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In most biological materials, free L-carnitine is present together with short-chain and long-chain carnitine esters. These are differentiated mainly according to their solubility in aqueous solvents. A standardized extraction procedure is therefore essential for reproducible estimations of carnitine content. Assays of L-carnitine are based on the reaction of L-carnitine with acetyl CoA with formation of acetyl L-carnitine and free CoASH, catalysed by carnitine acetyl transferase (EC 2.3.1.7). The two main principles employed to monitor this reaction are a) measurement of the incorporation of radio-labelled acetyl groups derived from acetyl CoA into acetyl carnitine, and b) photometric determination of free CoASH formed in the reaction, using thiol-group colour reagents or an enzymatic reaction. To avoid the background due to thiol-compounds in the sample, we suggest the introduction of an oxidation step with hydrogen peroxide, which is then removed with catalase. Using this method, we have established reference ranges for total acid-soluble L-carnitine and free L-carnitine in serum (men: 44.2-79.3, and 34.8-69.5, women: 28.1-66.4 and 19.3-53.9 mumol/l, resp.), skeletal muscle (adults: 21.0-23.1, and 19.5-35.1, children: 16.1-39.0 and 12.1-25.5 mumol/g non-collagen protein, resp.), and urine. The concentration of long-chain acyl L-carnitine in serum is 2.0-4.0 mumol/l. Carnitine levels in serum, tissues and urine are age-dependent with lower levels in newborn children. The ratio of short-chain acyl carnitine to free carnitine is mainly a reflection of hepatic acetyl CoA production.(ABSTRACT TRUNCATED AT 250 WORDS)

This report deals with two sisters who died with eight, respectively ten weeks under the signs of respiratory failure caused by progressive muscular weakness. Only an elevated cerebrospinal fluid protein was suspicious of an additional disturbance of the central nervous system. Muscle biopsy revealed a vacuolar myopathy. Histochemistry showed lipid storage, increased mitochondrial enzyme activity, and to a lower degree, glycogen accumulation especially in type I muscle fibers. Electron microscopy confirmed elevated lipid content in combination with increased, enlarged and abnormally structured mitochondria. Biochemical studies on muscle biopsy, in comparison with normal children, showed a significant decrease of carnitine content and an increased activity of carnitine palmityltransferase. Retrospectively from a clinical point of view this disease is suggestive of "systemic carnitine deficiency", even if some symptoms (hepatomegaly, cardiomyopathy) were not present and serum- and liver carnitine was not measured because the children died before the diagnosis of muscle carnitine deficiency was confirmed. The clinical picture of these two fatal cases is compared with another observation of muscle caritine deficiency. This child shows only a mild course of muscle disorder, but very similar morphological changes in muscle biopsy. Biochemically, there was a clear decrease in muscular carnitine, while the serum levels were in the normal range. The activity of muscular carnitine palmityltransferase was also normal.

Renal carnitine metabolism was studied in isolated kidney cortex tubules from fed rats. The tubular distribution of free carnitine (C), acid-soluble short chain acylcarnitine (AcC), and total acid-soluble carnitine was measured. The content of the last-mentioned in rat cortical tubule suspensions was 2.85 +/- 0.15 nmol/mg protein, 46% representing AcC. In the absence of metabolic substrates the AcC/C ratio declined from 0.84 to 0.48 during incubation. The administration of 2mM acetoacetate or 2mM 3-hydroxybutyrate caused an increase in AcC by 45% and 51%, respectively. The rise in AcC was paralleled by a decrease in C, resulting in an increase of the tubular AcC/C ratio to 1.69 and 1.85, respectively. In the presence of 1 mM exogenous L-carnitine 35 +/- 6 nmol AcC/(mg protein X h) was formed. The addition of acetoacetate and 3-hydroxybutyrate led to a 3.5 to 3.8-fold rise in AcC formation. Other substrates which are likewise metabolized by proximal tubules were less effective. More than 90% of the formed AcC was recovered in the extracellular fluid. The results suggest that proximal renal tubule cells are the intrarenal site of carnitine acylation and may be involved in the regulation of blood and/or urinary carnitine acylation state.

A 47-year-old male patient suffered from recurrent myalgia, induced by fasting or physical exercise. Later, he developed progressive muscular weakness. Serum levels of creatine phosphokinase (CPK) were elevated to approx. 400 U/1. Muscle biopsies showed lipid storage myopathy and signs of acute fiber necrosis, muscle carnitine was decreased to below 20% of controls, carnitine palmitoyl transferase (CPT) activity was normal. Carnitine was also moderately decreased in a liver biopsy and in plasma. Urine excretion of carnitine was low, no elevation of short-chain dicarboxylic acids could be found. The patient was also found to suffer from a lymphocytic malignant non-Hodgkin lymphoma, a monoclonal immunoglobulin (Ig) G-k in plasma and lymphocytic infiltration of bone marrow were demonstrated. At that time no evidence had been obtained to indicate that these two diseases could be related to each other. Autoantibodies against skeletal muscle could not be demonstrated. Absorption of L-carnitine p.o. was normal, however, plasma levels of carnitine fell again rapidly. Administration of 3 X 2 g L-carnitine per day normalized the patient's plasma carnitine levels and led to an increase of plasma short-chain acylcarnitine and ketone bodies, particularly beta-hydroxybutyrate (B-HOB). However, no significant clinical improvement could be seen. Additional application of prednisone led to normalization of CPK serum levels.

Two newborn female siblings fell ill with apathy, failure of suckling and a generalized progressive muscular hypotonia. Death occured at the age of 7 weeks, obviously caused by impairment of respiratory musculature. Biochemical studies in one child revealed carnitine deficiency especially in skeletal muscle; hepatic encephalopathy was absent. Both children had a generalized hyperaminoaciduria, an unusual finding in primary carnitine deficiency. Besides fatty metamorphosis of the liver, bilateral hydroureters and tubular calcifications of both kidneys, morphological studies showed a generalized lipid storage myopathy which predominated in Type-I-fibres and was accentuated in the muscles of the neck. Enzymehistochemical electron microscopy in longterm frozen muscle demonstrated that cytochrome-c-oxidase activity was absent not only in myopathic but also in most of the morphological unchanged muscle fibres. Only some fibres and endothelial cells displayed normal activity of mitochondria. Biochemically no cytochrome aa3 (cytochrome-c-oxidase) could be found in skeletal muscle; cytochrome b was almost undetectable. --In newborns with fatal lipid storage myopathy and carnitine deficiency it seems necessary to look for additional defects in the respiratory chain. Enzyme histochemical electron microscopy is a sensitive method in identifying cytochrome-c-oxidase even after a 12 months period of storage.