Detection of the Neurotoxin Psychosine in Samples of Peripheral Blood: Application in Diagnostics and Follow-up of Krabbe Disease

To the Editor.–The biochemical diagnosis of Krabbe disease (KD), a lysosomal storage disease with deficiency of galactosylceramidase (GALC), can be carried out by determination of the residual enzymatic activity in a variety of biologic samples, such as fibroblasts, dried blood spots, leukocyte pellets, and even amniotic fluid cells. However, the correlation between the clinical phenotype of affected individuals and the level of GALC activity is difficult to determine. Many late-onset KD cases have GALC activities similar to early onset cases, and even low GALC activity may be found in some asymptomatic individuals.1 This lack of correlation may be relevant at the time of treating and following up patients.Galactosylsphingosine (psychosine) is a neurotoxic sphingolipid degraded by GALC. Predictably, affected individuals show progressive accumulation of psychosine in central and peripheral nerve tissue. The accumulation of this sphingolipid has been proposed to trigger neuropathogenic mechanisms leading to the neurologic phenotype in KD2,3 and, consequently, may be a better endpoint to correlate with clinical phenotype.Blood analyses, including the use of dried blood spots, are routine procedures in neonatology. We examined whether psychosine was present in detectable quantities in the blood of the twitcher mouse, a natural occurring murine model of KD. Blood (approximately 150 µL/animal) was collected at various postnatal ages. Psychosine was analyzed from methanol/chloroform extracts purified on a cation exchanger column. Aliquots from each extract were passed through a Waters (Milford, Massachusetts) XTerra C18 analytical column and then through an Applied Biosystems (Carlsbad, California) PI 4000 triple quadrupole mass spectrometer.4 Results were expressed as mean femtomoles of psychosine per milligram of protein from 3 samples per group. The Figure shows that psychosine is detectable (at low levels) in blood samples from newborn mutants but becomes statistically significant after 10 days of postnatal age. Psychosine levels were essentially at trace levels in all control samples. Blood levels of psychosine reach their maximum at 40 postnatal days. At that age, Twitcher mice develop the full array of neurologic symptoms with ataxic movements, strong twitching and muscle wasting, and eventually, full paralysis and death. Residual GALC activity was measured in triplicate aliquots of each sample (20 µg protein) by incubation with N-lissamine rhodaminyl-6-aminohexanoylgalactosyl ceramide with results expressed as mean nanomoles per hour per milligram of protein from 3 individual samples per group. The GALC activity ranged between 0.059 and 0.294 nmol/h/mg in mutant white blood cell irrespective of age. The GALC activity in wild-type blood cells was approximately 4 nmol/h/mg. No significant correlation was determined between the GALC activity in mutant blood and the clinical phenotype at any age. However, psychosine increases in the mutant blood paralleled the progression of the neurologic disease.To test whether this method may be applied on dried blood spots, blood (50 µL) from 20-day-old mice was spotted and dried onto 1.5- by 1.5-cm Whatman qualitative filter strips (Sigma-Aldrich, St. Louis, Missouri). Dried blood spots were then maintained frozen for about a week before psychosine quantification. Our experience with quantifying psychosine shows that this is a very stable compound (we have successfully quantified psychosine in several decade-old, autopsied, brain materials). Using these filters as the starting material, we were able to statistically identify mutants, which had about a 10-fold increase in psychosine compared with control samples (Figure).These results suggest that measuring circulating blood levels of psychosine in patients with KD may serve as endpoints for correlation with their neurologic phenotype. Further, because human myelination starts during the last trimester of pregnancy, babies with KD are likely to be born with detectable psychosine in their blood, facilitating their identification in dried blood spot samples in reference centers for screening of metabolic, inherited diseases. Likewise, the procedure described herein may be of more rapid application to follow-up levels of correction in cases of KD subjected to hematopoietic replacement.5The authors have no relevant financial interest in the products or companies described in this article.