Phenylketonuria and the peoples of Northern Ireland

The comparison of regional patterns of recessive disease mutations is a new source of information for studies of population genetics. The analysis of phenylketonuria (PKU) mutations in Northern Ireland shows that most major episodes of immigration have left a record in the modern genepool. The mutation I65T can be traced to the Palaeolithic people of western Europe who, in the Mesolithic period, first colonised Ireland. R408W (on haplotype 1) in contrast, the most common Irish PKU mutation, may have been prevalent in the Neolithic farmers who settled in Ireland after 4500 BC. No mutation was identified that could represent European Celtic populations, supporting the view that the adoption of Celtic culture and language in Ireland did not involve major migration from the continent. Several less common mutations can be traced to the Norwegian Atlantic coast and were probably introduced into Ireland by Vikings. This indicates that PKU has not been brought to Norway from the British Isles, as was previously argued. The rarity in Northern Ireland of IVS12nt1, the most common mutation in Denmark and England, indicates that the English colonialisation of Ireland did not alter the local genepool in a direction that could be described as Anglo-Saxon. Our results show that the culture and language of a population can be independent of its genetic heritage, and give some insight into the history of the peoples of Northern Ireland. Received: 23 October 1996 / Accepted: 26 February 1997     

Phenylketonuria variants in Ontario.

Since mass screening of the newborn population for phenylketonuria (PKU) by the Guthrie test was begun in Ontario in July 1965 many variants of PKU have been recognized in the 96 to 97% screened. Seventy-one cases of classic PKU were detected (four were missed). Of 48 cases of persistent hyperphenylalaninemia discovered, 18 were classified as atypical PKU and 30 as persistent benign hyperphenylalaninemia. Numerous infants with transient hyperphenylalaninemia (initial values over 10 mg/dl in 12), in many instances the result of transient neonatal tyrosinemia, were discovered. There was a slight predominance of males. Serum phenylalanine values of up to 15 mg/dl seemed to be harmless to the developing brain. A survey of 67 247 adults in the general population revealed 1 person with PKU and 1 with persistent benign hyperphenylalaninemia; both had normal intelligence quotients. Of 1548 mothers of retarded children tested, none had hyperphenylalaninemia.     

Mouse Models of Human Phenylketonuria

Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine. Although this inborn error of metabolism was among the first in humans to be understood biochemically and genetically, little is known of the mechanism(s) involved in the pathology of PKU. We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein. Two of these have reduced PAH mRNA and display characteristics of untreated human PKU patients. A low PHE diet partially reverses these abnormalities. Our success in using high frequency random germline point mutagenesis to obtain appropriate disease models illustrates how such mutagenesis can complement the emergent power of targeted mutagenesis in the mouse. The mutants now can be used as models in studying both maternal PKU and somatic gene therapy.     

Diet and retarded growth.

The diets of 36 children below the third centile for height but with no organic disease were compared with the diets of a control group. In most cases retarded growth was associated with a long-continued deficiency in calorie intake. When the diets were reassessed about a year later the shortfall in calorie intake was significantly reduced. This improvement, which tended to be followed by an increase in the rate of growth in height, might have been due to alteration in the child''s circumstances or improvement in the family attitudes and feeding habits or both. Advice given at the clinic is thought to have played a part in bringing these changes about.     

Phenylketonuria and its variants: observations on intellectual functioning.

The age at diagnosis, dietary treatment and intelligence quotient (IQ) of 119 patients with phenylketonuria (PKU) and its variants who were under long-term observation were studied. In 27 of the 79 patients with classic PKU the diagnosis had been made and treatment begun late (after 2 months of age). The mean IQ of these 27 patients was 57.6 when they were in their early 20s (although 2 had normal IQs). In contrast, among the 52 patients with classic PKU who were not treated late the mean IQ was 93.6 for the 27 who were still receiving dietary therapy. The mean IQs were 99.3 and 92.7 at ages 5 (when the diet was discontinued) and 15 years respectively for the 12 who had been treated "adequately". It was 76.0 for the 13 who were "over-treated" (malnourished) in the first 6 months of life. Among the patients with atypical PKU, who were treated early, the mean IQs were 110.0 for the 7 who were still receiving dietary therapy and 102.7 for the 12 who were not. The 21 patients with persistent benign hyperphenylalaninemia, who were not treated, had a mean IQ of 104.2. The most important factor in the ultimate IQ of patients with classic PKU is very early diagnosis (by 2 weeks of age) along with immediate initiation of dietary therapy.     

Fish oil-enriched diet is mucosal protective against acetic acid-induced colitis in rats.

Products of arachidonic acid metabolism are elevated in patients with inflammatory bowel disease and this elevation is correlated with disease activity. Eicosapentaenoic acid competes with arachidonic acid and alters eicosanoid biosynthesis. In this experiment, the possibility that eicosapentaenoic acid could be used in the treatment of inflammatory bowel disease was investigated by determining the effect of 6 weeks of a fish oil-supplemented diet, enriched in eicosapentaenoic acid, on colonic and ileal morphology, histology, and in vivo fluid absorption in rats with 4% acetic acid-induced colitis. The results of an eicosapentaenoic acid-enriched diet were compared with results of saturated and polyunsaturated fatty acid-enriched diets. In rats with misoprostol pretreated acetic acid-induced colitis, an eicosapentaenoic acid-enriched diet reversed net colonic fluid secretion to absorption and prevented macroscopic and histologic injury, compared with saturated and poly-unsaturated fatty acid-enriched diets, which did not. The fish oil mucosal protective effect occurred in the presence of a 30-fold enhancement of PGE2 synthesis. In rats with non-misoprostol pretreated acetic acid-induced colitis, an eicosapentaenoic acid-enriched diet returned ileal fluid absorption to control levels, as compared with saturated and polyunsaturated fatty acid-enriched diets, which did not. In conclusion, a fish oil (eicosapentaenoic acid)-enriched diet, but not a saturated- or a polyunsaturated-enriched diet, protected colonic and ileal net fluid absorption in an experimental model of inflammatory bowel disease.     

The pathology of the Mongolian Gerbil (Meriones unguiculatus): a review.

Both naturally occurring disease processes and experimental models of human disease in the Mongolian gerbil were reviewed. The gerbil was highly susceptible to cerebral infarction following unilateral ligation of one common carotid artery and was useful in studies of the pathogenesis of stroke. Spontaneous epileptiform seizures mimicked those of human idiopathic epilepsy, and both seizure-sensitive and resistant strains have been bred. Perhaps because of its more efficient nephron, the gerbil accumulated four to six times as much renal lead as the rat, and the gerbil has been proposed as an experimental model of lead nephropathy. On standard diets, about 10% of the animals became obese, and some showed decreased glucose tolerance, elevated serum immunoreactive insulin and diabetic changes in the pancreas and other organs. Some breeders exhibited hyperactivity of the adrenal cortex associated with hyperglycemia, hyperlipidemia and degenerative vascular disease. Although dietary supplements of cholesterol were toxic and did not induce atherosclerosis, the gerbil was useful in other studies of cholesterol absorption and metabolism. Spontaneous, insidious periodontal disease became evident after about 6 months on standard diets, and dental caries were induced by cariogenic diets or by pathodontic streptococci. Spontaneous neoplasia occurred in 8.4--24% of gerbils, usually after 2 years of life. Adrenal cortical, ovarian and cutaneous tumors were the most consistently reported neoplasms.     

Soporte nutricional basado en la evidencia en la enfermedad inflamatoria intestinal

Patients with inflammatory bowel disease are at risk for malnutrition. Consequently all patients with inflammatory bowel disease should undergo nutritional screening to identify those who require thorough nutritional evaluation. When nutritional support is indicated, enteral nutrition (oral or through a tube) should be used. There are no significant differences between elemental and non-elemental enteral diets in inducing remission of Crohn's disease (CD). Nevertheless, given that non-elemental diets are better tolerated, most authors prefer polymeric diets. Enteral nutrition should not be used as the primary treatment of choice in patients with CD, since they are less effective in inducing remission than steroid therapy. Although dietary fat (quantity and type of fat) might influence the course of the disease, recommendations cannot be made on the basis of the available studies. Equally, there is insufficient evidence that glutamine is effective in inducing remission in CD. The use of probiotics presents a high level of evidence in maintenance treatment and in the prevention of postoperative pouchitis, although the level of evidence is lower in ulcerative colitis and CD. Further studies are required to investigate several issues such as dose, treatment duration, the separate or combined use of several strains, as well as the concomitant use of prebiotics, symbiotics or antibiotics.     

Phenylketonuria in a patient with cystinuria.

During routine screening procedures for amino-acid disorders by thin-layer chromatography, a 16-year-old boy was found to have phenylketonuria and cystinuria. A phenylalanine and a cystine loading were carried out. The patient was found to be homozygous for phenylketonuria and heterozygous for cystinuria type II. His father was heterozygous for phenylketonuria and cystinuria, while his mother proved to be heterozygous only for phenylketonuria.     

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.     

3. Protein diets for obesity: metabolic and clinical aspects

The alleged benefits of protein diets remain unproven, since research data on the safety and long-term utility of protein products as the principal or only source of nutrients for weight reduction programs are as yet insufficient. First, it is uncertain whether the decreases in body protein turnover occurring with these diets are consistent with normal function over long periods, though net balance of protein is obtained. Second, the main advantage to the patient is the suppression of appetite by the ketoacidosis, but it is the ketoacidosis that causes many of the untoward effects. Third, the addition of carbohydrate to a protein diet does not mitigate the benefit of the protein and prevents most of the untoward effects. Fourth, there is clearly no advantage of “predigested” proteins (which are generally poorer in quality than normal high-protein foods) except for the psychologic factor of being given “medication” for the “disease” of obesity. Fifth, there is a distinct danger of deficiencies of micronutrients developing with prolonged consumption of unsupplemented diets. Sixth, the cardiac disorders associated with death in persons taking these diets have not been shown to be coincidental rather than a direct consequence of the diets.In the present state of understanding of protein diets, they should be supervised only by specially trained physicians in rigorous multidisciplinary programs, preferably those with ongoing research. Only individuals free from contraindications should be so treated. Until compelling data proving the safety and efficacy of these diets are forthcoming, the general public should be counselled against their use.     

Molecular Genetics of Phenylketonuria in Inhabitants of Novosibirsk Region

Phenylketonuria is a wide-spread autosomal-recessive hereditary disease due to a deficient activity of the enzyme phenylalanine hydroxylase (EC 1.14.16.1). A decrease of the enzyme activity results from mutations in structure of the phenylalanine hydroxylase gene, whose incidence has pronounced regional and ethnic peculiarities. We have carried out a search for mutations in structure of exons of the phenylalanine hydroxylase gene in the group of 34 phenylketonuric patients, inhabitants of the Novosibirsk region, and evaluated frequencies of the alleles in comparison with other populations. The performed study has shown that the spread of mutant alleles in Siberia seems to be affected by gene flows from Eastern Europe (mutations R408W and R252W) and, to a lesser degree, from Scandinavia (mutations IVS12ntl and Y414C), Western (mutations E280K, R158Q, and R261Q) and Southern Europe (P281L). Alleles have been revealed also characteristic of Southeast Asia (R243Q) and Turkey (R261Q).     

Dietary fat and immune function. I. Antibody responses, lymphocyte and accessory cell function in (NZB x NZW)F1 mice

The influence of dietary fat on autoimmunity in lupus-prone (NZB x NZW)F1 mice has been demonstrated. In defining further the effects of dietary lipid on the immune system of this strain, female weanling mice were placed on four diets differing in quantity and type of fat. Their immunologic response was then studied by a variety of tests at 4 and 7 mo of age. Few differences were seen among the four groups at 4 mo of age. At 7 mo of age, however, the mice receiving diets high in saturated and unsaturated fats had a reduced mitogenic response to T cell mitogens and an enhanced response to the B cell mitogen LPS. Immunoglobulin levels and delayed hypersensitivity responses did not show any consistent differences among the diet groups. At 7 mo, however, mice receiving diets high in unsaturated fat demonstrated hyperresponsiveness to injected sheep red blood cells as measured by the hemolytic plaque technique. In addition, peritoneal leukocytes from the same diet group exhibited an increased response to bromelain-treated autologous erythrocytes which was decreased after treatment with anti-Thy-1 antiserum and complement. Phagocytosis by peritoneal macrophages was significantly decreased in the animals fed high-fat diets, particular high saturated fat. Similarly, natural killer cell activity was markedly reduced in the mice with a high intake of saturated lipid, a finding which correlated with the in vitro production of interferon. These results indicate that diets high in fat influence immune responses and thus can affect the onset and severity of autoimmune disease. A low-fat diet can reduce the development of disease by maintaining normal immune responses. The data also suggest that unsaturated fat may influence T helper cell activity and therefore antibody production, whereas saturated fats may affect cellular immune responses which are dependent on membrane contact.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Dietary n-3 (omega-3) polyunsaturated fatty acid effects on animal tumorigenesis.

Environmental variables influence the incidence and expression of disease. Dietary fat is one environmental variable that has been associated experimentally and epidemiologically with alterations in certain types of tumorigenesis. Recently, detailed biochemical analyses have shown that not all fatty acid families possess the same tumor-promoting potential. In general, diets containing high levels of the n-6 polyunsaturated fatty acids have routinely enhanced tumorigenesis in lipid sensitive carcinogen-induced and tumor transplant tumor models, whereas diets with equivalent levels of n-3 polyunsaturated fatty acids have diminished tumorigenesis. At present, there is no definitive biochemical mechanism that fully explains these observations, but several possibilities have been proposed. One of the most attractive of these hypotheses is that each polyunsaturated fatty acid family has an individual effect on eicosanoid metabolism which determines its tumor-promoting potential. Regardless of current uncertainties about mechanisms of action, however, results of numerous animal models affirm the importance of qualitative, as well as quantitative, dietary lipid differences on tumorigenesis. This knowledge strengthens the probability that further advances in our understanding of lipid-tumor interrelationships will have important preventive and therapeutic medical benefits.     

An effect of zinc deficiency on dental caries.

Rat pups suckled by dams fed a zinc-deficient diet developed higher levels of dental caries following a caries-test challenge than pups suckled by dams fed a zinc-adequate diet. The zinc deficient treatment was administered during the mineralization stage of tooth development before the molars erupted. The data shows that suboptimal zinc nutriture was associated with an increase in dental caries.Fluoride, a trace element, is recognized as a factor in preventing dental caries and its use has had a significant impact on dental health in this country. However, the possible roles of other trace elements have not been closely examined. Zinc is a trace element important to bone mineralization as well as in general nutrition.The effects on dental caries produced by the addition of zinc to rat diets have been reported (1, 2, 3). In those investigations, however, zinc supplements were added to diets that were adequate in zinc. Moreover, zinc supplements were fed after the teeth had appeared in the oral cavity. Because the critical period of tooth maturation occurs before eruption (4), post-eruptive zinc supplements probably should not markedly influence the development and, specifically, the mineralization of the tooth. Therefore, we investigated the influence of pre-eruptive zinc deficiency on caries development in rats.     

Nutritional investigations and management of captive moose

Historically, moose have been difficult to maintain in captivity when on diets of grass or legume hays and grain due to enteritis that frequently leads to chronic diarrhea/wasting disease. The development of wood-fiber diets has increased the lifespan of moose in captivity, but these diets do not completely prevent chronic wasting. Purina Mills (St. Louis, MO) hypothesized that captive moose are unable to digest starch that escapes the rumen, and therefore abnormal bacterial fermentation in the hindgut causes chronic diarrhea. An earlier study found no evidence of a digestive problem, so we tested the hypothesis that moose have difficulty metabolizing excess propionate produced from the fermentation of starch found in traditional cervid rations and high-grain wood-fiber diets. When challenged with an i.v. propionate load, moose metabolized propionate similar to healthy mule deer and domestic livestock. We then tested the hypothesis that grass forage is an initiating factor to chronic diarrhea/wasting and further hypothesized that grass, alfalfa, and other agriculture-based forages in association with an anaerobic bacteria produce inflammatory bowel disease (IBD) in moose. Captive moose that had ad libitum access to a wood-fiber pelleted moose diet and grazed in grass pastures developed chronic wasting symptoms at 2–4 years of age and died at 4.7 ± 0.3 years unless restricted from grass before the development of advanced symptoms. We isolated Bacteroides vulgatus in the feces and successfully treated a moose with chronic diarrhea/wasting disease with long-term metronidazole therapy, suggesting that the chronic enteritis causing wasting disease arises from a bacteria-associated defective immunosuppressive response similar to IBD in other species. Further support for the IBD cause of wasting in moose is that this animal will relapse within hours if the metronidazole treatment is discontinued even after many months. We developed a highly palatable high-fiber, low-starch moose ration that can be fed as the sole source of nourishment, although additional research and dietary improvements are required. Zoo Biol 16:479–494, 1997. © 1997 Wiley-Liss, Inc.     

Nutritional considerations in the pathogenesis of hepatic veno-occlusive disease in captive cheetahs

Veno-occlusive disease (VOD) of the liver has been diagnosed in a large number of captive cheetahs. Some ingredients or contaminants present in the diet were suspected as possible causes for this noninfectious disease with high incidence. Eight different diets fed to cheetahs kept in North American zoos were analyzed for vitamin A levels and the presence or absence of plant estrogens, nitro-saminines, nitrites, and aflatoxins. Three of the eight diets were considered to contain toxic amounts of vitamin A. In humans and rats, hypervitaminosis A has been associated with hepatic vascular lesions, mainly perisinusoidal fibrosis, which progress eventually to occlusive lesions similar to VOD. Plant estrogens were detected in appreciable amounts only in one of the exotic carnivore diets. The role of plant estrogens in the pathogenesis of VOD in captive cheetahs is not clear at this time and needs further investigation. Based on the liver pathology and diet analyses, nitrosamines or their dietary precursors and aflatoxins can be excluded as possible causes of VOD in cheetahs kept in North American zoos.     

Domestic animals in the elucidation of zinc's role in nutrition

Parakeratosis in swine, a disease that caused severe economic losses in many commercial herds during the late 1940's and early 1950's, was caused by an inadequate intake of zinc. In addition to poor growth, the disease involves primarily the epidermal layer of the skin, which in severe cases becomes thickened and heavily encrusted. Finally, deep fissures develop in the encrusted areas. The low availability of zinc in plant protein sources and the increasing use of soybean meal in practical-type diets during the late 1940's contributed to the increasing incidence of the disease. Excessive levels of calcium also increased the incidence and severity of the disease. Prominent features of zinc deficiency in poultry were failure of normal development of long bones and the occurrence of severe lesions on the feet and legs. As with the pig, the low bioavailability of zinc from plant protein sources led to the observation that practical-type diets were limiting in this metal. Although parakeratosis can be produced in cattle fed diets low in zinc, it does not seem to have been a major nutritional problem in ruminants.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.