Plasma Inborn errors

Erythrocyte Entrapment of Enzymes. Erythrocytes have been used as carriers for therapeutic enzymes in the treatment of inborn errors (249). Exogenous enzymes encapsulated in erythrocytes may be useful both for dehvery of a given enzyme to the site of its intended function and for the degradation of pathologically elevated, diffusible substances in the plasma. In the use of this approach, it is important to determine that the enzyme is completely internalized without adsorption to the erythrocyte membrane. Since exposed protein on the erythrocyte surface may ehcit an immune response following repeated sensitization with enzyme loaded erythrocytes, an immunologic assessment of each potential system in animal models is required prior to human trials (250). 

Familial LCAT deficiency, an inborn error of metabolism that affects the levels of plasma cholesteryl esters, was recently discovered in Scandinavia (G7, G8, G9, GIO, Gll, N4). Patients with this disease have... 

Norum, K. R., and Gjone, E, Familial plasma licithin cholesterol acyltransferase deficiency biochemical study of a new inborn error of metabolism. Scand. J. Clin. Lab. Invest. 20, 231-243 (1967). 

The analysis of amino acids has been the first-line approach for the diagnosis of inborn errors of metabolism in most laboratories ever since the end of the 1950s, and it is expected to continue to play this role for a long time. Both the plasma and the CSF amino acid profile are now well known and interpretation should not pose any problems. A correct diagnosis requires adequate pattern recognition [7]. 

Because of these ever-widening interests, the measurement of plasma tHcy is undertaken in many clinical chemistry and routine laboratories. Various methods are employed, including high-performance liquid chromatography (HPLC) assays, conventional amino acid analysis, capillary electrophoresis, gas chromatography with or without mass spectrometry, liquid chromatography with tandem mass spectrometry, and in many routine clinical chemistry laboratories immunoassays. In this chapter, those methods that are often available in laboratories involved in the investigation of inborn errors of metabolism are described, namely HPLC and tandem mass spectrometry. 

Schmidt-Sommerfeld E, Penn D, Duran M, et al (1992) Detection and quantitation of acylcarnitines in plasma and blood spots from patients with inborn errors of fatty acid oxidation. Prog Clin Biol Res 375 355-362... 

Erythrocyte Entrapment of Enzymes. Erythrocytes have been used as carriers for therapeutic enzymes in the treatment of inborn errors. Exogenous enzymes encapsulated in erythrocytes may be useful both for delivery of a given enzyme to Ihe she of its intended function and for file degradalinn of pathologically elevuied. diffusible substances in the plasma. 

The NMR methods have been used in clinical medicine for many years, and metabonomic evaluation of human samples has been conducted for at least the past 10 years (28). Classical examples include the application of NMR to the evaluation of inborn errors of metabolism (29). More recent work has applied metabonomics to the evaluation of the clinical severity of coronary artery disease and to establish a relationship between serum metabolic profiles and hypertension (30,31). Because metabonomics is highly sensitive to environmental or dietary influences (discussed under toxicological applications), concern has been raised that the natural variation in the human population would preclude the application of metabonomics to clinical problems. However, such concerns have been dealt with directly, and recently, Lenz et al. (32) demonstrated that urine and plasma could be collected from human subjects and used successfully for metabonomic analyses. Furthermore, in addition to the disease states described above, metabonomics has been shown to a potentially useful tool for describing alterations associated with dietary and nutritional practices (33). 

In the majority of cases, a UCD can be distinguished from other inborn errors of metabolism by routinely available clinical chemistry tests such as blood gases, acid/base balance, plasma glucose, ammonium, or lactate. Urea production, and hence serum urea nitrogen, is decreased in UCDs. Respiratory alkalosis has few causes and is an important diagnostic clue of hyperammonemia that should trigger measurement of plasma ammonium. 

Consideration of other plasma amino acids also informs the diagnosis of inborn errors of urea synthesis. The plasma concentrations of glutamine and alanine are often elevated in parallel with or prior to the ammonium concentration as they act as a nitrogen buffer. Plasma arginine concentrations are low since the only synthetic route for arginine in humans is via the urea cycle. In contrast, the arginine concentration is elevated in ARG-1 deficiency. Hyperornithinemia and homocitrullinuria are the characteristic features of the hyperammonemia, hyperornithinemia, and homocitrullinuria (HHH) syndrome caused by a defect in the ornithine transporter (ORNT-1). 

Mild hyperhomocysteinemia is defined as a plasma tHcy concentration of 10-30 (tmol/L moderate hyperhomocysteinemia is classified as 30-100 tmol/L. A very severe form of hyperhomocysteinemia, which produces plasma tHcy concentrations greater than 100 tmol/L, can be caused by one of several inborn errors of methionine metabolism. Patients with these disorders also have high levels of tHcy in their urine, a condition known as homocystinuria. 

Measurement of blood tHcy is usually performed for one of three reasons (1) to screen for inborn errors of methionine metabolism (2) as an adjunctive test for cobalamin deficiency (3) to aid in the prediction of cardiovascular risk. Hyperhomocysteinemia, defined as an elevated level of tHcy in blood, can be caused by dietary factors such as a deficiency of B vitamins, genetic abnormalities of enzymes involved in homocysteine metabolism, or kidney disease. All of the major metabolic pathways involved in homocysteine metabolism (the methionine cycle, the transsulfuration pathway, and the folate cycle) are active in the kidney. It is not known, however, whether elevation of plasma tHcy in patients with kidney disease is caused by decreased elimination of homocysteine in the kidneys or by an effect of kidney disease on homocysteine metabolism in other tissues. Additional factors that also influence plasma levels of tHcy include diabetes, age, sex, lifestyle, and thyroid disease (Table 21-1). 

Since D-amino acids are poorly utilized, diets containing sufficient quantities of D-enanticmers will result in elevated levels of plasma and urinary amino acids. Urinary excretion of D-methionine by infants fed a formula supplemented with DD-methionine has led to misdiagnosis of inborn errors of metabolism (77). D-Amino acids derived from processed food proteins may confuse medical diagnoses. Determining D-amino acid contents of caimon foods would estimate the significance of this problem. Some preliminary results are shown in Table VI. 

Jellum, Stokke and Eldjarn have been very active in the metabolic profile approach over several years and routinely investigate perhaps the most comprehensive number of compound types [341]. They screen eight fractions from a urine extract (also plasma and CSF if necessary) by GC and submit samples which show irregularities to GC-MS for further analysis. Spectra are identified off line from a remote terminal of a large, fast central computer. They have had considerable success with their methods, independently reporting three new disorders [345, 355, 356] and are able to detect about 40 of the approximately 150 documented inborn errors of metabolism. 

Lactate in CSF normally parallels blood levels, but not in children. With biochemical alterations in the CNS, however, CSF lactate values change independently of blood values. Increased CSF concentrations are noted in cerebrovascular accidents, intracranial hemorrhage, bacterial meningitis, epilepsy, inborn errors of the electron transport chain, and other CNS disorders. In aseptic (viral) menmgitis, lactate concentrations in CSF are not usually increased hence, CSF lactate has been used to help discriminate between viral and bacterial meningitis,but the clinical utility has been questioned. In a few children with inherited metabolic diseases, CSF lactate concentrations may be increased despite a plasma lactate in the reference interval. 

The method of choice is ion exchange chromatography by automatic analysis (S14). Urine and cerebrospinal fluid can be applied directly to the column, but plasma must be first deproteinized. Since the accurate estimation of gultamine is of paramount importance in all inborn errors of the urea cycle, care must be taken to avoid the breakdown of glu-... 

Thus, the major abnormalities of plasma free hydroxyproline appear to result from (1) the ingestion of hydroxyproline, collagen, or gelatin in any form, and (2) an inability to metabolize hydroxyproline from either prematurity or an inborn error of metabolism. Theoretically, a... 

In single pulse and spin-echo spectra of normal human and animal plasma, there are few resonances in the chemical shift range to high frequency of 85.3 when measured in the pH range 3 to 8.5. However, on acidification of the plasma to pH <2.5, resonances from histidine and phenylalanine become detectable. In plasma from patients with Wilson s disease (liver degeneration secondary to an inborn error of caeruloplasin/copper metabolism), weak signals from histidine and tyrosine are seen in spin-echo spectra at pH 7.6, but... 

The clinical presentation of carnitine deficiency includes generalized skeletal muscle weakness, fatty liver, and fasting hypoglycemia. Carnitine status can be assessed by measurement of plasma, urine, or red blood cell total and free carnitine concentrations. Plasma and urine carnitine concentrations are most helpful in primary carnitine deficiency (an inborn error of metabolism). Plasma concentrations constitute less than 1% of the total body carnitine. ... 

The significance of the complex sequence of events involved in the formation, transfer, and clearance of plasma lipoprotein CE is demonstrated dramatically by several inborn errors of metabolism. One such error is familial LCAT deficiency [67]. In this disease, as well as in diseases associated with acquired LCAT deficiency, LCAT activity in the plasma is abnormally low, and many hpoprotein and tissue abnormalities are observed. The content of UC and PC is abnormally high, and the molar ratio of UC to PC in the hpoproteins is also high, sometimes reaching a value of nearly 2 1. In association with these abnormahties, most lipoproteins show an abnormally low content of CE. In addition, there are abnormahties in the distribution and/or concentration of apolipoproteins AI, All, B, C, and E disc-shaped HDL and unusually small spherical HDL are seen and multilamehar vesicles containing UC and PC are usually present in the LDL fraction obtained by preparative ultracentrifugation. These abnormahties all seem to depend on the LCAT deficiency they are altered toward normal when patient plasma is incubated with LCAT in vitro. 

One of the functions of hepatic P-oxidation is to provide ketone bodies, acetoac-etate and p-hydroxybutyrate, to the peripheral circulation. These can then be utilized by peripheral tissues such as brain and heart. Beta-oxidation itself produces acetyl-CoA which then has three possible fates entry to the Krebs cycle via citrate S5mthase keto-genesis or transesterification to acetyl-carnitine by the action of carnitine acetyltrans-ferase (CAT). Intramitochondrial acetyl-carnitine then equilibrates with plasma via the carnitine acyl-camitine translocase and presumably via the plasma membrane carnitine transporter. Human studies have shown that acetyl-carnitine may provide up to 5% of the circulating carbon product from fatty acids and can be taker and utilized by muscle and possibly brain." In addition, acyl-camitines are of important with regard to the diagnosis of inborn errors of P- oxidation. For these reasons, we wished to examine the production of acetyl-carnitine and other acyl-camitine esters by neonatal rat hepatocytes. 

The data presented in this study show that we have developed a quantitative analysis of free carnitine and acyl-camitine species in plasma or serum. The quantitative nature of this analysis enables good discrimination between normal and abnormal profiles and accurate therapy monitoring, alfiiou one should realize fiiat mild cases exist wfiidi may have normal profiles in clinically stable episodes. Compared to other methods for quantitative acyl-camitine analysis like HPLC or GC/MS, the method described here is less laborious and allows determination of a wide variety of ac l-camitine species, including hydroxylated acyl-camitine spedes, which is not the case for the (laborious) GC/MS method. Since for selective screening for inborn errors of metabohsm, apart from urine samples, mostly serum or plasma samples are send in, this method allows direct comprehensive acyl-camitine analysis in such materials, obviating the need for a separate blood-spot for acyl-camitine analysis which would allow qualitative or semi-quantitative acyl-camitine andysis. "... 

Besides hormonal steroid conjugates (Figure 2.7), bile alcohol and bile acid conjugates can also be analyzed by shotgun steroidomics. These metabolites are often elevated in abundance in plasma as a consequence of an inborn error of bile acid biosynthesis or as a consequence... 

The next part of this chapter will include a discussion of the interpretation of amino acid patterns, with particular emphasis on plasma The rationale behind this discussion is that most amino acid analyses are undertaken in children with a view to diagnosing inborn errors of metabolism. One is then faced with the difficulty of interpreting a particular ammo acid pattern to decide whether or not this pattern is normal and, if it is abnormal, to... 

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