Annals of Clinical Biochemistry: International Journal of Laboratory Medicine

The oxidative phosphorylation system (OXPHOS) is organized into five multi-protein complexes, comprising four complexes (I-IV) of the respiratory chain and ATP synthase (complex V). OXPHOS has a vital role in cellular energy metabolism and ATP production. Enzyme analysis of individual OXPHOS complexes in a skeletal muscle biopsy remains the mainstay of the diagnostic process for patients suspected of mitochondrial cytopathy. Practical guidelines are presented to provide optimal conditions for performance of laboratory investigations and a reliable diagnosis. A fresh muscle biopsy is preferable to a frozen muscle sample because the overall capacity of the OXPHOS system can be measured in a fresh biopsy. In about 25% of patients referred for muscle biopsy to our centre, reduced substrate oxidation rates and ATP+creatine phosphate production rates were found without any defect in complexes I-V and the pyruvate dehydrogenase complex. Investigation of frozen muscle biopsy alone may lead to false-negative diagnoses in many patients. In some patients, it is necessary to investigate fibroblasts for prospective diagnostic purposes. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counselling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.

Although many patients suspected of suffering disturbances of the mitochondrial energy metabolism have been investigated, only a fraction of these patients have been diagnosed at the molecular level. Introduction of new techniques like proteomics will be necessary to understand the various clinical and biochemical aberrations in the field of mitochondrial disorders. Two-dimensional electrophoresis is the first, important step in the proteomics strategy. Separation of soluble proteins is performed on the basis of isoelectric point (net charge) in one direction and on molecular mass in the other. The technique provides an overview of the majority of proteins expressed in a sample (e.g. muscle biopsy, muscle cell or mitochondrial fraction). Once an abnormal spot is observed in the gel the responsible protein can be identified by analysing a limited part of its amino acid sequence by mass spectrometry. We optimized two-dimensional (2D) gel electrophoresis to obtain high resolution 2D-maps and tested the reproducibility of the technique. Potentially, this new technique is capable of identifying novel mitochondrial diseases and defining their molecular basis.

Proton nuclear magnetic resonance (NMR) spectroscopy of body fluids has been successfully applied to the field of inborn errors of metabolism. This technique has the advantage of minimal sample pretreatment not requiring extraction or derivatization steps. Moreover, the spectrum provides a comprehensive metabolic profile of proton-containing, low-molecular-weight metabolites. The sensitivity limit is in the low micromolar range. This allows diagnosis of many inborn errors of metabolism. This review explains the key features of the NMR spectrum and reviews the available literature on metabolic diseases. Three novel diseases have been delineated with the technique. Relevant parts of the spectra from the urine samples of patients with these diseases are shown. NMR spectroscopy may develop to become a key tool in a metabonomics approach in clinical biochemistry.

Neurodegenerative disorders have traditionally been classified according to clinical criteria, e.g. as dementia syndromes (the best known is Alzheimer's disease) or as movement disorders (e.g. Parkinson's disease). Another subdivision is based on recent insights into the respective pathogenetic mechanisms, leading to the recognition of so-called tauopathies and α-synucleinopathies. It is this increased knowledge of the underlying (neuro)pathological mechanisms that has sparked interest in studies aimed at the identification of disease-specific biomarkers in cerebrospinal fluid (CSF) for this field of neurological disorders. This review deals with the recent progress that has been made in identification, quantification and subsequent validation of brain-specific proteins in CSF for the diagnosis of various neurodegenerative disorders. Development of disease-specific CSF biomarkers will undoubtedly add to the process of differential diagnosis early in the course of the disease.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). A deficiency of DPD is increasingly being recognized as the cause of an important pharmacogenetic syndrome. The importance of DPD deficiency in the aetiology of unexpected severe 5FU toxicity has been demonstrated by the fact that, in 39-;59% of cases, decreased DPD activity could be detected in peripheral blood mononuclear (PBM) cells. It was observed that 55% of the patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of the patients with a normal DPD activity (P = 0·01). Furthermore, toxicity developed significantly earlier in patients with low DPD activity than in patients with normal DPD activity (10·0 ± 7·6 versus 19·1 ± 15·3 days, P < 0·05). In patients suffering from severe 5FU-associated toxicity, 11 mutations have been identified in DPYD, including one splice-site mutation (IVS14 + 1G→A), one nonsense mutation (E386X), four missense mutations (M166V, V335L, I560S, D949V) and five polymorphisms (C29R, R21Q, S534N, I543V, V732I). Considering the common use of 5FU in the treatment of cancer patients, the severe 5FU-related toxicities in patients with a low DPD activity and the high prevalence of the IVS14 + 1G→A mutation, analysis of the DPD activity in PBM cells or screening for the IVS14 + 1G→A mutation should be routinely carried out prior to the start of treatment with 5FU.

Homocysteine, a sulphur amino acid, is a branch-point intermediate of methionine metabolism. It can be degraded in the transsulphuration pathway to cystathionine, or remethylated to methionine via the remethylation pathway. In both pathways, major genetic defects that cause enzyme deficiencies are associated with very high plasma homocysteine concentrations and excretion of homocystine into the urine. Mildly elevated plasma homocysteine concentrations are thought to be an independent and graded risk factor for both arterial occlusive disease and venous thrombosis. Genetic defects in genes encoding enzymes involved in homocysteine metabolism, or depletion of important cofactors or (co)substrates for those enzymes, including folate, vitamin B₁₂ and vitamin B₆, may result in elevated plasma homocysteine concentrations. Plasma homocysteine concentrations are also influenced by dietary and lifestyle factors. In the last decade, several studies have been conducted to elucidate the genetic determinants of hyperhomocysteinaemia in patients with cardiovascular disease. We report on both environmental and genetic determinants of hyperhomocysteinaemia and give a detailed overview of all the genetic determinants that have been reported to date.

Background: It has been suggested that serum cystatin C (cyst-C) concentration provides a better indication of changes in glomerular filtration rate (GFR) than does serum creatinine concentration.

Methods: Because of conflicting results as to the usefulness of cyst-C, we compared the GFRs calculated from serum cyst-C, inulin clearance and endogenous creatinine clearance in children. GFRs calculated from cystatin concentration, inulin clearance following a single injection and endogenous creatinine clearance using Jaffé and enzymic methods were compared in 66 children (1·3-21·9 years) with a variety of renal disorders. Receiver operating curve analysis was used to determine the cut-off value that would give the best discrimination between normal and decreased GFR.

Results: The serum cyst-C concentration ranged from 0·66 to 7·61 mg/L (median 1·94). Serum creatinine Jaffé concentration (creat-J) ranged from 38 to 871 µmol/L (median 105) and creatinine enzymatic concentration (creat-E) ranged from 28 to 862 µmol/L (median 126). The linear correlation coefficient (R) of 1/cyst-C versus GFR (R = 0·937) did not differ from either that of 1/creat-J versus GFR (R = 0·918) or that of 1/creat-E versus GFR (R = 0·901). These coefficients had overlapping confidence intervals. The areas under the curve for cyst-C, creat-J and creat-E were 0·967, 0·977 and 0·924, respectively, and were not significantly different from each other. For cyst-C, the optimal cut-off was 1·1 mg/L.

Conclusions: Serum cyst-C is equivalent to creat-J and creat-E as a marker for estimating the GFR in the paediatric population studied.

Background: The causes of cobalamin (vitamin B₁₂) deficiency in older people are only partly understood. We investigated the role of the cobalamin-binding proteins and tested the hypothesis that low saturated transcobalamin concentration is an early marker of cobalamin deficiency.

Methods: We measured saturated (holo) and unsaturated (apo) transcobalamin and haptocorrin concentrations in healthy middle-aged volunteers, healthy older volunteers, cobalamin-deficient older volunteers and cobalamin-deficient older patients.

Results: Holo and apo concentrations of transcobalamin and haptocorrin were similar in healthy middle-aged and older subjects. Holotranscobalamin concentrations were significantly decreased in cobalamin-deficient subjects but did not differ between healthy volunteers and patients. Furthermore, the relative amount of cobalamin on transcobalamin (i.e. holotranscobalamin/holotranscobalamin + holohaptocorrin) was similar in all four groups.

Conclusions: Abnormalities of the cobalamin-binding proteins are not a cause of cobalamin deficiency in the aged. Plasma holotranscobalamin concentration did not differ between stages of cobalamin deficiency in older persons. Therefore, plasma holotranscobalamin is not an early marker of cobalamin deficiency in older people and has no additional value in the diagnostic work-up of reduced plasma cobalamin concentrations in older people.

Background: Methotrexate (MTX) followed by 6-mercaptopurine (6MP) is one of the best known combinations for the treatment of childhood acute lymphoblastic leukaemia. Tiazofurin (TF) and 6-thioguanine (TG) are also used as chemotherapy agents in the treatment of malignancies. We have examined the induction of apoptosis by combinations of these drugs to gain more insights into their efficacy in the treatment of malignancies.

Methods: The induction of apoptosis was examined in Molt-4, a human malignant acute lymphoblastic T-cell line. The cells were exposed to increasing drug concentrations at various exposure times. Annexin V/FITC and propidium iodide (PI) were used as markers for apoptosis and cell death. Annexin V/FITC positive and PI positive cells were detected by flow-cytometric analysis.

Results: Sequential 24-h exposure with MTX (0·005-0·02 µmol) followed by 6MP (1-10 µmol) and 24-h exposure with TF (5·20 µmol) followed by TG (0·5-2 µmol) showed a more than additive induction of apoptosis compared with single-drug exposure. Simultaneous administration of the drugs does not show an additive effect on apoptosis.

Conclusions: The results of this study indicate that sequential administration of MTX before 6MP and of TF before TG may be essential for therapeutic success in the treatment of leukaemia.

Background: Statistical testing of assay linearity using replicate measurements made on binary mixtures of samples provides no information about the significance of any detected deviations from linearity, which may often be desirable.

Method: Performance of additional statistical analysis of the assay data.

Result: Objective measures for non-linearity and the minimum detectable deviation from linearity of the assay are obtained.

Conclusion: A few additional calculations from the available data make it possible to assess whether or not an assay method needs improvement to achieve adequate performance within its intended application.

Background: Myo-inositol plays a key role in an important intracellular signalling pathway. A deranged myo-inositol metabolism has been associated with neural tube defects. A myo-inositol loading test was performed to investigate the kinetics in healthy women of reproductive age.

Methods: Five healthy non-obese females {mean age (standard deviation: SD) 22·8 (2·2) years} were recruited at the University Medical Center Nijmegen. Blood samples were drawn fasting and at 20, 40, 60, 90, 180 and 270 min after ingestion of 100 mg/kg body weight of myo-inositol. Urine samples were collected before myo-inositol loading and at 180 and 270 min post-loading. Samples were analysed for serum myo-, epi- and scyllo-inositol and glucose concentrations by gas chromatography. Plasma insulin concentrations were determined by radio-immunoassay. Random intercept models were fitted to evaluate the data.

Results: The estimated myo-inositol and scyllo-inositol concentrations both reached maximum values at 180 min post-loading, respectively: mean (SD) 101·5 (9·2) µmol/L and 1·09 (0·11) µmol/L. The estimated plasma insulin and serum glucose concentrations decreased slightly but significantly during the experiment: P < 0·0001 and P < 0·05, respectively. At 180 and 270 min post-loading, urinary myo-inositol concentrations were increased and urinary glucose concentrations were unchanged.

Conclusions: Myo-inositol enters the bloodstream quickly after oral ingestion and a small amount of myo-inositol is converted to scyllo-inositol. The synthesis of glucose from myo-inositol could not be detected by serum measurements. These data can be used in further research into the association between myo-inositol and neural tube defects.

Background: Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity.

Methods: We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate.

Results: The K _m values calculated for 6MP were 0·54 (RBC), 0·85 (pMNC) and 0·65 (Molt-F4 cells) mmol/L. The K _m values for S-adenosylmethionine were 11·9 (RBC), 16·4 (pMNC) and 6·65 (Molt-F4 cells) µmol/L. The assay variation was 8·2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean ± standard deviation): female children and young adults, 15·1 ± 4·8 pmol/10⁷ cells per h (n = 44); male children and young adults, 15·8 ± 6·4 pmol/10⁷ cells per h (n = 59). No gender or age differences were found.

Conclusion: The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.

Background: As recurrences after treatment for differentiated thyroid cancer can occur many years after thyroidectomy, periodic monitoring of serum thyroglobulin (Tg) levels is performed in these patients. However, autoantibodies that can interfere with Tg immunoassays occur in the blood of approximately 25% of these patients. Several earlier reports suggest that measuring Tg mRNA by reverse-transcriptase polymerase chain reaction (RT-PCR) could be of value, especially in patients with Tg autoantibodies.

Methods: Using an earlier described, real-time quantitative Taqman RT-PCR assay, Tg mRNA concentrations were assessed in peripheral blood taken from 58 patients treated for thyroid cancer and from two healthy controls.

Results: In all tested samples Tg mRNA could be found. No correlation between serum Tg protein and Tg mRNA could be found. Tg mRNA concentrations did not differ between serum Tg-negative and Tg-positive patients. No differences in the number of patients with high or low Tg/β-actin ratios were found between the groups of patients without, ¹³¹I uptake on whole-body scan, or patients with thyroid bed uptake, uptake elsewhere in the neck, or distant metastases with or without regional uptake (P = 0·871).

Conclusions: We were not able to confirm earlier positive reports on the clinical value of Tg mRNA measurement for the monitoring of patients treated for thyroid cancer.

Background: Ubiquinol is a sensitive redox marker in the first line of the antioxidative defence mechanism and is increasingly being measured in oxidation studies. Because of its apparent instability during storage and processing, we compared various storage conditions.

Method: Blood was collected from three volunteers into tubes containing EDTA; it was then separated at 4°C and cryopreserved with saccharose (final concentration 6 g/L). Aliquots were stored with or without glutathione or butylated hydroxytoluene at -20°C and -80°C.

Results and conclusion: Ubiquinol in samples stored at -20°C was not stable; however, it was stable when stored at -80°C, even without addition of antioxidant. By contrast, α-tocopherol was stable under all conditions studied.

In a patient with microcephaly, feeding problems and restlessness, moderately increased serum and urine citrulline concentrations were observed. Protein and allopurinol loading did not result in additional indications for a urea cycle defect. The diagnosis of citrullinaemia was made at both the enzyme and DNA level, resulting from a novel mutation in the argininosuccinate synthetase gene. The fact that the patient has not suffered from severe deterioration, and that there were only minor abnormalities in metabolite concentrations, suggests that the argininosuccinate synthetase capacity was less affected in vivo than in vitro. In vitro nuclear magnetic resonance investigation suggested an active acetylation mechanism for citrulline. This case illustrates the importance of performing extensive biochemical and molecular investigations in order to reach a definitive diagnosis, particularly in instances of moderate citrullinaemia.

There is no definitive treatment protocol for alkaptonuria. A patient with alkaptonuria was treated with ascorbic acid (0·5 g/day) from the age of 4 years. He developed episodes of severe recurrent joint pain at 9·5 years of age after which a protein-restricted diet (1·3 g/kg/day) was started. Protein restriction in combination with ascorbic acid therapy (1 g/day in two divided doses) resulted in a significant decrease but not a normalization of the urinary homogentisic acid excretion. Joint pain resolved and the radiological evidence of 'moth-eaten' irregularities on the articular surface in both knees disappeared. He is currently well, growing normally and in nitrogen balance. Our findings document a reversal of bone abnormalities and clinical symptoms in a case of alkaptonuria. The results should be confirmed in a larger study. We suggest that protein restriction should be applied in combination with ascorbic acid in affected patients as soon as joint pain occurs.