Urinary Dolichol Research Articles

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus.

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.

Aberrant dolichol chain lengths as biomarkers for retinitis pigmentosa caused by impaired dolichol biosynthesis

We observed a characteristic shortening of plasma and urinary dolichols in retinitis pigmentosa (RP) patients carrying K42E and T206A mutations in the dehydrodolichol diphosphate synthase (DHDDS) gene, using liquid chromatography-mass spectrometry. Dolichol-18 (D18) became the dominant dolichol species in patients instead of dolichol-19 (D19) in normal individuals. The D18/D19 ratio was calculated and used as an index of dolichol length distribution. K42E/K42E and K42E/T206A patients have significantly higher plasma and urinary D18/D19 ratios than K42E and T206A carriers. The ratios of carriers are significantly higher than normal individuals. Receiver operating characteristic (ROC) analysis shows that plasma and urinary D18/D19 ratios can unambiguously discriminate patients from carriers, and carriers from normal individuals. Dolichol analysis also provides evidence that the T206A mutation is RP-causative. The methodologies and procedures used for dolichol profiling are reliable, high throughput, and cost effective. Dolichol profiling, complementary to genotyping, can be readily adapted as a test in the clinic not only for the diagnosis of patients but also for identification of carriers with DHDDS or other genetic mutations that may impair dolichol biosynthesis.

Hepatic Isoprenoid Metabolism in a Rat Model of Smith‐Lemli‐Opitz Syndrome

Elevated (4 to 7-fold) levels of urinary dolichol and coenzyme Q and substantially longer chain lengths for urinary dolichols have been reported in Smith-Lemli-Opitz Syndrome (SLOS) patients, compared to normal subjects. We investigated the possibility of similar alterations in hepatic, nonsterol isoprenoids in a well-established rat model of SLOS. In this model, the ratio of 7-dehydrocholesterol (7DHC) to cholesterol (Chol) in serum approached 15:1; however, total sterol mass in serum decreased by >80%. Livers from treated rats had 7DHC/Chol ratios of ~32:1, but the steady-state levels of total sterols were >40% those of livers from age-matched (3-month-old) control animals. No significant differences in the levels of LDL receptor or HMG-CoA reductase were observed. The levels of dolichol and coenzyme Q were elevated only modestly (by 64 and 31%, respectively; p<0.05, N=6) in the livers of the SLOS rat model compared to controls; moreover, the chain lengths of these isoprenoids were not different in the two groups. We conclude that hepatic isoprenoid synthesis is marginally elevated in this animal model of SLOS, but without preferential shunting to the nonsterol branches (dolichol and coenzyme Q) of the pathway and without alteration of normal dolichol chain lengths.

Increased nonsterol isoprenoids, dolichol and ubiquinone, in the Smith-Lemli-Opitz syndrome: effects of dietary cholesterol

Smith-Lemli-Opitz syndrome (SLOS) is an inherited autosomal recessive cholesterol deficiency disorder. Our studies have shown that in SLOS children, urinary mevalonate excretion is normal and reflects hepatic HMG-CoA reductase activity but not ultimate sterol synthesis. Hence, we hypothesized that in SLOS there may be increased diversion of mevalonate to nonsterol isoprenoid synthesis. To test our hypothesis, we measured urinary dolichol and ubiquinone, two nonsterol isoprenoids, in 16 children with SLOS and 15 controls, all fed a low-cholesterol diet. The urinary excretion of both dolichol (P < 0.002) and ubiquinone (P < 0.02) in SLOS children was 7-fold higher than in control children, whereas mevalonate excretion was comparable. In a subset of 12 SLOS children, a high-cholesterol diet decreased urinary mevalonate excretion by 61% (P < 0.001), dolichol by 70% (P < 0.001), and ubiquinone by 67% (P < 0.03). Our hypothesis that in SLOS children, normal urinary mevalonate excretion results from increased diversion of mevalonate into the production of nonsterol isoprenoids is supported. Dietary cholesterol supplementation reduced urinary mevalonate and nonsterol isoprenoid excretion but did not change the relative ratios of their excretion. Therefore, in SLOS, a secondary peripheral regulation of isoprenoid synthesis may be stimulated.

Biliary excretion of dolichols and beta-hexosaminidase--effect of ethanol and glucagon.

Alcohol has been reported to increase the urinary excretion of dolichols, and urinary dolichols are suggested to be derived from the lysosomes of the renal cells. In the present study we examined the effects of alcohol and glucagon on the biliary excretion of dolichols in rats. Chronic ethanol treatment decreased both biliary dolichol and beta-hexosaminidase excretion. The absolute amount of dolichol excreted into the bile correlated highly significantly with the absolute amount of biliary beta-hexosaminidase. Our results indicate that biliary dolichols are--at least in part--derived from hepatic lysosomes. Decreased biliary dolichol output during chronic alcohol administration suggests that urinary and biliary dolichol excretions are regulated independently of each other.

Gammaglutarayltransferase, aspartate and alamine aminotransferases and their ratio, mean cell volume and urinary dolichol in pregnant alcohol abusers

Gammaglutarayltransferase, aspartate and alamine aminotransferases and their ratio, mean cell volume and urinary dolichol in pregnant alcohol abusers

Partition of free dolichol in human urine.

We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol.

Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis.

The kinetics of the oligosaccharide transfer from oligosaccharyl pyrophosphoryldolichol to endogenous protein acceptors in human fibroblasts were studied. No alterations in the transferase activity and enzyme characteristics could be observed in fibroblasts from neuronal ceroid-lipofuscinosis (NCL) patients. Analysis of urinary dolichol of two NCL patients also did not reveal substantial differences with respect to controls.

Gamma-glutamyltransferase, aspartate and alanine aminotransferases and their ratio, mean cell volume and urinary dolichol in pregnant alcohol abusers.

To study the activities of serum gamma-glutamyltransferase (GGT), aspartate and alanine aminotransferases (AST, ALT), and their ratio (AST/ALT), mean erythrocyte cell volume (MCV) and urinary dolichol output in alcohol-abusing pregnant women, and compare the results to those of abstinent pregnant women. Prospective descriptive study. Special outpatient clinic for pregnant problem-drinkers in the department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Finland. 25 pregnant women referred to the special clinic at between 12 and 24 weeks gestation, they consumed at least 150 g of ethanol weekly, and a control group of 20 abstinent pregnant women matched for age, parity and smoking habits. The women were encouraged to visit the clinic at 2-4 week intervals. At each visit blood and urine samples were obtained, and the women were interviewed on their alcohol consumption during the previous weeks and encouraged to abstain in the future. Neonatal condition, fetal alcohol effects (FAE) in the newborn. Serum activities of GGT, AST and ALT, the AST/ALT ratio, the MCV, and the urinary concentration of dolichol in alcohol-abusing women with either healthy or FAE infants, compared with those of abstinent women with healthy infants. Of the 25 alcohol-abusing women 13 gave birth to infants with FAE and 12 to healthy infants. All the women in the control group gave birth to healthy infants. GGT, AST and ALT activities were increased in all alcohol-abusing women, regardless whether the infant had FAE or not. GGT was the best of these markers, GGT activities above the 95th normal centile were found in 33% of the samples from all alcohol-abusing women. The AST/ALT ratio, MCV and urinary dolichol concentration were poor indicators of abusive drinking. Maternal alcohol abuse is difficult to assess by laboratory tests. Of the commonly used and easily available tests, GGT proved to be the best in our study.

Strenuous physical activity, aspirin and heat stress increase urinary dolichols: evidence for lysosomal origin of urinary dolichols

Strenuous physical activity, aspirin and heat stress (Finnish sauna) were all found to significantly increase urinary dolichol excretion. In contrast, serum dolichol concentration studied before and after aspirin and sauna, was not affected. A similar aspirin-induced increase, as seen in urinary dolichol concentration, was also observed in the urinary excretion of two lysosomal enzymes — β-hexosaminidase and β-glucuronidase. In contrast, the excretion of two non-lysosomal enzymes — lactate dehydrogenase and leucine aminopeptidase — was not affected. The lack of correlation between serum and urinary dolichols, and the parallel increase in urinary dolichols and the activities of the lysosomal enzymes suggest that urinary dolichols may be derived from the lysosomes of the renal cells. We conclude that the finding of increased urinary dolichol concentrations in some relatively common conditions limits the clinical use of urinary dolichols as a diagnostic tool in neuronal ceroid lipofuscinosis or alcoholism.

Urinary Dolichol—A Doubtful Marker of Alcoholism

The purpose of this study was to replicate the results of Pullarkat and Raguthu and Roine et al. who found elevated levels of urinary dolichol (long-chain 2,3-dihydropolyprenols) in chronic alcoholic patients. We investigated a sample of 21 alcohol-dependent inpatients voluntarily entering detoxification treatment. Urinary dolichol was only slightly increased as compared to 21 healthy controls. When dolichol was related to urinary creatinine no differences between alcoholic patients and controls could be found. Under conditions of confirmed abstinence the slightly elevated levels of dolichol returned to normal within 2 weeks. Compared with the sensitivity of gamma-glutamyltransferase which ranges from 72-85%, the value of urinary dolichol (sensitivity 9-19%) as a biochemical marker of alcoholism must be doubted.

Serum dolichols in different clinical conditions

We studied the effect of seven different clinical diseases (viral infections, bacterial infections, malignant diseases, cardiovascular diseases, gastroenterological diseases, endocrinological diseases, and rheumatic diseases) as well as normal pregnancy on serum dolichol concentrations in 76 hospitalized patients and in 10 pregnant women. In contrast to urinary dolichols, serum dolichols were not significantly increased in any of these conditions, suggesting that dolichol levels in serum and urine are independently regulated. Furthermore, we found that serum dolichol concentration does not undergo diurnal variation, as in healthy volunteers time of blood sampling did not affect serum dolichols. Our results suggest that serum dolichol concentration, which has earlier been found to be exceptionally high in aspartylglucosaminuria and mannosidosis, might serve as a laboratory marker for these recessively inherited lysosomal storage diseases.

Effect of alcohol on blood dolichol concentration.

Serum dolichol levels were studied in 95 active alcoholics and 16 abstinent alcoholics (at the time of blood sampling) and compared to those of 41 social drinkers. Active alcoholics had a significantly higher mean serum dolichol concentration (182.7 +/- 5.1 ng/ml, p less than 0.001 than either abstinent alcoholics (138.7 +/- 5.4 ng/ml) or social drinkers (142.1 +/- 4.1 ng/ml). During weekend (48 hr) heavy drinking (5.5 g of alcohol per kg b.w.) no significant changes were seen in mean serum dolichol concentrations in 10 healthy, nonalcoholic volunteers. Neither did moderate drinking for 10 days (60 g of alcohol daily)--preceded and followed by a period of abstinence--produce any significant changes in serum dolichol levels in 10 nonalcoholic subjects. During detoxification treatment of 12 alcoholics, mean serum dolichol concentration stayed constant for the first 7 days; on entering treatment it was 227.7 +/- 15.0 ng/ml, on the 3rd day 238.5 +/- 15.9 ng/ml, and on the 7th day of treatment 222.6 +/- 18.6 ng/ml. Our results show that as well as increasing urinary dolichol excretion, chronic alcohol abuse also produces increased serum dolichol concentrations. However, contrary to urinary dolichols, serum dolichol levels do not react significantly to heavy drinking lasting for 48 hr in nonalcoholic volunteers. Furthermore in alcoholics increased serum dolichol concentrations do not decrease as rapidly during abstinence as urinary dolichol concentrations do.

Significant increases in urinary dolichol levels in bacterial infections, malignancies and pregnancy but not in other clinical conditions.

The effect of different clinical conditions on urinary dolichols was studied in 219 hospital patients and in 24 pregnant women. Significantly increased urinary dolichol levels were found in patients with severe bacterial infections (mean +/- SEM, 37.5 +/- 8.0 micrograms/mmol creatinine, P less than 0.001), in patients with haematological or metastatic (23.3. +/- 5.1, P less than 0.05) as well as localised (15.4 +/- 1.8, P less than 0.01) malignancies and in pregnant women (22.2 +/- 1.8, P less than 0.001) as compared to healthy controls (6.6 +/- 0.4). These results show that urinary excretion of dolichols may be increased, not only in alcoholics and patients with some rare neurodegenerative storage diseases, but also in patients suffering from various other diseases.

Effects of moderate drinking and alcohol abstinence on urinary dolichol levels

Urinary dolichols were studied during a period of 10 days of moderate drinking (60 g of alcohol daily), preceded and followed by a period of abstinence, in 10 healthy volunteers. No significant changes were observed in dolichol levels during this time. Thus, moderate alcohol consumption does not affect urinary dolichols. Furthermore, to establish the time of abstinence needed for elevated urinary dolichol levels to return to normal, 17 alcoholic patients entering a detoxification unit and with initially increased urinary dolichols, were followed for seven days. Mean urinary dolichol level, which was clearly elevated in the beginning of detoxification, returned to normal by the 5th day of the treatment. Half-life decay for increased urinary dolichols was found to be about 3 days.

Urinary dolichol--a new marker of alcoholism.

Urinary dolichol levels of 31 skid-row alcoholics and 49 alcoholics entering a detoxification unit were compared to those of 51 nonalcoholic controls (social drinkers). The mean urinary dolichol content as related to urinary creatinine was significantly (p less than 0.001) higher in the two groups of alcoholics than in the controls. In this material the sensitivity of increased urinary dolichol in the detection of alcoholism was 68% as compared to 44% sensitivity of serum gamma-glutamyltransferase. The percentage of false-positives in the control group was 3.9%. Urinary dolichol is suggested as a potential tool for the detection and follow-up of alcohol abuse.

Effect of heavy weekend drinking on urinary dolichol levels

The effect of acute heavy alcohol consumption on urinary dolichol levels was studied in 10 healthy volunteers during 2 separate weekends (48 hr). During one of the weekends the subjects received alcohol 5.5 g of per kg body weight in 4 different sessions. The other weekend was otherwise identical, but no alcohol was served. During the weekend when the subjects consumed alcohol, urinary dolichol levels began to rise significantly on the second day of the experiment reaching more than double of the baseline values 42 hours after the beginning of the experiment. During the control weekend, no such increase in urinary dolichol levels was observed. Thus, heavy drinking lasting more than one day is capable of increasing urinary dolichol levels.

Elevated urinary dolichol excretion in the hermansky-pudlak syndrome. Indicator of lysosomal dysfunction

The Hermansky-Pudlak syndrome, a triad of albinism, platelets lacking dense bodies, and storage of ceroid-like material in tissues, occurs approximately once in 2,000 northwestern Puerto Ricans. The manifestations of storage disease are variable and include granulomatous colitis, restrictive lung disease, kidney failure, and cardiomyopathy. The autofluorescent material stored in the Hermansky-Pudlak syndrome is histochemically similar to that stored in neuronal ceroid/lipofuscinosis. The material in neuronal ceroid/lipofuscinosis contains dolichols, which are components of lysosomes, and patients show increased urinary excretion of dolichols. This study of 49 patients with the Hermansky-Pudlak syndrome found that urinary dolichol levels are increased in those patients with evidence of ceroid storage in the kidneys but are not elevated when storage occurs in tissues other than the kidneys. The excretion of ceroid was not influenced by the saturation state of dietary fat. A defect in processing of membranes of lysosomes, melanosomes, and dense bodies may be involved in the syndrome.

Urinary dolichols, alcohol and liver disease

Urinary dolichols, alcohol and liver disease

Elevated urinary dolichol levels in chronic alcoholics.

Urinary dolichol (long chain 2,3-dihydropolyprenol) levels were determined in 23 chronic alcoholics and 15 normal subjects using high performance liquid chromatography. Normal dolichol levels were 4.7 +/- 2.8 ng/ml whereas, in chronic alcoholics, this value was increased to 61.1 +/- 44.6 ng/ml. Stopping the intake of alcohol for a few weeks brought the urinary dolichol gradually to the normal level.