UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
Isovaleric acidemia: an integrated approach toward predictive laboratory
medicine
Dercksen, M.
Publication date
2014
Link to publication
Citation for published version (APA):
Dercksen, M. (2014). Isovaleric acidemia: an integrated approach toward predictive laboratory
medicine.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)
and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open
content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please
let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material
inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter
to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You
will be contacted as soon as possible.
Chapter 7
Polyunsaturated fatty acid and vitamin B12 status
in treated isovaleric acidemia patients
M. Dercksen 1,2, W. Kulik1, L.J. Mienie2, C.J. Reinecke2; R.J.A. Wanders 1, M.Duran1
Laboratory Genetic Metabolic Diseases, Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands1; Centre for Human Metabonomics, North-West University (Potchefstroom Campus), South Africa 2
Abstract
Nutritional deficiencies are frequently observed when treating patients with inborn errors of metabolism (IEMs) due to an unbalanced diet. We recently found evidence of vitamin B12 deficiency in a metabolomics investigation of urine of treated isovaleric acidemia (IVA) patients. We subsequently examined the nutritional status by reporting on the haematological profile as well as potential deficiencies in polyunsaturated fatty acids (PUFAs) and functional vitamin B12 in treated IVA patients. A complete blood count was performed as well as gas chromotography flame ionization detector (GC-FID) analysis to determine PUFAs in plasma of 10 IVA patients. In vivo markers of functional vitamin B12 deficiency namely methylmalonic acid, homocystine and total homocysteine in urine and plasma were assayed via GC-MS, MS/MS and UPLC-MS/MS. The general clinical chemistry tests did not indicate severe hematological abnormalities or nutritional insufficiencies except for the presence of a mild thrombocytosis. We identified a significant reduction of plasma PUFA levels, especially in omega-3 (all groups, p < 0.001) and omega-6 (in particular 20:3n-6 p < 0.001) fatty acids. In addition, an elevation in omega-9 fatty acids, with the exception of 20:3n-9 and C22:1n-9, was suggestive of an essential fatty acid deficiency (EFAD). Furthermore, individual patients showed elevated methylmalonic acid, total homocysteine and homocystine levels, but the overall assessment of functional vitamin B12 in the patient group was normal. This study emphasizes the potential nutritional insufficiencies which may occur due to therapeutic intervention in isovaleric acidemia.
Introduction
The classical management and intervention of patients with inborn errors of amino acid catabolism encompasses a specific dietary regimen. Isovaleric acidemia (IVA), a genetic defect of leucine catabolism as a result of deficient isovaleryl-CoA dehydrogenase (E.C.1.3.99.10), is a treatable condition, which requires a diet low in protein, or more specifically leucine. The consequence of such a diet is the limited intake of animal products (meat, fish, milk, eggs), putting these patients at risk of acquiring deficiencies in several vitamins, trace elements, and polyunsaturated fatty acids (PUFAs) (Giovannini et al., 1995; Vockley et al., 2012).
Vitamin B12 deficiency has been reported in patients with phenylketonuria, who changed from a strict phenylalanine-free diet, with additional vitamins and minerals, to a relaxed dietary regimen. Eventually, shortage of vitamin B12 may be associated with megaloblastic anemia, spastic paralysis, tremors and disorientation (Robinson et al., 2000). In addition to potential vitamin insufficiency, several investigations of patients treated for phenylketonuria, urea cycle defects, maple syrup urine disease, methylmalonic acidemia and homocystinuria have demonstrated deficiencies in omega-3 and omega-6 fatty acids and their related biological products, as a result of an exclusion dietary regimen (Vlaardingerbroek et al., 2006; Fekete and Decsi, 2010). This may also hold true for our IVA patients who follow a low-protein diet, without notable nutrient supplementation.
The PUFA group includes the omega-3 and omega-6 fatty acids, which are derived from the essential fatty acids linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3), as well as omega-7 and omega-9 fatty acids, which are non-essential and synthesized from saturated fatty acids (Jeppesen et al., 1997). The production of PUFAs from essential fatty acids takes place in the endoplasmic reticulum, mitochondria and the peroxisomes of the cell (mostly in the liver) and includes processes of elongation, desaturation and β-oxidation (Sprecher, 2000; Duran and Wanders, 2008). The intricate participation of multiple enzymes including elongases and desaturases (Δ5, Δ6, Δ9) is shared in omega-3, omega-6 and omega-9 fatty acid synthesis. The desaturases are the rate-limiting steps in the pathway and are strongly regulated by substrate availability (Nakamura and Nara, 2004). Numerous PUFAs, including dihomo-gamma-linolenic acid (DGLA, 20:3n-6), arachidonic acid (AA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA, C22:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), have important clinical functions in the cell (Nakamura and Nara, 2004; Vognild et al., 1998; Benatti et al., 2004).
PUFAs play an intricate role in membrane fluidity since they are incorporated into phospholipids, thereby contributing to the structure and function of cell membranes.
They are also involved in several signal transduction processes as well as the production of biomolecules that are vital in immune response, blood clotting (platelet aggregation), gestation and blood pressure regulation (Von Schacky et al., 1985). A deficiency in EFA may lead to dermatitis, liver anomalies, impaired chylomicron synthesis, severe fat malabsorption, dysregulation of cholesterol metabolism and irregularities in the immune system as a result of hematological disturbances (Benatti et al., 2004). It has also been reported that PUFAs, especially DHA and AA, are highly concentrated within the structural lipids of the central nervous system, in particular the brain and retina, and are vital in the development and maintenance of neurological function e.g. myelination (Lauritzen et al., 2001; Peters et al., 2013). They are particularly important for the pregnant mother and her growing fetus as well as for the development of neonates up to childhood (Uauy et al., 2000). For this reason, an adequate intake and a fine balance in the regulatory production of PUFAs are needed.
Our recent IVA metabolomics study showed moderately increased methylmalonic acid in urine (Dercksen et al., 2013), potentially associated with vitamin B12 deficiency, and prompted us to investigate the patients' nutritional status. A complete blood count (CBC) from each patient was obtained to investigate their basic clinical and nutritional status. In addition, we studied the PUFA levels as well as the concentrations of functional vitamin B12 markers i.e. methylmalonic acid, total homocysteine and homocystine, in plasma and urine of the treated IVA patients.
Patients and methods
Collection of samples from isovaleric acidemia patients
Ten patients with isovaleric acidemia as a result of a homozygous c.367G > A (p.G123R) mutation of the isovaleryl-CoA dehydrogenase gene, with ages ranging from 2 to 24 years, were initially diagnosed by the Potchefstroom Laboratory for Inborn Errors of Metabolism, South Africa (Dercksen et al., 2012). The patients and their families attended an informative one-day session in which all aspects of the investigation were discussed. Informed consent was given by all participating individuals or their legal guardians. Non-fasting blood specimens and random urine samples were collected on the same day, in a controlled environment, which included the consumption of a low protein meal. Whole blood was collected to perform a CBC on each patient. Plasma samples were collected and kept frozen (-80 °C). Urines samples were stored at -20 ºC. None of the patients suffered from any metabolic decompensation at the time of sample collection. Control groups consisted of 20 and
54 comparable hospital-based subjects for the studies on vitamin B12 parameters and PUFAs, respectively.
Full blood count determination
A CBC was conducted on EDTA blood, collected from patients, within 90 minutes after collection. The ADVIA 2120 (Siemens, Diagnostics: New York) analyzer was used to obtain a full blood panel for each patient.
Analysis of PUFAs in plasma
We analyzed the PUFAs in plasma samples using a modification of the method initially described by Muskiet et al. (1983) and validated by Duran and Wanders (2008). A volume of 50 µl of plasma was pipetted into a 2 ml glass tube. Subsequently, an internal standard (100 µl), containing 72 µmol/l of 18-methyl-C19:0-methylester dissolved in chloroform, was added to the samples. Simultaneous hydrolysis and methylation of the fatty acids was achieved with 1 ml methanolic hydrochloric acid (3 M). The samples were vortexed and subsequently incubated at 90 °C for 4 hours. After cooling, 2 ml hexane was added and vortexed for 10 seconds. The hexane layer was collected and dried under nitrogen at room temperature. The dried residue was resuspended in 100 µl hexane. The derivatized PUFAs were analyzed on a GC-FID (HP GC5890 series II, Agilent: Palo Alto, CA) equipped with a combined capillary free fatty acid pre-column (Hewlett-Packard FFAP, Agilent: Palo Alto, CA) and a DB17 column (J & W Scientific, Agilent: Palo Alto, CA). All concentrations were expressed as µmol/l, and significance was given by p-value calculation.
Analysis of urinary metabolites related to vitamin B12 deficiency
Methylmalonic acid in the urine of treated IVA patients and control subjects was measured via GC-MS (Agilent, 7890A GC, 5975C MS, Palo Alto, CA). The methylmalonic acid was identified and quantified using the Automated Mass Spectral Deconvolution and Identification System software (AMDIS, version 2.66 from the National Institute for Standards and Technology) (Reinecke et al., 2012). Urinary homocystine was determined by tandem mass spectrometry with a dedicated stable-isotope sulphur-containing amino acid analysis, based on a protocol described by Pitt et al. (2002) and performed with an Agilent 1200 LC and 6410 triple Quad MS (Agilent, Palo Alto, CA) in the positive electrospray ionization mode. Data were quantified using Agilent MassHunter (B.03) data analysis software.
Analysis of plasma metabolites related to vitamin B12 deficiency
Methylmalonic acid (MMA) and total homocysteine in plasma were analyzed with stable isotope UPLC-MS/MS and HPLC-MS/MS methods, respectively. Calculations were performed with Masslynx version 4.1 software (Waters, Milford, MA). Plasma MMA was determined essentially as described by Blom et al. (2007) with minor modifications. A volume of 10 μl internal standard [10 μM methylmalonic acid-d3 (Cambridge Isotope Lab, Andover, MA)] followed by 100 μl plasma was pipetted into a filtered tube (Microcon YM-30, Merck Millipore, Billerica, MA). The samples were mixed and centrifuged for 40 minutes at 11,000 g (room temperature). The deproteinized samples were acidified with 10 µl of a 4% formic acid solution, vortexed and 10 μl of the sample was injected into the UPLC-MS/MS (Acquity UPLC with Quattro Premier XE mass spectrometer, Waters, Milford, MA). The product separation was achieved with the Acquity UPLC HSS T3 column (Waters, Milford, MA).
Total homocysteine in plasma was recorded essentially as described by Tuschl et al. (2005) and Fowler (2008). A volume of 50 μl plasma and 20 μl internal standard [25 μM d8-dl-homocystine (Cambridge Isotope Lab, Andover, MA)] were pipetted into a 2 ml tube. An additional 10 μl DTT solution (1,4-dithiothreitol) was added and the samples were vortexed. The samples were left for 15 minutes at room temperature, after which 500 μl acetonitrile was added. The samples were vortexed and centrifuged for 15 min at 11,000 g (4 °C). Ten microliters of the supernatant was injected on the HPLC-MS/MS (Waters, Milford, MA). The product was separated on a Supelco LC-CN column (Supelco, Bellefonte, PA).
Statistical analysis
The data were analyzed using the STATISTICA data analysis software system, version 10 (StatSoft, Inc, Tulsa, OK). The PUFA data and vitamin B12 markers of the IVA patients and control group were statistically compared and correlated.
Results
Hematological status of treated IVA patients
A CBC was recorded for all patients in order to obtain information on their general health and condition including nutritional and infectious status. The results did not reveal any obvious aberrations for the patients as a group. However, mild thrombocytosis was observed in half the patients and neutropenia was present in one patient. Two patients had slight anemia, but no indication of megaloblastic anemia,
associated with vitamin B12 deficiency, was present, i.e all MCV values were below 96 fl. Their MCHC levels exceeded 32 g/l, which implied that patients did not have an iron deficiency, associated with anemia. Detailed information, including reference ranges of the CBC can be viewed in supplementary Table 1 (Wu, 2006).
Polyunsaturated fatty acid status of IVA patients
In order to ascertain possible insufficiencies in PUFAs, we measured the mono- and polyunsaturated fatty acids in plasma of the IVA patients. All plasma omega-3 fatty acids were significantly decreased compared to the levels in control subjects. Some omega-6 fatty acids (C20:3n-6, C20:4n-6) were significantly lower than controls whereas C22:5n-6 was higher (Table 1). Most of the other PUFA values followed a downward trend in the IVA group, but no significant difference from controls was observed. We unexpectedly found a significant reduction in C18:1n-7, C20:3n-9 and C22:1n-9, which contrasted with essential fatty acid deficiency described previously (Jeppesen et al., 1997).
Table 1: Individual mono- and polyunsaturated fatty acids of control and IVA groups
Fatty acid IVA (n = 10) Control subjects
(n = 54)
p-value (t-test)
Omega-3 fatty acids
C18:3n-3 24.8 ± 13.3 73.0 ± 45.6 0.0016*
C20:5n-3 13.9 ± 17.9 67.6 ± 50.2 0.0015*
C22:5n-3 18.7 ± 10.4 34.8 ± 12.2 0.0002*
C22:6n-3 64.3 ± 28.5 128.5 ± 46.1 0.0001*
Omega-6 fatty acids
C18:2n-6 2796.7 ± 493.9 3217.0 ± 630.1 0.051 C18:3n-6 35.0 ± 17.7 46.1 ± 21.7 0.1332 C20:2n-6 16.9 ± 4.8 20.3 ± 6.8 0.1361 C20:3n-6 84.8 ± 30.8 157.0 ± 44.1 < 0.001* C20:4n-6 415.6 ± 114.3 594.5 ± 144.9 0.0005* C22:4n-6 16.1 ± 6.5 14.5 ± 4.6 0.3549 C22:5n-6 13.9 ± 5.2 10.0 ± 4.1 0.0107*
Omega-7 fatty acids
C16:1n-7 206.0 ± 92.7 244.4 ± 108.9 0.2997
C18:1n-7 115.3 ± 25.6 186.5 ± 45.8 < 0.001*
Omega-9 fatty acids
C16:1n-9 67.3 ± 75.2 50.6 ± 15.8 0.1353 C18:1n-9 2365.1 ± 844.5 2135.4 ± 665.5 0.3403 C20:3n-9 6.3 ± 3.8 10.7 ± 4.4 0.005* C22:1n-9 3.4 ± 2.2 20.2 ± 6.8 < 0.001* C24:1n-9 77.5 ± 21.6 68.5 ± 18.2 0.1692 Omega-6/omega-3 ratio 32.7 ± 13.2 14.75 ± 4.3 < 0.001*§
EFA values are in µmol/l and represent mean ± standard deviation observed in patients and control groups. * Significant differences between patient and control groups.
§ An elevated omega-6 (sum)/omega-3 (sum) ratio is a good indicator of EFAD deficiency (Simopoulos, 2006).
As can be seen from Table 1, individual PUFA values (with some only marginally decreased in the IVA patient group) did not give a clear indication of a fatty acid deficiency. Consequently the sum of the fatty acids belonging to each PUFA group was used to present a potentially improved profile for the evaluation of the PUFA status of IVA patients. We observed a statistically significant decline in omega-3 and omega-6 fatty acids with a non-significant accumulation of omega-9 fatty acids, consistent with a mild EFAD (Fig. 1) (Jeppesen et al., 1997). In addition, the omega-6/omega-3 ratio value in the patients was more than twice the ratio value observed in the control group (p < 0.001) and an additional indicator of EFAD (Simopoulos, 2006).
Fig. 1: The sum of omega-3, omega-6 and omega-9 fatty acids in control (indicated by the white
bar) and treated IVA patients (indicated by the gray bar). A significant decline in omega-3 and omega-6 fatty acids (p < 0.001 and p < 0.005, respectively) was observed with a concomitant moderate increase (p = 0.34) of the omega-9 fatty acids in the IVA patient group.
Metabolites indicative of vitamin B12 deficiency
To investigate the possibility of functional vitamin B12 deficiency, as suggested by the observations in the metabolomics study, we determined methylmalonic acid as well as total homocysteine and free homocystine in plasma and urine, respectively (Figs 2 and 3). A non-parametric comparison was made in view of the non-Gaussian distribution of data within the IVA group. No statistical difference in functional vitamin B12 indicators between the IVA group and controls was found. However, two patients had elevated urinary MMA levels (7.6 and 6.1 mmol/mol creatinine, respectively) and one patient showed an increased plasma MMA level (0.7 µmol/l). The reference ranges for MMA in urine (0–5 mmol/mol creatinine) and plasma (0.09–0.34 µM) were used to evaluate MMA levels of patients (Vugteveen et al., 2011; Hoffman and Feyh, 2002). These results are not conclusively indicative of a functional vitamin B12 deficiency.
Fig. 2: Vitamin B12 markers in the IVA patient group (1; n = 10) and control group (2; n = 20).
Fig. 3: Vitamin B12 markers in IVA patient group (1: n = 10) and control group (2; n = 20). (A)
We identified three patients who had total homocysteine levels above the normal reference range (3.3–10.3 µM) (Fowler, 2008). No statistical difference between the two groups was found with regard to total homocysteine levels in plasma. Urinary homocystine was within normal reference ranges (0–1 mmol/mol creatinine) and no significant difference between patients and control subjects was observed (Skovby, 2002). These results are complementary to our MMA observations and argue against a generalized vitamin B12 deficiency in this group of IVA patients.
Discussion
The main purpose of this study was to investigate whether there are nutritional deficiencies in metabolically stable, treated IVA patients. Individuals with similar amino acid disorders have shown nutritional deficiencies (Fekete and Decsi, 2010). Consequently, we assessed biochemical parameters that are indicative of nutritional status in this IVA group. More specifically, we included a basic hematological profile, the determination of PUFAs and the measurement of biochemical indicators of functional vitamin B12 deficiency.
We compared the polyunsaturated fatty acid profiles of 10 IVA patients with control subjects and found a significant reduction in all the individual omega-3 fatty acids in IVA patients. Furthermore, significant deficiencies of individual PUFAs were found in the omega-6 and omega-9 fatty acid classes, with the exception of C18:2n-6, C18:3n-6, C20:2n-6, C22:4n-6, C16:1n-9, C18:1n-9 and 24:1n-9. The significantly reduced 22:1n-9 found in IVA patients is noteworthy. This has also been reported in adrenoleukodystrophy patients with aberrant neurological function (Rasmussen et al., 1994). Essential fatty acid and PUFA levels in plasma are a reflection of their intake during a limited period before blood sampling as well as of their interconversion in the liver, which is an ongoing process (Nettleton, 1995). Normal plasma levels of C18:2n-6 and C18:3n-C18:2n-6 as observed in our patients may indicate that their dietary intake was not necessarily insufficient.
However, the summation of the groups of omega-3 (significantly lower), omega-6 (significantly lower) and omega-9 (higher) fatty acids, depicted in Fig. 1, revealed an overall depletion of PUFA/EFA (Jeppesen et al., 1997). It is important to note that the measurement of PUFAs in erythrocytes, which was not feasible in our study, may provide even more exact information with regard to the PUFA status. Nevertheless, our data are in good agreement with those in previous reports, who studied patients with various defects of amino acid metabolism, including those with urea cycle defects (Vlaardingerbroek et al., 2006; Fekete and Decsi, 2010).
PUFAs play a vital role as precursors of immunological factors such as thromboxane, leukotriene and prostaglandins (Nettleton, 1995). IVA patients may present with thrombocytopenia, neutropenia or even pancytopenia due to the accumulation of the toxic isovaleryl-CoA, during metabolic decompensation, which results in bone marrow suppression (Vockley et al., 2012). To our surprise we observed that half of the metabolically stable IVA patients exhibited unexplained mild thrombocytosis (supplementary Table 1). In view of the involvement of PUFAs as precursors of important immunological factors, we sought a correlation between precursors (PUFAs) and hematological parameters, specifically platelets. However, no significant correlation between hematological markers and the PUFA levels was found.
Methylmalonic acid, total homocysteine and homocystine levels in urine and plasma of our IVA patients were mostly within the normal ranges, except for one or two individual patients, which did not indicate a generalized functional vitamin B12 deficiency. Moreover, no hematological or clinical irregularities associated with vitamin B12 insufficiency were observed, although symptoms associated with IVA and vitamin B12 deficiency may overlap. In this respect there is discussion on the feasibility of various biochemical markers for vitamin B12 deficiency. Unfortunately, the blood levels of vitamin B12 were not assessed in our patient group; neither did we measure the levels of holotranscobalamin, potentially the best vitamin B12 marker (Heil et al., 2012). The starting point of our search for vitamin B12 anomalies was the metabolomics finding of urine MMA elevation (Dercksen et al., 2013) primarily observed in treated IVA patients. This finding should be interpreted with caution, as it has been reported that isovaleryl-CoA inhibits the enzyme succinyl-CoA ligase (Bergen et al., 1982). Such an inhibition might explain the unusual presence of MMA in the urine of IVA patients. Nevertheless, we propose the future monitoring of vitamin B12 markers in order to improve the personalized nutritional treatment of these patients.
Conclusion
The depletion of PUFAs (indicated by a significantly increased omega-6/omega-3 fatty acid ratio) was evident in treated IVA patients. We suggest that dietary supplementation of DHA and AA may be required to overcome nutritional deficiencies imposed by the protein-restricted diet prescribed to IVA patients. The monitoring of nutritional markers should be included to optimize the treatment regimen of IVA patients and provide continuous individualized health care.
Acknowledgements
The study was financially supported as a Carolina MacGillavry PhD Fellowship awarded by "Koninklijke Nederlandse Akademie van Wetenschappen". We thank the staff at the Laboratory Genetic Metabolic Diseases (GMD) at the Academic Medical Center, University of Amsterdam, for their expert advice and assistance. The study was also made possible with the logistical, scientific and statistical contribution of the staff of the Centre for Human Metabonomics and Wilma Breytenbach, the statistical consultant at the North-West University in Potchefstroom, South Africa. We are very grateful to the patients and their families for participation in this study
P a ti e n t in fo rm a ti o n P a ti e n t 1 (f a m il y 1 ) P a ti e n t 2 (f a m il y 1 ) P a ti e n t 3 (f a m il y 2 ) P a ti e n t 4 (f a m il y 2 ) P a ti e n t 5 (f a m il y 3 ) P a ti e n t 6 (f a m il y 3 ) P a ti e n t 7 (f a m il y 4 ) P a ti e n t 8 (f a m il y 5 ) P a ti e n t 9 (f a m il y 6 ) P a ti e n t 1 0 (f a m il y 7 ) P h e n o ty p e c la ss if ic a ti o n m ild m ild se ve re se ve re se ve re se ve re m ild m ild m ild m ild 0– 2 yr 3– 5 yr 6– 15 y r > 15 y r C u rr e n t a g e 6. 5 yr 8 yr 12 y r 16 y r 7 yr 4y r 12 y r 6 yr 2 yr 24 y r F u ll b lo o d c o u n t H em og lo bi n (g /d l) ↓ 1 0 .9 12 .5 14 ↓ 1 3 .4 12 .8 12 .8 14 .3 13 .2 12 .1 14 .4 11 .1 –1 4. 1 11 –1 4 11 .5 –1 5. 5 14 .0 –1 7. 5 R ed c el l c ou nt (1 0 12/l) ↓ 3 .9 8 4. 45 4. 88 4. 94 4. 71 4. 8 4. 83 4. 69 4. 41 5. 24 3. 9– 5. 1 4– 5. 2 4– 5. 3 4. 9– 5. 8 H em at oc rit ( % ) ↓ 3 1 .8 37 .3 40 .6 ↓ 3 9 .7 38 38 .3 41 39 .6 36 .3 43 .5 30 .0 –3 8. 0 34 –4 0 35 –4 5 41 .5 –5 3. 5 M C V ( fl) 80 83 .8 83 .3 ↓ 8 0 .4 80 .7 79 .3 85 84 .4 82 .4 ↓ 8 3 72 .0 –8 4. 0 76 –8 7 77 –9 5 84 –9 8 M C H ( pg ) 27 .5 28 28 .8 ↓ 2 7 .2 27 .3 26 .5 29 .6 28 27 .3 ↓ 2 7 .5 25 .0 –2 9. 0 24 –3 0 25 –3 3 28 –3 2. 5 M C H C ( g/ l) 34 .4 33 .4 34 .6 33 .8 33 .8 33 .4 34 .8 33 .2 33 .2 33 .1 31 .0 –3 6. 0 31 –3 6 31 –3 6 31 –3 7 R D W ( % ) 13 12 .2 13 .6 14 .2 12 .6 13 .6 12 .3 12 .9 13 .2 13 .3 11 .6 –1 4. 0 11 .6 –1 4 11 .6 –1 4 12 –1 4. 5 W hi te b lo od c el ls ( 10 9/l) 8. 78 12 .1 1 7. 41 5. 22 9. 15 11 .6 3 6. 21 10 .8 6 9. 27 6. 01 6. 00 –1 6. 00 5– 15 5– 13 4. 6– 11 .5 N eu tr op hi ls ( 10 9/l) 5. 9 5. 61 3. 79 2. 53 6. 43 4. 73 ↓ 1 .9 4 4. 83 2. 54 3. 35 2– 8 1. 5– 8 2– 8 2– 6. 5 Ly m ph oc yt es ( 10 9/l) ↓ 1 .9 4. 88 2. 62 2. 13 1. 49 5. 77 3. 54 4. 62 5. 63 1. 83 3. 5– 11 6– 9 1– 5 1. 3– 3. 7 M on oc yt es ( 10 9/l) 0. 67 0. 87 0. 47 0. 32 0. 99 0. 56 0. 36 0. 75 0. 94 0. 46 0. 20 –1 .0 0 0. 2– 1 0. 2– 1 0. 25 –0 .8 E os in op hi ls ( 10 9/l) 0. 14 0. 33 0. 34 0. 05 0. 07 0. 19 0. 16 0. 21 0. 13 0. 21 0. 00 –1 0– 1 0– 1 0– 0. 4 B as op hi ls ( 10 9/l) 0. 02 0. 06 0. 03 0. 01 0. 02 0. 03 0. 02 0. 05 0. 05 0. 03 0. 00 –0 .1 0 0. 00 –0 .1 0 0– 0. 1 0– 0. 07 La rg e un st ai ne d ce lls ( ac tiv at ed ly m ph oc yt es ) (1 0 9/l) 0. 16 ↑ 0 .3 5 0. 16 0. 18 0. 15 ↑ 0 .3 5 0. 2 ↑ 0 .4 1 0. 01 0. 13 0– 3 0– 3 0– 0. 3 0– 0. 3 P la te le tt e co un t (1 0 9/l) 30 1 ↑ 7 1 7 29 6 31 4 ↑ 4 9 6 ↑ 5 8 6 22 4 ↑ 4 4 6 ↑ 4 7 7 19 2 20 0– 55 0 20 0– 45 0 18 0– 40 0 15 0– 40 0 * H e m a to cr it : m e as ur e s th e p er ce n ta g e o f re d b lo o d ce lls i n a g iv e n vo lu m e o f w h ol e b lo o d. * M C V (M ea n c or p u sc u la r vo lu m e) : is a m e as ur e m e n t o f th e a ve ra ge s iz e o f yo ur R B C s. * M C H (M ea n c or p u sc u la r h em og lo bi n ): is a c a lc u la tio n o f th e a ve ra ge a m o un t of o xy ge n -c ar ry in g h em o gl o bi n i ns id e a r e d b lo o d c el l. * M C H C ( M e an c o rp u sc u la r h em o gl o bi n c o nc e n tra ti on ): is th e c al cu la ti o n o f th e a ve ra g e c on ce nt ra tio n o f h em og lo bi n in r ed b lo o d ce ll s. * R D W (R ed b lo o d ce ll d is tr ib u ti on w id th ) a c a lc u la ti on o f t h e va ry in g s iz e s o f re d b lo od c e ll (R B C ) v ol u m e i n a b lo od s a m p le . R e fe re n c e v a lu e s ( W u , 2 0 0 7 ) S u p p le m e n ta ry d a ta S u p p le m e n ta ry T a b le 1 : B lo od p an el o f i nd iv id ua l t re at ed IV A p at ie nt s an d re le va nt r ef er en ce v al ue
References
Benatti P, Peluso G, Nicolai R, Calvani M. 2004. Polyunsaturated fatty acids: Biochemical, nutritional and epigenetic properties. J Am Coll Nutr 23:281-302.17.
Bergen BJ, Stumpf DA, Haas R, Parks JK, Eguren LA. 1982. A mechanism of toxicity of isovaleric acid in rat liver mitochondria. Biochem Med 27:154-160. Blom HJ, van Rooij A, Hogeveen M. 2007. A simple high-throughput method for the
determination of plasma methylmalonic acid by liquid chromatography-tandem mass spectrometry. Clin Chem 45:645-650.
Dercksen M, Duran M, IJlst L et al. 2012. Clinical variability of isovaleric acidemia in a genetically homogeneous population. J Inherit Metab Dis 35:1021-1029. Dercksen M, Koekemoer G, Duran M, Wanders RJA, Mienie LJ, Reinecke CJ. 2013.
Organic acid profile of isovaleric acidemia: a comprehensive metabolomics approach. Metabolomics 9:765-777.
Duran M, Wanders RJW. 2008. Plasmalogens and polyunsaturated fatty acids. In: Blau N, Duran M, Gibson M. Eds. Laboratory Guide to the Methods in Biochemical Genetics. Springer-Verlag Berlin, Heidelberg pp. 207-220. Fekete K, Decsi T. 2010. Long-chain polyunsaturated fatty acids in inborn errors of
metabolism. Nutrients 2:965-974.
Fowler B. 2008. Homocysteine, S-adenosylmethionine and S-adenosylhomo-cysteine. In: Blau N, Duran M, Gibson M. Eds. Laboratory Guide to the Methods in Biochemical Genetics. Springer-Verlag Berlin, Heidelberg. pp. 91-114.
Giovannini M, Biasucci G, Luotti D, Fioir L, Riva E. 1995. Nutrition in children affected by inherited metabolic diseases. Ann 1st, Super Sanità 31:489-502.
Heil SG, De Jonge R, De Rotte MCFJ et al. 2012. Screening for metabolic B12 deficiency by holotranscobalamin in patients suspected of vitamin B12 deficiency: a multicentre study. Ann Clin Biochem 49:184-189.
Hoffman GF, Feyh P. 2002. Organic acid analysis. In Blau N, Duran M, Blaskovics ME Eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. Springer, Heidelberg. pp. 27-44.
Jeppesen PB, Christensen MS, Høy C, Mortensen PB. 1997. Essential fatty acid deficiency in patients with severe fat malabsorption. Am J Clin Nutr 65:837-843.
Lauritzen L, Hansen HS, Jorgensen MH, Michaelsen KF. 2001. The essentiality of long-chain n-3 fatty acids in relation to development and function of the brain and retina. Prog Lipid Res 40:1-90.
Muskiet FA, van Doormaal JJ, Martini IA, Wolthers BG, van der Slik W. 1983. Capillary gas chromotographic profiling of total long-chain fatty acids and cholesterol in biological materials. J Chromatogr 278:231-244.
Nakamura MT, Nara TY. 2004. Structure, function and dietary regulation of ∆6, ∆5, and ∆9 desaturases. Annu Rev Nutr 24:354-376.
Nettleton JA. 1995. Omega-3 fatty acids and health. New York: Chapman and Hall. Peters BD, Machielsen MW, Hoen WP et al. 2013. Polyunsaturated fatty acid
concentration predicts myelin integrity in early-phase psychosis. Schizophr Bull 39:830-838.
Pitt JJ, Eggington M, Kahler SG. 2002. Comprehensive screening of urine samples for inborn errors of metabolism by electrospray tandem mass spectrometry. Clin Chem 48:1970-1980.
Rasmussen M, Moser AB, Borel J, Moser HW. 1994. Brain erucic and very long chain fatty acid levels in adrenoleukodystrophy patients treated with glyceryl trioleated and trierucate oils (Lorenzo's oil). Neurochem Res 19:1073-1082. Reinecke C, Koekemoer G, Van der Westhuizen F et al. 2012. Metabolomics of
urinary organic acids in respiratory chain deficiencies in children. Metabolomics 8:264-283.
Robinson M, White FJ, Cleary MA, Wraith E, Lam WK, Walter J H. 2000. Increased risk of vitamin B12 deficiency in patients with phenylketonuria on an unrestricted or relaxed diet. J Pediatr 136:545-547.
Simopoulos AP. 2006. Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother 60:502-507.
Skovby F. 2002. Disorders of sulfur amino acids. In Blau N, Duran M, Blaskovics ME Eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. Springer, Heidelberg pp. 243-260.
Sprecher H. 2000. Metabolism of highly unsaturated n-3 and n-6 fatty acids. Biochim Biophys Acta 1486:219-231.
Tuschl K, Bodamer O A, Erwa W, Mühl A. 2005. Rapid analysis of total plasma homocysteine by tandem mass spectrometry. Clin Chem Acta 2005; 351:139-141.
Uauy R, Mena P, Rojas C. 2000. Essential fatty acids in early life: structural and functional role. Proc Nutr. Soc 59:3-15.
Vlaardingerbroek H, Hornstra G, de Koning TJ, et al. 2006. Essential polyunsaturated fatty acids in plasma and erythrocytes of children with inborn errors of amino acid metabolism. Mol Genet Metab 88:159-165.
Vockley J, Zschocke J, Knerr I, Vockley CW, Gibson KM. 2012. Branched chain organic acidurias In: Valle D, Beaudet A, Vogelstein B, Kinzler KW, Eds.
The Online Metabolic and Molecular Bases of Inherited Disease, Part 8, McGraw-Hill, 2012: www.ommbid.com (accessed on 2 October 2012). Vognild E, Elveoll EO, Brox J, et al. 1998. Effects of dietary marine oils and olive oil
on fatty acid composition, platelet membrane fluidity, platelet responses, and serum lipids in healthy humans. Lipids 33:427-436.
Von Schacky C, Fischer S, Weber PC. Long term effects of dietary marine ω-3 fatty acids upon plasma and cellular lipids, platelet function and eicosanoid formation in humans. J Clin Invest 76:1626-163112.
Vugteveen I, Hoeksma M, Bjorke Monsen A et al. 2011. Serum vitamin B12 concentrations within reference values do not exclude functional vitamin B12 deficiency in PKU patients of various ages. Mol Genet Metab 102:13-17.
Wu A. 2006. Tietz Clinical Guide to Laboratory Tests. 4th ed. St. Louis, Missouri:
