CLINICAL CMMISTRY
Diseases of Metabolism (Disorders of Amino Acid Metabolism) Steven C. Kazmierczak Department of Pathology and Laboratory Medicine, East Carolina University School of Medicine, Greenuille. North Carolina 27858-4354 Amino acids play a variety of roles in metabolism, as components of proteins, and some even serve as neurotransmitters. Much of the interest in amino acid metabolism is centered around the intermediary steps involved in catabolism. the regulation of biosynthesisand transport, and genetic disorders resulting in abnormal urinary and or plasma concentrations of amino arids and their derivatives. The concentrations of amino acids in plasma in normal subjects have heen well-defined and show relatively little inter- or intraindividualvariation. Maintenanceof these steady-state concentrations is dependent upon the delicate balance between production, utilization. and elimination. This review covers new information which has appeared in the literature between January 1987 and October 1992, on the diagnosis and management of disorders of amino acid metabolism.
SPECIFIC AMINO ACID DISORDERS Homocysteine Homocysteine, a thiol-containing amino acid, isan intermediateofthetranasulfurationpathwaywhirh converta the sulfur-containing amino acid methionine to cysteine. The clinical condition of homocystinuria has stimulated interest in the role of homocysteine in rellular functions. Several abnormalities in enzymes controlling the metabolism of homocysteine have been descrihed. Impairment of cystathionine 8-synthase. which regulates a step in the transsulfuration of homocysteine to cysteine, and deficiency of 5,lO-methylenetetrahydrofolatereductase. which provides substrate for the remethylation of homocysteine to methionine, are the most common abnormalities seen. Recently, substantial improvementq in techniques used for determination of homocysteine in biologir material has enabled quantitation of homocysteine from normal individuals. from atients withdiseases other than homocystinuria. in variousieficiency states, and following exposure to various drugs tG11. One possible consequence of inrreased plasma homocysteine whirh has attracted murh attention is the increased risk of premature vascular disease and thrombosis ( C I - G ~ I .Even patients with mild homocysteinemia may be at risk for premature vascular disease. These patients may be heterozygous for homocystinuria; theestimated prevalence of the heterozygous state heing 1 - 2 7 of the population ( C ~ J . The exact mechanism by which homocysteine promotes atherogenesis and thrombosis is uncertain. I n vitro studies haveshown that high conrentrationsof homorysteinedamage cultured endothelial cells. The toxiceffects of homocysteine on these cells apparently results from the fnrmation of hydrogen peroxide (L'6,. Workers have shown that the oxidation of homocysteine to homocystine is oxygen.dependent and is catalyzed by micromolar amounts of copper or ceruloplasmin. Catalase, a scavenger of hydrogen peroxide, prevents the effert of homocysteinemediated injury. Homocysteinemia has not been found to show any significant correlation with other factonassociated with atherosclerosis, including serum cuncentrations of total cholesterol. LDL and HDL cholesterols. triglycerides, hypertension. cigaretu smoking. and diabetes ( G 2 . G7-G9). Some rerent reports suggest that homocysteine may exist in plasma in forms not detected by ordinary screening proceduresand may accumulate in large amounts in patients with coronary disease. Very high roncentrations of homocysteine thiolactone have heen found in patients with atherir sclerosis when compared to controls (GIUJ. In addition M homocysteine, cyst hionine and n-aminoadipic acid have also been found 10 be marked1 increased in those patients with coronary artery disease , $ I l l . The present state of knowledge suggesta a role for mild homocysteinemia in the development of atherosclerotic lesions. Theexact mechanism bywhich homcqsteineinduces the lesion, however. requires further study. Other factors which may help modulate plasma homocysteine concentrations.surh asfolate and vitamin B12concentrations,alsoneed
Steven C. Kamkrcrsk is an Aaslstam prolessor in ths C e p a m n t of Pathobgy
7-
and Laboratow Medicme at East Carolina
UniverritvSdmlolMedkmeandScannRc , .-..~. Director 01 me Clinical Chemistry Laba ratories at Pin County Memwbl Hosp& and the E.C.U. School 01 Medicine. H, ~~
~~
~
~~~
~
received hls B.S. degree in biology hon Youngstown State University In 1982 an, his Ph.D. degree in clinical chemistry hon The Ohlo State University in 1986. Fror 1986 to 1988 he was employed as i Postdoctoral Fellow in blochemistry at T h ClevelandClinicFoundation. In 1988. DI Kazmierczak joined the faculty at Eas. Carolina University. His research interests Ile in ths areas of enzymology. method development and automatlon. and artlRcial Intelligence.
to he considered. In addition, precautions must be taken when measuring lasma homocysteineconcentrations in order to avoid redistrifution between the free and protein-bound homocysteine fractions. Individuals who are obligatory heterozygotes for cystathionine @-synthase deficiency have a 50% or greater reduction in enzyme activity. Challengingthese patientswith a loading dose of methionine yields increased plasma concentrations of sulfur amino acids, which are useful for the identification of such individuals. Unfortunately, considerable overlap between heterozygous and control subjects is found. At present, measurement of protein-hound homocysteine may be a more convenient and useful for the identification of individuals with heterozygous homocystinuria (G12, G23).
TYROSINEMIA (HEREDITARY TYPE I) Hereditary tyrosinemia type I is a metabolic disorder of autosomal recessive inheritance. The incidence of the disease can vary according to region. In Sweden the reported incidence is about 1 per 100 OOO births (G24), while in a restricted area of Canada, the incidence is approximately 1 per 1800 births (GI5). The clinical course of the disorder can vary significantly. Asa result,acuteand chronic forms ofthe disorder have heen identified. The acute form of the disease is the most frequently reported. Symptoms usually appear within the first months of life with vomiting, diarrhea, failure to thrive, and lethargy (G16). Liver disease with hypoproteinemia, hyperbilirubinemia, and defective coagulation capacity are consistent findings. In the acute form, death from hepatic failure usually occurs during the first year of life. The acute form of tyrosinemia may be difficult to distinguish from neonatal hepatitis, sepsis, and some metabolic disorders such as galactosemia and hereditary fructose intolerance (G26). Specific biochemical parameters are employed for establishingthecorrectdiagnosisoftyrosinemia type I. Succinylacetoneis excreted in largequantitiesin these patients. Normalindividuals bavealso beenshown toexcrete this metabolite, although in very small amounts. Other biochemical abnormalities suggestive of the disease include increased plasma concentrations of tyrosine and methionine along with decreased cholesterol and increased urinary 6-aminoleuclinic acid and cathecolamines (GZ7). Recently, an infant with typical clinical and biochemical findings consistent with hereditary tyrosinemia type I was discovered, except that no excess of succinylacetone was present (G18). This individual was found to have complete deficiency of maleylacetoacetate isomerase activity in liver. The lesssevere chronic formoftyrosinemiausuallypresents as hepatomegaly and/or rickets early in childhood. In these patients, death from primary liver cancer in the cirrhotic liver usually occurs in the first two decades of life. The many dysplastic cells found in the liver of these patients may be ANALYTICAL CHEMISTRY, VOL. 65, NO. 12. JUNE 15, 1993
401R
CLINICAL CHEMISTRY
related to the high tumor risk (G19-G21). Serum a-fetaprotein is often raised in these atients. Attacks of severe abdominal pain, resembling tfose of acute intermittent porphyria, may occur and cause death from respiratory paralysis (G22). Until 1977, the enzyme defect causing this disorder was thought to be 4-hydroxyphenylpyruvatedioxygenase. At this time, however,it was found that patients secretelarge amounts of succinylacetoacetateand succinylacetone,which identified the primary defect as a deficiency of fumarylacetoacetase (G23). Treatment of tyrosinemia involves maintenance of a diet restricted in henylalanine and tyrosine. Although this may prevent or a l h i a t e the kidne damage which usually occurs, it does not prevent the fadoutcome (G19). Liver translantation is currently the most effective therapy. Recently, owever, therapy aimed at the prevention of the formation of hepatotoxic and nephrotoxic compounds and succinylacetone has been attempted (G19). This new approach involves the use of 2-[2-nitro-4-(trifluoromethyl)benzoyl]-l,3-cyclohexandione (NTBC) to inhibit the enzyme 4-hydroxyphenylpyruvate dioxygenase. Inhibition of this enzyme, which is at a point preceding the fumarylacetoacetate hydratase deficiency, prevents the formation and subse uent accumulation of compounds thought to be responsib e for the liver and kidney damage (G24). Also, inhibition of the formation of succinylacetone leads to an increase in the activity porphobilinogen synthase. In the limited number of patients who have been treated with NTBC therapy, results have been encour Improvements in liver function and declines in a etoprotein concentrations have been noted. Decreases in a-fetoprotein may be regarded as prognostically favorable. The decline in a-fetoprotein may indicate that the normal process of cell differentiation and maturation has resumed, reducing the risk of cancer. Failure to achieve a normal decline in a-fetroprotein may indicate a prognostically unfavorable course. Failure to achieve a normal decline in a-fetoprotein concentrations followingthe startof therap with NTBC may indicate that malignant disease is alread; resent at the initiation of therapy (G19). Treatment with N h C may prove to be an alternative to liver transplantation for this otherwise fatal disease. Further studies are needed.
E
P
ying
DISORDERS OF ORGANIC ACID METABOLISM These disorders are characterized by the presence of shortchain acids resulting from the incom lete oxidation of carbohydrates, amino acids, and fatty aci s. Some disorders, such as maple syrup urine disease, can be classified as either an amino acidopathy or organic acidopathy. Maple syrup urine disease, in this review, will be classified as a disorder of organic acid metabolism. Maple Syrup Urine Disease Maple syrup urine disease (MSUD)is an inherited metabolic disorder of branched-chain amino acids. Several phenotypic variants of MSUD have been described classical, intermittent and intermediate. In the classical type of the disease, failure to thrive, acidosis, and neurological sym toms are the usual findings. Left untreated, the patient Seteriorates rapidly. MSUD is caused by a deficiencyof branched-chain a-ketoacid dehydrogenase; the result of which is very lar e increases in plasma and urine concentrations of the brancted-chain amino acids leucine, isoleucine, and valine and accumulation of branched-chain keto acids and 2-hydroxy acids. All three of the above groups of compounds are instrumental in establishing the correct di osis (G25). Additional metaboliteswhich may be d e t e x n increasedamounts from the urine of patients with MSUD include N-acetyl derivatives of valine, leucine, isoleucine,and alloisoleucineand have been recently described (G25). Another report of increased excretion of 3-hydroxyisovaleratein a patient with MSUD is surprisin since 3-hydroxyisovalerate is a metabolite from leucine t%at is located distally to the enz e block (G26). metabolite in The formation of increased amounts of patients with MSUD might possiblybe explained by the action of cytoplasmic a-ketoisocaproate oxy enase, which is present in human liver and has been suggestedito function as a “safety valve”to prevent excessiveaccumulation of a-ketoisocaproate (G27). In all probability, additional compounds will be
B
tE
402R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
detected in the serum and urine of these atients as the biochemical pathways of patients with M8UD is further elucidated. Treatment strategies to reduce the acute increases in amino acid concentrations during crisis episodes typically involve administration of glucose with or without insulin in order to increase the deposition of amino acids in muscle as protein (G28). These treatment strategies have been based on the assumption that the only important route of removal of branched-chainamino acids in patients with MSUD is through protein synthesis. Recent findings, however, indicate that significant clearance of these amino acids, particularity leucine, ma take place through routes other than protein synthesis (829). Valine, leucine, and isoleucine stimulate rotein synthesis and inhibit protein catabolism in vitro. Tiese findings can be reproduced by leucine given alone, but not isoleucine or valine, sug eating a uni ue anabolic regulatory role for branched-ciain amino aci%s,particularly leucine. However, the actual physiological importance of these findings is, at present, uncertain (G30, G31). Phenylketonuria Phenylketonuria (PKU) is an autosomal recessive disorder and one of the most common inborn errors of metabolism in man, with a frequency of approximately 1 per 10 OOO live births in the United States. The frequency in Europe, however, has been found to vary considerably (G32). The condition is characterized by a lack of phenylalanine hydroxylase, which is normally present in significant quantities only in the liver. Affected individuals excrete large quantities of phenylpyruvate in the urine. The disease,if left untreated, leads to hyperphenylalaninemia and mental retardation. Dietary treatment consisting of phenylalanine restriction leads to marked improvementsin behavior and motor development if implemented early in life. Hyperphenylalaninemia is clinically and biochemically a hetero enous condition. The most severe form, in which little or no pfenylalanine hydroxylase activity is present, is referred to as classical PKU. The benign form of the disease, in which hydroxylase activity is present in significant amounts, is referred to as benign hyperphenylalaninemia. Collaborative studies for the treatment of children with PKU has established the following biochemical parameters, each of which must be met, as diagnostic: (1)two measurements of blood phenylalanine greater than 20 mg/dL, made 24 h apart while the patient is on a normal diet; (2) blood tyrosine concentrations of less than 5 mg/dL; and (3) the presence of metabolic excretion products of henylalanine in the urine (G33). Efforts to improve early bi)agnosis of PKU have led to the use of other techniques. Measurement of phenylalanine hydroxylase activity in liver tissue has enabled the discrimination of some phenotypes (G34). However, the invasiveness of the procedure precludes the technique from routine use. Several in vivo methods which attempt to assess the ability of the individual to remove phenylalanine from plasma or to hydroxylate it to tyrosine also have been attem ted (G35). Unfortunately, these tests fail to discriminate getween normal and heterozygous individuals in many instances. Much better discrimination can be obtained using deuterated phenylalanine loading techniques (G35, G36). A number of physiological variables, a art from phenylalanine hydroxylase activity itself, can inluence the results of loading tests with labeled and unlabeled substrates. Changes in protein metabolism, urinary excretion, transamination rates, phenylalanine distribution, and transport and hormonal influences can effect test results (G35). Further studies on the effects of these fadors are needed to evaluate their effects. Most recently, advancements in molecular genetic techniques for gene analysis may provide a sensitive and convenient means of detection of PKU. The polymerase chain reaction for DNA amplification has been automated to screen up to 100 samples for PKU at a time (G32, G37). Further refinements in these techni ues and additional characterization of the phenylalanine%ydroxylase gene should make carrier detection of PKU technologically feasible. The neuropathologic effects that characterize PKU are attributed to the accumulation of toxic metabolites of phenylalanine. However, evidence also exists that excess phenylalanine may interfere with the uptake into the brain of large neutral amino acids. Thus, phenylalanine, by itself,
CLINICAL CHEMISTRY
may be the “toxic”compound causingneuropathologiceffects (GW. A large number of other metabolites suspected of being the toxic compound have also been identified and characterized. Concentrations of 3-hydroxyphenylacetic acid and 4-hydroxyphenylacetic acid were found to be increased in the urine of PKU atienta (G39). However, evidencesuggests that neither meta%olite is derived from tissue metabolism of phenylalanine, but instead is derived from the action of intestinal bacteria. Thus, given their probable intestinal origin, it is unlikely that blood and tissue concentrations of these metabolites would correlate with blood concentrations of phenylalanine (G38). Increased excretion of 4-hydroxyphenyllactic acid has previously been reported in carriers of PKU following administration of phenylalanine derivatives (G39). Only recently, however, has reliable methods for measurement of 4-hydroxyphen llactic acid and its precursor, 4-hydroxyphenylpyruvic a c i i been established (G40). Measurement of these compounds using specific methodologies suggests that these metabolites do not play a role in the pathology of PKU. Phenylpyruvic acid and phenylacetic acid are unique in that both demonstrate toxicity to cells in culture. Phenylpyruvic acid has been shown to inhibit a number of enzymes in vitro (G41). While initial findings suggest a role for those com ounds in development of the lesion in PKU (G39), furtfer studies appear to discount these claims (G42). In light of the recent data showing lack of support for phenylalanine metabolites as mediators of the neuropathologic changes seen in patients with PKU, the suggestion that phenylalanine is the mediator of the disease, as previously discussed, warrants further study (G38). Monitoring of phenylalanine metabolites in PKU and making dietary adjustments on the basis of urinary concentrations of these markers has also been advocated by some (G39). Until the controversy as to what the toxic compound(s) in PKU is settled, treatment modalities based on each of the above hypotheses for phenylalanine toxicity will probably be practiced. Treatment for PKU has traditionally been based upon the dietary restriction of phenylalanine, while maintaining adequate protein and caloric intake. Properly treated PKU children exhibit normal growth and development. A number of alternative methods for treatment of PKU have been proposed (G34). One technique involves the use of multitubular enzyme reactions with the enzyme immobilized into asymmetric hollow fibers (G43). The use of L-phenylalanine ammonia lyase has been proposed due to the fact that no coenzyme is required. In this system, which is similar in construction to the hollow-fiber cartridges used in hemodialysis, the enzyme is immobilizedat the outer wall of a hollow fiber and is separated by a porous membrane from the blood that circulates through the lumen of the fibers. The extracorporeal use of enzyme reactors for enzyme replacement therapy may prove to be an effective treatment modality for PKU. This technology may also be applicable to a wide spectrum of other diseases characterized by enzyme deficiency.
UREA CYCLE DISORDERS Urea cycle disorders have varied clinical expressions, but hyperammonemia is characteristic of all inherited urea cycle disorders presenting in the neonatal period (G44). The urea cycle serves two primary functions: (1)the incorporation of excess nitrogen not needed for biosynthetic purposes into urea and (2) it contains most of the biochemical pathways required for the biosynthesis and degradation of arginine. One additional function that the urea cycle may serve is in the regulation of acid-base balance via the dis osal of bicarbonate. Arguments both for and against this afditional feature have been made. Arguments against this function include the finding that patients with complete or essentially complete defects in one of the urea cycle enzymes have little evidence of disorders of pH homeostasis other than the respiratory alkalosis related to the stimulatory affect of ammonium on respiration (G45, G46). The hyperammonemia which is seen in atients with urea cycle disordersvaries in severit . Enzyme Aficiencies leading to hyperammonemia include Lficiencies of carbamoyl phosphate synthetase, ornithine transcarbamylase, arginosucci-
nate synthethase, arginosuccinate lyase, and arginase. The resultin hyperammonemia varies in severity according to where t%e enzyme deficiency occurs within the cycle. Deficiency of ornithine transcarbamylase (OTC) is the most common hereditary urea cycle enzyme defect. The clinical diagnosis of OTC deficiency in a hyperammonemic patient is suggested by decreased or absent plasma citrulline with orotic aciduria (G47-G49). OTC deficiency is an x-linked disorder. Clinical manifestations thus are most severe in hemizygous males who may present with hyperammonemic coma leading to death in the neonatal period. Milder forms of OTC deficiency may present during infancy, childhood, or adulthood and presumably are the consequence of heterogeneity in the mutations a t the OTC gene locus (G50). The frequency of new mutations in OTC deficiency appears to be hi h, accounting for as much as one-third of new cases (G51). 8hese atients have various degreesof mental retardation and epi&c hyperammonemia. Females who are heterozygous for the mutant allele are usually phenotypically normal; however, they may have clinical manifestations similar to those in males with milder forms of OTC deficienc (G52). It is sometimes possible to diagnose symptomatic d T C deficiency by measuring plasma ammonium, citrulline, and glutamine and urinary orotic acid excretion (G53). However, the methods most commonly used for the identification of heterozygotes utilize the metabolic consequence of OTC deficiency (G51, G54-G56). The oral administration of a nitrogen bolus stimulates the intramitochondrial accumulation of carbamoyl phosphate in the abnormal hepatocytes. The excess carbamoyl hosphate diffuses into the cytosol, where it functions as a sugstrate for the synthesis of pyramidine, resulting in the accumulation and excretion of orotic acid. Unfortunate1 ,nitrogen loading has several d i s a d v a n t y s The protein golus can produce lethargy and vomiting ue to the resulting hyperammonemia (G54). More recently, investigators have tried all0 urinol as a substitute for the nitrogen load. Metabolism of Jopurinol to oxypurinol ribonucleotide inhibits the enzyme orotidine monophosphate decarboxylase which in turn leads to the accumulation of orotidine monophosphate and its precursor orotic acid. This accumulation eventually results in orotic aciduria and orotidinuria. In cells with mutations a t the OTC locus, inhibition in the pathway by allopurinol was found to result in increased urinary excretion of orotidine (G53). This suggests that measurement of urinary excretion of orotidine in women after the administration of allopurinol may be a simple, inexpensive, and noninvasive method of assignin carrier status to women suspected of carrying a mutant O T 8 allele. The definitive diaposis, however, can only be established by enzymatic analysis (G57). Unfortunately, s ectrophotometric methods, which measure citrulline profuction, have several disadvantages. These methods lack sensitivity to accurately measure low enzyme activities. Also, the low sensitivity of these assays is limiting with respect to the amount of tissue available for analysis. This in turn precludes use of these methods for measurement of enzyme activities in tissues other than liver which have low enzyme activities compared to that found in liver. Measurement of enzyme activities in tissues other than liver would allow prenatal diagnosisof urea c cle enzyme disorders and would also allow for tissue, other tgan liver, to be analyzed. Recently, highly sensitive and specific radiochromatographic assays for diagnosis of enzyme deficiencies have been reported (G57). This method can reliably measure OTC activity in as little as 0.05 mg of tissue. The hi h reproducibility of this method at low enzyme activities &ows for the prediction of prognosis in patients with OTC deficiency. In addition to hyperammonemia, another manifestation of urea cycle disorders is the presence of hyperglutaminemia (G47,G58). Ammonia, by itself, is believed to be an im ortant cause of the cerebral dysfunction of hepatic encephdpathy and other diseases in which hyperammonemia occurs (G59). Pathophysiological changes due to the hyperammonemia include depressed brain function, increased transport of neutral amino acids across the blood-brain barrier, increased brain content of aromatic amino acids, and increased rate of 5-hydroxytryptaminemetabolism (G60). Identical chan es can be observed when plasma ammonia is increasedeby artificialmeans (e.g., by urease injections) in otherwiee healthy ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
40m
CLINICAL CHEMISTRY
animals (G61, G62). One important effect of hyperammonemia is a decrease in the rate of metabolism of glucose by brain cells by as much as 30% (G63). The decreased rate of ener consumption, reflecting a decrease in the activi of brain ce%, is maintained indefinitely thereafter (G59, 62, G63). One interesting finding, however, is that the decreased cerebral rate of glucose metabolism caused by hyperammonemia correlates more closely with plasma glutamine,a metabolite of ammonia, than with ammonia itself (G61). This finding suggests that incorporation of ammonia into glutamine is responsible for the toxic effects of hyperammonemia. Recent reports sug est that glutamine may be more closely related to the pa&+ physiology of the ence halo athy of hyperammonemia than is ammonium (G60, $64, &5). The idea that ammonia must be metabolized to become toxic runs contrary to current thinking that ammonia is detoxified by metabolism to glutamine. It has long been known that increased ammonia can cause neurological disorders. It has been assumed that the synthesis of glutamine is a rotective mechanism and that once formed, lutamine is reyeased by the brain for metabolism or disposa by other or ana (G66). While this concept is valid for some tissues, intrain, glutamine is not simply an end product of ammonia metabolism. Glutamine is also used as a precursor of neurotransmitters. Followin the release of these neurotransmitters from neurons, ttey are taken up by astrocytes and reconverted into glutamine (G67). Glutamine synthesis plays an important role in the metabolic cycle between astrocytes and neurons. It is unlikely that the primary role of glutamine synthesis in brain is to protect against hyperammonemia. A more reasonable hypothesis su gests that high rates of glutamine synthesis, as a result o f increased ammonia concentrations, disturb the normal metabolic balance between astrocytes and neurons (G61, G65). The exact mechanism by which increased glutamine synthesis in response to hyperammonemia leads to cerebral dysfunction is at resent unknown. Interference withcerebral production of A!'P has been suggested to explain cerebral dysfunction in chronic hyperammonemia(G68);however, data supporting this are not very strong (G62). It has also been suggested that ammonia may impede cerebral function by inhibiting glutaminase, resultin in decreased supply of lutamate for neuronal function (869). Recent experiments, [owever, do not support this h othesis either (G61). The most likely mechanism which hyperammonemia mediatescerebral dysfunction involves ita effect on the carrier s stem that transports neutral amino acids across the blood -train barrier and the increase in the brain content of aromatic amino acids (G62). The concentration of lutamine in the brain is closely correlated with the ermeabhty of the bloodbrain barrier to neutral amino aci& and to the accumulation of aromatic amino acids. Thus, glutamine synthesis may be linked to the stimulation of neutral amino acid trans ort caused by h erammonemiaas well as the decrease in cere7Jral energy m e a o l i s m (G61, G62). Whatever the exact mechanism, the relationship between changes in the permeability of the blood-brain barrier and cerebral dysfunction needs further clarification.
2
6
LITERATURE CITED (01) Veiand, P. M.; Refsum, H. J. lab. CHn. Med. 1969, 114, 473-501. (G2) Manilow, M. R. Ckcul~tkn1990, 81, 2004-2006. (03) Clarke, R.; Dab, L.; Roblnson, K.; et ai. N. .€ng/. J. Med. 1991, 324, 1149-1155. (Q4) Kang, S. S.; Wong. P. W. K.; Manllow, M. R. Annu. Rev. Nub. 1992, 12, 279-298. (G5) Manlbw, M. R.; Kang, S. S.; Taybr, L. M.; et al. Ckc&bbn 1969, 70, 1180- 1188. (08)Whorton. A. R.: Montaomw, . M. E.; Kent, R. S. J. Cffn. Invest. 196S. 76, . 295-302. (G7) Israelsson, B.; Brattstr&n, L. E.;Hultberg, B. L. AlhwaPckKosls 1986, 71, 227-233. (a) Berg, K.; Manilow, M. R.; Klerult,; Upson, B. CUn. &net. 1992, 41, 315-
-- .. !29i
(G9) Stampfer, M. J.; Manllow, M. R.: Wllbtt, W. C.; et ai. JAMA, J. Am. M. ASS=. 1992, 268, 877-881. (GlO)McCuHy, K. S.; Vezerfdls, M. P. Res. Commun. Chem. PeM. phemcd. 1986, 50, 107-120. (Q11) Olszewdtl. A. J.; Szostak. W. B. AthsrasckKosis 1987, 80, 109-115. (G12) Wlby, V. C.; Dudman, N. P. B.; Wllcken, D. E. L. MeteboKsm 1986, 37, 191-195. (G13) Sartwk, R.; Cenouo, R.; Corbo, L,; Andria, 0 . J. InherltedMeteb. Dkr. 1966, 0, 25-29.
404R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 199
(G14) Landrigan, P. J. Am. J. AnbMc HISSMI 1992, 82, 941-943. (GIs) Epstsln, S. S.; Blngham,B.; Ran, D,; B r a , I . D. Int. J. HISSllh Sew. 1992, 22,455-489. (G16) KVmengen, E. A. Sc8nd. J. Clh. Lab. Inwst. 1986, 46, 2 7 3 4 . (G17) W e , C. F.; Zuoweete, E. L.; h4eett, E. A.; Fh~elll,V. N.; Petering, H. G.; H. K. J. Pscuatr. 1977, 00, 400-406. (GIB) WsF,R.; Mlchals, K.; Galbraeth,J.; Matabn, R. Ped&&. Re$. 1986,23, 320-3332. (GIS)UndatSdt, S.; Holme,E.; Lock, E. A.; Hjalmarwn,0.; S t r a M . B. Lancet 1992, 340, 813-817. ((320) Manowski, Z.; Slhrer, M. M.; Roberts, E. A.; Superlna, R. A,; PMHps, M. J. Iwod. Pew.1990, 3, 694-701. ((321) Welnbetg, A. Q.; Mke, C. E.; Worthen, H. G. J. m t r . 1976, 88,434438. ((122) Mlchell, G.; Larochelie J.; Lambert, M.; et al. N. En@. J. M.1000,322, 432-437. (G23) UndMad, 8.; UWtedt, S.; Steen. G. Roc. Ne#. Aoed. So/. U.S.A. 1977, 74, 4641-4845. (G24) Editorial. lancet 1002, 340, 822-823. ((325) Lehmnt,W.; Werk, E. C h Wh. A& 1986, 172, 123-126. ((326) HaWfeM, L.; Ne*, A. S. Wh, A& 1987, 16#, 77-84. d e IOOS, 32,160-164. (ci27) Sabourln, P. H.; Bleber, L. L. M ((328) wendel, v.; v.; L m , 1.; &mer, H. J. Eu. J. Pwyetr. 1982. 1s.172-175. (029) GI& ~ . f ~ m p l e b A. y , M.; ~orou~endi, M. A.; Lenoard, J. v.; Sonkmn, P. H. Po&&. Res. 1967. 27, 10-13. (030)Thompson,G.N.;Bre~n,J.L.:Pacy,P. J.;etal. Am. J.~%ys/cf. 1980, 258. E6544660. (031) Schakwaldt, P.; w e u , V. AW. Medeb. BO/. 1969,41,105116. (032) WOO, S. L. 6 k b d ~ t r 1969, y 28, 1-7. ((333)WllllamMN1, M. L.; DobSOn, J. C.; Koch, R. psd[eblw 1977,60,815-621. (034) Largllkre, C.; Dhondt,J. L.; Farrlaux, J. P. J. InherltedMemb. 1xs.1968, 0, 207-211. ((335) RKnnpson, G. N.; HaH#ey, D. J. Clh. Invest. 1990, 86, 317-322. ((336) Koch, R. Annu. Rev. Nub. 1987, 7, 117-135. Muiils, K. B.; Erlich, H. A. N e w ((337) Saki,R. K., Bugewan, T. L.; Horn, G. 1.; 1986, 324, 163-166. ((338) Kauhnen, S. J. pedlb&. 1989, 114, 895-900. ((339) Matalon, R.; Mlchals, K.; Sullivan. D.; Levy, P. Roce&#@ of Ltrs fht IntcKnedbnel h4eeW m D k w Phenyhhkm end lkrrkr Centsr fw mk, SckMCes end MebboRem. Charttabk, TWt; 1987; pp 82-93. (040)Tuchmen, M.; Flsch, R. 0.; Ramnaralne, M. L.; K M , W. #ochm. kkd. I O M , 34, 203-206. (641) Kaufman, S. Advences In MwuchSmbbyl Pbnum: New York, 1977; VOI. 2, pp 1-132. (G42) Loo, Y. H.; Potempska, A.; Wlsniewskl, H. M. J. Namchem. 1 9 1 , 4 5 , 1596-1600. (G43)A m h , C. M.: Anthone, S.: Hcfvath, C.; et ai. Ann. Intom. M.1967, 108, 531-537. ((344) Appb@rth, D. A.; Dimmlck, J. E.; Toone, J. R. Ped&*. Cfn. Mwih Am. 1989, 36,49-65. ( ~ 4 5 ) ~ ~ e m ww, s. .; ~ , A . L m . s ~ t m r n ~ o 6th ed.; W a w - W H New York, 1969 pp 629-663. (046) W a l w M. Am. J. mys/cf. 1966, 250, F181-Fl86. (G47)Am.P.H.;Hauesr,E.R.;Thomas,Q.H.;~n,G.;Hesa,D.;BrusHow, S. W. N. €@. J. Med. 1990, 322, 1652-1655. (G46) Alon~O,E.; Rublo, V. J. Nub.. 1969. 1 IS, 1188-1 195. (G49) BeuchtTIaflfl, C. E m 1067, 38, 233-241. ((350)FlnkdsWn, J. E.; Hatmer, E.; Brudbw, S. W. Am. J. Hum. Qenet. 1988, 45 (Suppl. AS). ((351) MecKenzle, A. E.; MacLeOd, H. L.; Hdck,H. M. C.; Kornduk, R. G. J. mlr.1969, 115, 605-606. ((352) Rowe, P. C.; Newman, S. L.; Bruslbw, S. W. N. fn@. J. Med, 1986, 314, 54 1-547. ((353)Hatmer, E. R.; Flnkelsteln,J. E.; Vaiie, D.; Brudlow, S. W. N. €@. J. W . 1990. 322,1641-1645. ((354) kO8trl;M. L.; Hauser, E. R.; Barthokmew, D.; Brusilow,S. W. J. &$ab: 1991, 1 1 5 923-928. ((355) Betshaw, M. L.; Neylor, E. W.; Themes, Q. H. J. pwlbtr. 1969, 175, 595-598. ((356) Carstens, R. P.; Fenton, W. A,; Rosenberg, L. R. Am. J. Hum Qenef, 1991, 48,1105-1114. ((357) Tuchmen, M.; Tsel, M. Y.; HoWtnecM,R. A.; Brusllow,S. A. pedkrtr. Res. 1980, 26, 77-82. ((158) Brudbw, S. W. pwlbtr. Res. 1991, 20, 147-150. ((359) Mans. A. M.; Daloseph, M. R.; Davls, D. W.; VI&, J. R.; Hawklns, R. A. Am. J. mys/cf.imo. 258, EIOCEIIO. (-0) Hawkins, R. A.; J%ssy, J. J. 1991, 277, 897-703. (~1)~yJ.;Mans,A.M.;DeJossph,M.R.;Hawklns,R.S.~.J.1990, 272,311-317. (G82) JOW, J.; Daloeeph, M. R.; Hawklne. R. A. J. 1991, 277, 693-696. (ma)Hawklna, R. A.; Mans, A. M. MpaMC Encephblkpalhy: PethophVskkoy and Tmbnent; H w n e ~Rsss: CiHtOn, KI, 1989: p~ 195-230. (084) M a d , N. E.; McOowan, K. D.; Bruslbw. S. W. J. P6detr. 1992, 121. 259-261. (W5) Takehashi, H.; Koehler, R. C.; Bruslbw, S. W.; Traystman, R. J. Am. J. mYSM 1991, 261, H825-H829. (086) Krebs, H. A. Bkahem. J. 1986, 20, 1951-1969. (-7) A l b r m , J. M u o p h e m r e w 1969, 28, 885-887. (088) Cooper, A. J.; Plum, R. f71y~bl. Rev. 1967, 67, 440-519. (-9) Buttworth, R. F.; Lavole, J.; Qlgvere, J. F.; Layrargues. G. P.; Bergeron. M. P8M. 1967, 6, 131-144.
a.
2.
.-
.-
f ~ ~
