

We quantified vitamin A-transporting retinol-binding protein (RBP) in serum or plasma by size-exclusion "high-performance" liquid chromatography, using a TSK 2000 column and fluorescent detection of the bound retinol. Serum or plasma samples filtered through a 0.20-microns (pore size) Millex filter were applied directly to the column. The pH 7.0 mobile phase contained sodium phosphate, disodium EDTA, and mercaptoethanol. Two peaks with RBP immunological activity were eluted: the smaller peak containing at least 86% of the vitamin A, which was identified as transthyretin-bound RBP; the larger peak containing a small amount (less than 14%) of a highly fluorescent vitamin A-containing protein, identified as free RBP. Both free and transthyretin-bound RBP can be quantified by this method.

The prenatal diagnosis and prenatal treatment of a fetus with the neonatal-onset variant of biotin-responsive multiple carboxylase deficiency are described. Pre-amniocentesis maternal urinary organic-acid profiles were normal. Amniotic-fluid methylcitrate (17 weeks' gestation) was ten standard deviations above the control mean. Amniotic-fluid-cell propionyl CoA carboxylase, pyruvate carboxylase, and 3-methylcrotonyl CoA carboxylase activities were indistinguishable from activities in controls after cell growth in biotin-supplemented media. In contrast, growth in biotin-restricted media resulted in carboxylase activities significantly below control values. The fetus was considered to be affected, and the mother was begun on oral biotin at 231/2 weeks' gestation. The full-term baby girl exhibited no clinical or chemical aberrations during the first 4 days of life, before the start of postnatal biotin supplementation. Cord-blood biotin was 34 times the upper normal neonatal concentration. The diagnosis was confirmed by carboxylase assays on cultured skin fibroblasts obtained after birth; by slightly raised urinary organic-acid levels later in infancy; and by demonstration of· fibroblast holocarboxylase synthetase with an elevated Km(biotin) and a depressed Vmax. Growth and development of the baby have been normal to age 11/4 years on oral biotin. The prenatal diagnosis of neonatal-onset biotin-responsive multiple carboxylase deficiency can be made by means of enzymatic studies of amniotic-fluid cells. Amniotic-fluid methylcitrate should be a useful adjunct to enzymatic studies. Prenatal treatment was effective in our patient, and there was no apparent toxicity to mother or baby from biotin supplementation during the last 16 weeks of pregnancy.

The assembly of proteins of the intrinsic activation complex has been partially elucidated. In the present study we examine the association of gamma-carboxylated serine proteinase zymogens factors IX and X, and their proteolytically activated counterparts factors IXa and Xa to unilamellar lipid vesicles of defined composition using three types of physical measurement. Utilizing relative light scatter to estimate the dissociation constants for binding in the presence of calcium ions, it appears that factor IXa (0.93 +/- 0.37 microM) may preferentially associate with phospholipids relative to factor IX (0.35 +/- 0.08 microM). In contrast, factor X (0.34 +/- 0.14 microM), the substrate for factor IXa, appears to bind to phospholipid with a higher affinity than factor Xa (0.58 +/- 0.13 microM). These observations are compatible with the hypothesized dynamics where the forward 'traffic' is facilitated by favoring the association of factor IXa with factor X. The dissociation constants were estimated by molecular exclusion chromatography (1.1 - 2.5 microM) and do not reflect these relative and ordered differences in association with lipid vesicles. Quasi-elastic light scatter analyses indicate that each protein appears to saturate the same vesicle surface, consistent with competition for similar surface lipids, although the molecular shell formed by factor Xa (36 A) is smaller, suggesting that it has a different packing on the phospholipid surface than the other proteins (64-79 A). The pattern of preferential affinities for phospholipid is consistent with a kinetically functional forward traffic through the reaction precursors to products, and suggests that these preferential affinities may assist in the ordering of the four proteins in the intrinsic activation complex.

Biotin-responsive multiple carboxylase deficiency is an inherited disorder of organic acid metabolism in man in which there are deficiencies of propionyl-coenzyme A (CoA), 3-methylcrotonyl-CoA, and pyruvate carboxylases that can be corrected with large doses of biotin. It has been proposed that the basic defect in patients with the early infantile form of the disease is in holocarboxylase synthetase, the enzyme that covalently attaches biotin to the inactive apocarboxylases to form active holocarboxylases. We have developed an assay for holocarboxylase synthetase in extracts of human fibroblasts using as substrate apopropionyl-CoA carboxylase partially purified from livers of biotin-deficient rats. Fibroblasts from the initial patient with the infantile form of biotin-responsive multiple carboxylase deficiency were shown to have abnormal holocarboxylase synthetase activity with a maximum velocity about 30-40% of normal, a Km for ATP of 0.3 mM similar to the normal Km of 0.2 mM, and a highly elevated Km for biotin of 126 ng/ml, about 60 times the normal Km of 2 ng/ml. These results show that the primary defect in this patient is a mutation affecting holocarboxylase synthetase activity, and thus a genetic defect of the metabolism of biotin.