SNCA, MAPT, and GSK3B in Parkinson disease: a gene-gene interaction study.

BACKGROUND AND PURPOSE: Recent evidence suggests that variation in the SNCA, MAPT, and GSK3B genes interacts in affecting risk for Parkinson disease (PD). In the current study, we attempt to validate previously published findings, evaluating gene-gene interactions between SNCA, MAPT, and GSK3B in association with PD. METHODS: Three Caucasian PD patient-control series from the United States, Ireland, and Norway (combined n = 1020 patients and 1095 controls) were genotyped for SNCA rs356219, MAPT H1/H2-discriminating SNP rs1052553, and GSK3B rs334558 and rs6438552. RESULTS: Our findings indicate that as previously reported, the SNCA rs356219-G allele and MAPT rs1052553 (H1 haplotype) were both associated with an increased risk of PD, whilst contrary to previous reports, GSK3B variants were not. No pair-wise interaction was observed between SNCA, MAPT, and GSK3B; the risk effects of SNCA rs356219-G and MAPT rs1052553-H1 were seen in a similar manner across genotypes of other variants, with no evidence suggesting synergistic, antagonistic, or deferential effects. CONCLUSIONS: In the Caucasian patient-control series examined, risk for PD was influenced by variation in SNCA and MAPT but not GSK3B. Additionally, those three genes did not interact in determining disease risk.

Metabolic, idiosyncratic toxicity of drugs: overview of the hepatic toxicity induced by the anxiolytic, panadiplon.

Preclinical drug safety evaluation studies, typically conducted in two or more animal species, reveal and define dose-dependent toxicities and undesirable effects related to pharmacological mechanism of action. Idiosyncratic toxic responses are often not detected during this phase in development due to their relative rarity in incidence and differences in species sensitivity. This paper reviews and discusses the metabolic idiosyncratic toxicity and species differences observed for the experimental non-benzodiazepine anxiolytic, panadiplon. This compound produced evidence of hepatic toxicity in Phase 1 clinical trial volunteers that was not predicted by rat, dog or monkey preclinical studies. However, subsequent studies in Dutch-belted rabbits revealed a hepatic toxic syndrome consistent with a Reye's Syndrome-like idiosyncratic response. Investigations into the mechanism of toxicity using rabbits and cultured hepatocytes from several species, including human, provided a sketch of the complex pathway required to produce hepatic injury. This pathway includes drug metabolism to a carboxylic acid metabolite (cyclopropane carboxylic acid), inhibition of mitochondrial fatty acid beta-oxidation, and effects on intermediary metabolism including depletion of glycogen and disruption of glucose homeostasis. We also provide evidence suggesting that the carboxylic acid metabolite decreases the availability of liver CoA and carnitine secondary to the formation of unusual acyl derivatives. Hepatic toxicity could be ameliorated by administration of carnitine, and to a lesser extent by pantothenate. These hepatocellular pathway defects, though not directly resulting in cell death, rendered hepatocytes sensitive to secondary stress, which subsequently produced apoptosis and hepatocellular necrosis. Not all rabbits showed evidence of hepatic toxicity, suggesting that individual or species differences in any step along this pathway may account for idiosyncratic responses. These differences may be roughly applied to other metabolic idiosyncratic hepatotoxic responses and include variations in drug metabolism, effects on mitochondrial function, nutritional status, and health or underlying disease.

Attitudes toward colon cancer gene testing: factors predicting test uptake.

OBJECTIVES: Genetic discoveries in hereditary nonpolyposis colorectal cancer (HNPCC) have made possible genetic testing to determine susceptibility to this form of colorectal cancer (CRC). This study measured the uptake of genetic testing for HNPCC among first-degree relatives of CRC patients and conducted a preliminary analysis of the predictors of test uptake. MATERIALS AND METHODS: We compared 77 test acceptors and 181 decliners on demographic, medical history, and psychological characteristics, controlling for distance from the testing center. The psychological factors studied were risk perception for CRC, frequency of cancer thoughts, and perceived ability to cope with unfavorable genetic information. RESULTS: In the final regression model, after accounting for all variables, the significant predictors of test uptake were increased risk perception, greater perceived confidence in ability to cope with unfavorable genetic information, more frequent cancer thoughts, and having had at least one colonoscopy. The association between risk perception and uptake was dependent on frequency of cancer thoughts. Among those who thought about getting CRC more often, the probability of testing increased as perceived risk increased to approximately 50% likelihood of getting CRC and then leveled off. In contrast, among those who never or rarely thought about getting CRC, risk perception was unrelated to testing decision. CONCLUSIONS: Our findings are consistent with the associations reported between psychological factors and other cancer screening behaviors.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells.

PNU-87407 and PNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals. The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria. PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H. contortus. Each compound reduced motility and ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner; however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H. contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to > or = 0.25 microM PNU-87407, > or = 1 microM closantel or > or = 10 microM PNU-88509. Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407 were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 microM, respectively.

Disruption of mitochondrial activities in rabbit and human hepatocytes by a quinoxalinone anxiolytic and its carboxylic acid metabolite.

The quinoxalinone anxiolytic, panadiplon, was dropped from clinical development due to unexpected hepatic toxicity in human volunteers. Subsequent experimental studies in rabbits demonstrated a hepatic toxicity that resembled Reye's syndrome. In the present studies, we examined the effects of panadiplon and a metabolite, cyclopropane carboxylic acid (CPCA) on hepatic mitochondrial activities in vitro and ex vivo. Acute inhibition of beta-oidation of [14C]palmitate was observed in rabbit and human hepatocyte suspensions incubated with 100 microM panadiplon. Panadiplon (30 microM) also reduced mitochondrial uptake of rhodamine 123 (R123) in cultured rabbit and human, but not rat hepatocytes, following 18 h exposure. CPCA also impaired beta-oxidation and R123 uptake in rabbit and human hepatocytes. R123 uptake and beta-oxidation in cells from some donors was not impaired by either agent, and cell death was not observed in any experiment. Hepatocytes isolated from panadiplon-treated rabbits had reduced palmitate beta-oxidation rates and inhibited mitochondrial R123 uptake; R123 uptake remained inhibited until 48-72 h in culture. Rabbit mitochondrial respiration experiments revealed a slightly lower ratio of ATP formed/oxygen consumed in panadiplon-treated animals: direct exposure of normal rabbit liver mitochondria to panadiplon did not have this effect. Hepatocytes isolated from panadiplon-treated rabbits showed reduced respiratory control ratios and lower oxygen consumption compared to controls. Our results indicate that panadiplon induces a mitochondrial dysfunction in the liver, and suggest that this dysfunction may be attributed to the carboxylic acid metabolite.

Phenotypic expression of disease in families that have mutations in the 5' region of the adenomatous polyposis coli gene.

BACKGROUND: Germline mutation in a gene on chromosome 5 (the adenomatous polyposis coli gene) causes familial adenomatous polyposis of the colorectum. Phenotypic manifestations of this condition vary, but the exact relation of the phenotype to the mutation site along the gene has not been fully described. OBJECTIVE: To determine how the location of mutations along a gene that is associated with multiple colorectal polyps (the adenomatous polyposis coli gene) is related to the phenotypic expression of the syndrome in families. DESIGN: Prospective cohort study. SETTING: Polyposis registry. PATIENTS: 20 patients from 7 families that had mutations in the adenomatous polyposis coli gene that were located toward the 5' end of codon 158 (proximal 5' families), were compared with 52 patients from 7 families that had mutations downstream from codon 158, in codons 179 to 625 (distal 5' families). MEASUREMENTS: Sex, age at diagnosis of familial adenomatous polyposis, number of polyps at first examination of the colon, distribution of polyps, age at diagnosis of colorectal cancer, and location of colorectal cancer. RESULTS: Mutations that were proximal to codon 158 were found in 7 of 112 families (6%). At the first examination of the colon, 8 of 17 (47%) patients in proximal 5' families and 9 of 48 (19%) patients of similar ages in distal 5' families were found to have fewer than 100 adenomas (P = 0.029). The distribution of polyps was frequently right-sided in patients in proximal 5' families (P = 0.001). The cumulative probability of survival without colorectal cancer was greater for patients in proximal 5' families (P = 0.041). CONCLUSIONS: Families with adenomatous polyposis that have proximal 5' mutations of the adenomatous polyposis coli gene are more likely to have a heterogeneous phenotype with delayed development of colonic polyposis and colorectal cancer than are families with distal 5' mutations of the gene. Management should include genotyping of patients who are at risk, colonoscopic surveillance of genotypically positive persons, and prophylactic colectomy if several adenomas are found.

The use and interpretation of commercial APC gene testing for familial adenomatous polyposis.

BACKGROUND: The use of commercially available tests for genes linked to familial cancer has aroused concern about the impact of these tests on patients. Familial adenomatous polyposis is an autosomal dominant disease caused by a germ-line mutation of the adenomatous polyposis coli (APC) gene that causes colorectal cancer if prophylactic colectomy is not performed. We evaluated the clinical use of commercial APC gene testing. METHODS: We assessed indications for APC gene testing, whether informed consent was obtained and genetic counseling was offered before testing, and the interpretation of the results through telephone interviews with physicians and genetic counselors in a nationwide sample of 177 patients from 125 families who underwent testing during 1995. RESULTS: Of the 177 patients tested, 83.0 percent had clinical features of familial adenomatous polyposis or were at risk for the disease-both valid indications for being tested. The appropriate strategy for presymptomatic testing was used in 79.4 percent (50 of 63 patients). Only 18.6 percent (33 of 177) received genetic counseling before the test, and only 16.9 percent (28 of 166) provided written informed consent. In 31.6 percent of the cases the physicians misinterpreted the test results. Among the patients with unconventional indications for testing, the rate of positive results was only 2.3 percent (1 of 44). CONCLUSIONS: Patients who underwent genetic tests for familial adenomatous polyposis often received inadequate counseling and would have been given incorrectly interpreted results. Physicians should be prepared to offer genetic counseling if they order genetic tests.

Evidence against genetic anticipation in familial colorectal cancer.

Anecdotal reports of hereditary colorectal cancer suggest that genetic anticipation (earlier appearance in successive generations) occurs, but ascertainment bias and cohort effects confound this interpretation. Using approaches that correct for such biases, we examined the age at diagnosis of colorectal cancer from family history questionnaires completed by subjects in the Johns Hopkins Hereditary Colorectal Cancer Registry; 475 parent-offspring pairs in 308 pedigrees were studied. We observed the expected cohort effect among offspring, in that the mean ages at diagnosis of those born before 1921, between 1921 and 1930, and after 1930 were 63 +/- 13 (SD), 57 +/- 10, and 42 +/- 10 years, respectively, while their parents' mean ages were 65 +/- 14, 66 +/- 14, and 58 +/- 15 years, respectively. In the cohort born before 1921, in which observation periods for both parents and offspring were comparable, there was no difference in age at diagnosis by pairwise comparison or life table analysis (P = 0.15 and 0.23, respectively; r = 0.32). Subgroup analysis of 67 pairs from 38 families that met the International Collaborative Group (ICG) criteria for hereditary nonpolyposis colorectal cancer (HNPCC) and of 14 pairs from 7 families with known germline mutations of DNA mismatch repair genes also showed no significant differences (mean age at diagnosis: 56 +/- 14 years for parents and 57 +/- 16 years for offspring from ICG families, and 45 +/- 10 years for parents and 44 +/- 12 years for offspring in families with known mutations). We also found no evidence for effect of parental gender on age at diagnosis in offspring of either gender, nor a secular trend toward younger onset colorectal cancer in this sample. In conclusion, there is no evidence for genetic anticipation or genomic imprinting of age at diagnosis in this sample of colorectal cancer families. Apparent anticipation appears to reflect a birth cohort bias of ascertainment.

Potentiation of hypoxic injury in cultured rabbit hepatocytes by the quinoxalinone anxiolytic, panadiplon.

The quinoxalinone anxiolytic, panadiplon, produces hepatic metabolic inhibition (mitochondrial impairment), microvesicular steatosis and centrilobular necrosis in rabbits. Metabolic inhibition occurs in cultured hepatocytes without cytotoxicity, suggesting that hepatic injury is influenced by additional factors. The present experiments were conducted to determine if metabolic inhibition by panadiplon predisposed hepatocytes to hypoxic injury. Injury (cell death) was evaluated by lactate dehydrogenase (LDH) release from cells; ATP and glycogen levels were also evaluated. Under hypoxic conditions, control cultures showed a 6.5-fold increase in LDH release compared to normoxic controls, with a coincident 80% decrease in ATP and 50% decrease in glycogen levels. Under normoxic conditions 10 microgram/ml panadiplon treatment for 48 h reduced ATP and glycogen levels by 40% but did not cause an increase in LDH leakage. Cells treated with panadiplon, then exposed to hypoxia conditions, showed a significant level of injury compared to normoxic control cultures, and a further reduction in ATP. No additional decrease in glycogen ws observed. In an attempt to prevent panadiplon-mediated injury, glycolytic substrates (dihydroxyacetone or pyruvate) were included during normoxic and hypoxic incubations. Both cotreatments reduced the level of LDH leakage produced by panadiplon during hypoxia. Cotreatment did not generally increase ATP or glycogen levels (compared to panadiplon treatment groups) during hypoxia, though individual experiments showed a slight increase in ATP levels. During normoxia both cotreatments with panadiplon resulted in significantly higher glycogen levels than in panadiplon cultures alone. These results suggest that cellular glycogen and subsequently ATP levels are reduced during panadiplon exposure, metabolically predisposing hepatocytes to hypoxic injury.

Cultured hepatocytes as investigational models for hepatic toxicity: practical applications in drug discovery and development.

Drugs can fail at any phase during discovery, preclinical or clinical development due to unacceptable levels of toxicity, and liver is commonly the principle target organ. Investigational toxicology methods, using appropriate models and hypotheses, can often resolve problems, identify toxic chemical substituents and salvage therapeutic discovery programs. While in vivo models are used to investigate hepatic drug effects in the context of toxicokinetics and systemic influences, cell culture models provide in vitro systems for investigating specific mechanisms in a precisely controlled environment. Using primary hepatocytes isolated from laboratory animals, we have explored several drug-induced hepatic disorders that surfaced during different phases of drug discovery and development. Additionally, the use of human hepatocytes has allowed us to address concerns for human exposure, examine human relevance of animal data, and provide perspective on problems encountered in clinical trials.

Induction of a hepatic toxic syndrome in the Dutch-belted rabbit by a quinoxalinone anxiolytic.

The non-benzodiazepine anxiolytic, panadiplon, was discontinued from clinical development due to evidence of hepatic toxicity in human volunteers that was not predicted by rat or monkey preclinical development studies. The present study was conducted to examine potential toxicity in the rabbit. Three groups of female rabbits were administered vehicle, 10 mg/kg per day or 20 mg/kg per day of panadiplon by oral gavage for 14 days. Animals in the 20 mg/kg group lost weight, and 6/10 developed a profound lethargy. Hepatic toxicity was observed in treated animals, evidenced by dose- and time-related increases in serum transaminase activities, gross hepatic lesions and multifocal centrilobular necrosis. Hepatic microvesicular steatosis was evident in treated animals; lipid analysis revealed a 123% increase in hepatic triglyceride. A time-dependent increase in serum triglyceride levels was observed in the high-dose group beginning on day 4. Hepatic glycogen was reduced, and histochemical examination revealed the reduction to be heterogeneous across the lobule with some areas showing a complete absence of glycogen. One rabbit in each drug-treated group showed mild hypoglycemia at day 12, and 4/10 rabbits in the high-dose group showed hyperglycemia at days 12-14. We conclude that panadiplon produced a microvesicular steatosis and hepatic toxicity in the rabbit. The observed toxicity resembled a Reye's syndrome-like toxicity produced by a variety of mitochondrial fatty acid oxidation inhibitors.

Nucleotides-induced cytosolic calcium transient and plasma membrane permeability in Chang human liver cells.

Extracellular nucleotides induce changes in cytosolic free Ca++ and also increase plasma membrane permeability to Ca++ ions in Chang human liver cells. Ca++ permeability induced by nucleotides is reversible and inactivated immediately upon removal of agonist. Stimulated cells transferred into the fresh medium and re-exposed to nucleotides demonstrate reopening of Ca++ channel without stimulation of Ca++ transient. Relative potencies of nucleotides to induce membrane permeability and Ca++ transient were: UTP > ATP > gamma-S-ATP > ADP (non-hydrolyzable ATP analogues and AMP had no effect). The permeability is not affected by specific inhibitors of voltage-operated calcium channels (verapamyl,cis-diltiazem and nifedipin). Nucleotides do not produce plasma membrane damages at concentrations up to 1 mM, as shown by exclusion of the propidium iodide. There are at least two types of nucleotides receptors in Chang cell membrane: P2y subtype receptors which is responsible for generation of the Ca++ transient, and P2x subtype receptors which lead to the opening of plasma membrane Ca++ channels upon activation.

In vitro assessment of trospectomycin and gentamicin sulphate in the LLC-PK(1) cell line.

Concurrent use of more than one antibiotic in clinical treatment of serious infections is common. Trospectomycin sulphate, a semi-synthetic aminocyclitol, may be used in combination with the aminoglycoside gentamicin sulphate. To investigate any potential interaction, trospectomycin and gentamicin toxicity were assessed in vitro using a two-factor response surface design study with each factor at four levels. Cultures of the LLC-PK(1) cell line (proximal renal epithelium) were treated with 0, 125, 250 or 500 mug/ml of the drugs, alone or in combination, and cytotoxicity was evaluated at 3, 7, 10 and 14 days of continuous exposure. Cytotoxicity was assessed by morphological and biochemical criteria (lactate dehydrogenase, LDH; alkaline phosphatase, ALP; gamma-glutamyl transpeptidase, GGT; aspartate transaminase, AST; alanine transaminase, ALT; acid phosphatase, ACP). ALP, GGT, LDH and AST activity increased in the control cultures over the experimental period. Cytoplasmic vacuolation, increased number of detached cells and reduced dome formation occurred at 250 and 500 mug gentamicin/ml and in combination with these levels of trospectomycin at study days 10 and 14. There was a statistically significant (P < 0.05) interaction (decrease) for ALP and GGT at study day 14. In summary, the LLC-PK(1) culture system provides a useful in vitro screen to evaluate potential xenobiotic interactions to assess nephrotoxicity.

Iodine in milk and meat of dairy cows fed different amounts of potassium iodide or ethylenediamine dihydroiodide.

Relationships between I intake by lactating Holstein cows and iodine concentrations in milk and meat were investigated. Six treatment groups with seven cows assigned to each treatment were fed a basal diet containing .8 mg I/kg alone or supplemented with I at 1, 2, or 4 mg/kg in four 5-wk periods. Basal alone was fed in the first and third periods and the I supplement was potassium iodide in the second period and ethylenediamine dihydroiodide in the fourth period. Iodine concentrations in milk increased with each increase in dietary I from 205 ng/ml for basal periods to 404, 477, and 757 ng/ml when 1, 2, or 4 mg/kg I was fed as potassium iodide; and 467, 535, and 869 ng/ml when 1, 2, or 4 mg/kg I was fed as ethylenediamine dihydroiodide. Concentrations of I in skeletal muscle after the fourth period were not affected by 2 mg/kg I and only increased from 166 to 199 ng/g when supplemental I was 4 mg/kg. Moderate changes in dietary I are quickly reflected in milk I, but I in meat is relatively stable.

Effect of magnesium administration route on plasma minerals in Holstein calves receiving either adequate or insufficient magnesium in their diets.

Rates of increase in plasma Mg following rectal or oral administration of solutions containing 30 g MgCl2.6H2O were compared in 10 Holstein bull calves receiving wheat straw (.07% Mg) and concentrates (.04 or .24% Mg) fed separately for ad libitum consumption. Treatments were administered in a sequence, which involved each calf with all combinations of MgCl2.6H2O dosing routes and dietary Mg within a 6-wk period. Plasma Mg concentration averaged 1.95 mg/dl initially but fell below 1 mg/dl within 2 wk after supplemental Mg was omitted. Maximum increases in plasma Mg concentration following oral or rectal dosing were 16 or 47% when dietary Mg was adequate and 48 or 124% when Mg was deficient. Calves fed either diet responded maximally to rectal infusion within 10 min, but plasma Mg of deficient calves increased throughout 160 min after oral dosing. Plasma Mg of deficient calves responded quicker and reached higher concentrations after rectal infusion, but the response was sustained longer after oral administration.

In vitro cytotoxicity of tetracyclines and aminoglycosides in LLC-PK(1), MDCK and Chang continuous cell lines.

The objective of this study was to determine if cell lines isolated from particular organs retain their sensitivity to xenobiotic toxicity in vitro. The toxicity of chlortetracycline, demeclocycline and tetracycline was examined in the proximal kidney (LLC-PK(1)), distal kidney (MDCK) and human liver (Chang) cell lines. The toxicity of amikacin, gentamicin and neomycin was studied in the LLC-PK(1) and MDCK lines. Cytotoxicity was assessed by cytoplasmic LDH leakage. Kidney cells treated with tetracyclines displayed minimal toxic response. The Chang cell line showed greater sensitivity to these compounds and ranked them as follows: demeclocycline > chlortetracycline > tetracycline. The kidney lines produced the rankings as follows: LLC-PK(1): amikacin > neomycin > gentamicin; MDCK: neomycin > amikacin > gentamicin. The results from the tetracyclines are consistent with the expectation that these compounds would not be nephrotoxic in vitro since in vivo investigations suggest hormonal mediation is required, and would be hepatotoxic in vitro because of direct action of these xenobiotics on liver cells in vivo. Similarly, the aminoglycosides were more toxic to the proximal kidney cells than to the distal cells as seen in vivo. These results suggest that continuous cell lines may provide important information in the assessment of xenobiotic cytotoxicity.

The effects of high and low energy density diets on satiety, energy intake, and eating time of obese and nonobese subjects.

The concept of time-energy displacement is based on the hypothesis that unrefined complex carbohydrates of low energy density will prolong eating time and induce satiety at a low energy intake. The present study compared the effects of diets low in energy density and high in energy density on satiety, energy intake, and eating time among 20 obese and nonobese subjects. Each diet was served over a 5-day period, and subjects were allowed to eat to satiety. With equal acceptance ratings of the diets, satiety was reached on the diet low in energy density at a mean daily energy intake one-half that of the diet high in energy density (1570 versus 3000 kcal). Eating time was significantly longer on the diet low in energy density by an average of 33%/day. Obese and nonobese subjects were comparable in their satiety ratings, energy consumption, eating time, and food acceptance. These data support the concept of time-energy displacement, which should therefore have applicability to the treatment and prevention of obesity.

Time-calorie displacement diet for weight control: a prospective evaluation of its adequacy for maintaining normal nutritional status.

An unsupplemented 1000 kcal (4186 kJ) diet emphasizing large quantities of unrefined complex carbohydrates was evaluated for nutritional adequacy in a 20-week weight-control program for obese adults. Assessment of nutritional status, routinely obtained upon admission, was repeated at approximately 10-week intervals on 26 patients who were losing weight while adhering to the dietary guidelines. At least one repeat assessment was obtained on all 26 patients: 13 had follow-up assessment at 10 weeks, three at 20 weeks, and ten at both 10 and 20 weeks. Weight loss averaged 0.7 kg/wk over an average of 15 weeks with a mean energy intake of 1020 kcal (4270 kJ), 55 percent of baseline. At each follow-up assessment mean skinfold thickness fell significantly whereas muscle mass was maintained according to arm muscle circumference and creatinine-height index. Mean blood levels of retinol, beta-carotene, folate, vitamin B12, ascorbic acid, thiamin, riboflavin, pyridoxine, iron, transferrin saturation and calcium excretion remained within normal limits throughout treatment. Ascorbic acid levels rose significantly. The results indicate that the experimental diet, without supplementation, can fulfil nutritional requirements while restricting energy intake for weight reduction.