Glycerol Kinase Deficiency with Increased Triglycerides and Weight Gain: Pseudo or Real?

A 20-year-old male was found to have increased triglycerides (TG) (777 mg/dL [8.78 mmol/L], reference interval: <150 mg/dL [<1.7 mmol/L]), with normal total cholesterol (159 mg/dL [4.1 mmol/L], reference interval: <200 mg/dL [<5.2 mmol/L]) and normal glucose (80 mg/dL [4.4 mmol/L], reference interval: 65–99 mg/dL [3.6–5.5 mmol/L]) concentrations. The patient was diagnosed with glycerol kinase deficiency (GKD) and adrenal hypoplasia congenita (AHC) at two-and-a-half years of age during an admission for severe hypoglycemia. He has a contiguous Xp21 microdeletion (Xp21.3-p21.2), a 256 Mb deletion resulting in AHC (due to a nuclear receptor subfamily 0 group B member 1 [NR0B1] gene deletion), GKD (due to a glycerol kinase [GK] gene deletion), and a small 3′ terminal deletion of the dystrophin gene (DMD), the latter of unknown clinical significance. He was followed by pediatric endocrinology and received treatment with daily fludrocortisone (0.1 mg in the morning and 0.05 mg at night) and hydrocortisone (5 mg, 3 times a day), and weekly testosterone (26 mg). He had an 8.5 kg weight gain over the previous year, resulting in a current body mass index (BMI) of 32.2 kg/m2 (approximately 97th percentile; in Class 1 [low-risk] obesity range). A lipid panel was ordered and repeated, which demonstrated markedly increased TG concentrations on both occasions, and decreased high density lipoprotein (HDL) cholesterol on the repeated test (35 mg/dL [0.9 mmol/L], reference interval: >40 mg/dL [>1.0 mmol/L]) (Table 1). Patient laboratory results. Bolded values highlight results outside of the reference intervals. Patient laboratory results. Bolded values highlight results outside of the reference intervals. Visual inspection of the specimen showed clear plasma with slight turbidity (Fig. 1A). The lipemia index (L-index) of the specimen, measured by bichromatic turbidimetry, was 30 (Roche Cobas), which was considered inappropriately low and inconsistent with such a markedly increased TG concentration. Suspecting pseudohypertriglyceridemia due to glycerol interference with the TG assay, the glycerol concentration was measured directly by enzymatic assay (Randox Laboratories), and showed increased glycerol at 736.5 µmol/L (reference interval: 28 to 108 µmol/L). Despite being clearly increased, the concentration of measured glycerol did not fully account for the measured TG. Furthermore, the calculated TG/L-index ratio (log-transformed TG concentration divided by the log-transformed L-index, 0.66) was lower than that previously reported in patients with GKD (0.80) (1). Patient plasma sample and lipoprotein metabolism profile. (A), Plasma sample visualization; (B), lipoprotein metabolism profile (patient Lanes 1 to 3, control Lane 4). Lane 1: whole plasma; Lane 2: plasma d > 1.006 fraction (LDL, HDL); Lane 3: plasma d < 1.006 fraction (chylomicron, VLDL); Lane 4: control. Cholesterol mmol/L = mg/dL × 0.02586; triglycerides mmol/L = md/dL × 0.01129. Color figure available at https://academic.oup.com/clinchem. Due to the presence of GKD and 2 features of metabolic syndrome in this patient (increased BMI, decreased HDL cholesterol), additional specimens were collected for lipoprotein metabolism profile assessment, which included ultracentrifugation, selective precipitation, electrophoresis, and direct measurement of cholesterol and TG in isolated lipoprotein fractions. Cholesterol was measured by a cholesterol ester hydrolase–cholesterol oxidase–peroxidase chromogenic method. TG was measured by hydrolysis to glycerol, followed by phosphorylation (by glycerol kinase) and oxidation (by glycerol phosphate oxidase) to dihydroxyacetone phosphate and hydrogen peroxide; hydrogen peroxide then reacted with 4-aminophenazone and 4-chlorophenol in the presence of peroxidase in a Trinder end point reaction to form 4-(p-benzoquinone-monoimino)-phenazone, which was measured spectrophotometrically at wavelengths (primary/secondary) 700/505 nm. Figure 1 shows that in addition to hyperglycerolemia, true hypertriglyceridemia was also present. Distinct bands for chylomicrons, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and HDL were noted on electrophoresis. Increased VLDL-TG, LDL-TG, and chylomicron-TG indicated the presence of true hypertriglyceridemia components, most consistent with hyperlipoproteinemia type IV (increased VLDL together with a minor chylomicron band in otherwise clear specimens could be due to insufficient fasting or slow clearance of a fatty meal; however, all specimens collected from our patient were fasting, per provider) or type V (increased VLDL and chylomicrons). As shown in Table 1, and in agreement with previous findings, the total cholesterol concentration was within the reference interval, while the HDL cholesterol concentration was decreased. Although there was an increased HDL-TG concentration at 539 mg/dL (6.09 mmol/L), we did not observe a strong HDL electrophoresis band. Since the TG measurement in the HDL fraction was also based on measurement of glyceride after ultracentrifugation (to isolate the HDL component), the increased HDL-TG in the absence of strong HDL electrophoresis bands was consistent with hyperglycerolemia. The following questions will be addressed as part of the discussion of this case: How is TG measured in serum? What are common causes of pseudohypertriglyceridemia? How can TG concentration be determined without the interference from endogenous glycerol? The most commonly used assay for TG measurement is based on the hydrolysis of TG to free fatty acids and glycerol, and the measurement of glycerol by a sequence of enzymatic steps resulting in color formation and spectrophotometric measurement (2). Consequently, conditions presenting with increased concentrations of endogenous or exogenous free glycerol will result in an overestimation of serum TG. As the most common cause of pseudohypertriglyceridemia due to hyperglycerolemia, GKD is a rare X-linked recessive disorder due to pathogenic variants in the GK gene in which the phosphorylation of glycerol to glycerol-3-phosphate is impaired. In GKD, glycerol cannot be used as a substrate for hepatic gluconeogenesis, or be esterified to free fatty acids, resulting in increased serum and urine glycerol concentrations (3). There are 3 existing phenotypes: the complex infantile form, the juvenile form, and the benign or adult form. The juvenile and adult forms are due to isolated GK deficiency caused by pathogenic variants in the GK gene, while the complex infantile form, as seen in our case, is an Xp21 contiguous gene deletion involving the GK and NR0B1 genes, with or without DMD genes (4). Other causes of pseudohypertriglyceridemia include the presence of exogenous glycerol from excessive oral intake of glycerol-containing products (5, 6). It is important to recognize pseudohypertriglyceridemia, especially in isolated GKD patients without other symptoms. It prevents overestimation of cardiovascular risk and exposure of the patient to unnecessary lipid-altering agents, and heightens the awareness of the potential for symptomatic GKD in male offspring (7). Unlike previous case reports in the literature that were clear GKD cases with pseudohypertriglyceridemia, our patient’s clinical presentation and laboratory results suggested coexistent true hypertriglyceridemia which could not be accounted for by interference from glycerol. This case presented a challenge in determining the true TG concentration in the presence of increased glycerol concentration. There are 3 strategies to measure TG without interference from endogenous glycerol: first, by internally “blanking” glycerol; second, by measuring free glycerol directly and subtracting glycerol from total TG; and third, by performing a full lipoprotein profile analysis. In the “blanking” method, the specimen initially reacts with reagents to oxidize and inactivate free glycerol. Then, lipase and additional enzymes are added to hydrolyze TG to glycerol, which undergoes further oxidation to generate chromogenic products that are measured photometrically (8). Unfortunately, commercial glycerol-blanking assays are no longer available in the United States due to low demand from clinical laboratories (Alan Remaley, personal communication, January 29, 2024). In our case, the enzymatically measured glycerol concentration was increased, confirming the presence of hyperglycerolemia in GKD; however, this glycerol concentration only accounted for approximately 10% of total TG (Table 1). Additional testing (data not shown) revealed nonlinearity for the upper range of the assay, which restricted the use of this value to back-calculate the concentration of total TG after subtracting the glycerol component. However, the use of the calculated TG/L-index ratio, and further lipoprotein metabolism profiling, led to the discovery of concurrent hyperlipoproteinemia with a primary increase in VLDL and a small increase in chylomicrons (not enough to raise the L-index significantly or cloud the appearance of the sample). The strongest evidence that glycerol did not account for all of the TG observed in this patient is found in the lipoprotein metabolism profile and gel electrophoresis results (Fig. 1). In lane 3, clear bands were visible for chylomicrons and VLDL, which were also supported by the TG measurements after ultracentrifugation showing 291 mg/dL (3.3 mmol/L) of VLDL-TG and 102 mg/dL (1.2 mmol/L) of chylomicron-TG. This was also supported by the slight turbidity observed in the plasma specimen (Fig. 1). In this case, the hyperglycerolemia alone could not have resulted in these effects on either the sample (turbidity) or lipoprotein metabolism profile/gel electrophoresis results. This data clearly demonstrates that this case represents a mix of “pseudo” and “true” hypertriglyceridemia. Our case is consistent with the observation that hyperglycerolemia and true hypertriglyceridemia can coexist in GKD patients (7, 9) and supports the known role of GKD in altering expression of glucose and lipid metabolism genes (10). It should be noted that the risk of acute pancreatitis increases progressively with serum TG concentrations above 500 mg/dL (5.6 mmol/L). In lieu of having “blanking” TG assays available, it is important that GKD patients with apparently increased TG concentrations undergo full lipoprotein metabolism profiling to provide an accurate assessment of TG concentration and definitively rule out the possibility of true hypertriglyceridemia. Commercially available TG assays are based on the measurement of glycerol and cannot differentiate between endogenous glycerol and glycerol derived by hydrolytic action of lipase on glycerides. GKD is the most commonly reported cause of pseudohypertriglyceridemia due to increased glycerol concentrations in patient blood. Awareness of pseudohypertriglyceridemia due to hyperglycerolemia in GKD is important to prevent iatrogenic effects from the use of TG-lowering drugs. In lieu of unavailable commercial glycerol-blanked TG assays, lipoprotein metabolism profiling is a viable approach to measure TG concentration without interference from endogenous glycerol. Hyperglycerolemia and true hypertriglyceridemia can coexist in GKD patients. The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Jingjing Jiao (Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Paul E. Bernard (Data curation-Equal, Formal analysis-Equal, Investigation-Equal, Writing—original draft-Equal, Writing—review & editing-Equal), Jeffrey Meeusen (Formal analysis-Equal, Investigation-Equal, Methodology-Equal, Writing—review & editing-Equal), Leah Militello (Investigation-Equal, Writing—review & editing-Equal), and Joe El-Khoury (Conceptualization-Equal, Formal analysis-Equal, Investigation-Equal, Project administration-Equal, Resources-Equal, Supervision-Equal, Writing—original draft-Equal, Writing—review & editing-Equal) Upon manuscript submission, all authors completed the author disclosure form. None declared. J.M. El-Khoury disclosed receiving consulting fees from Siemens Healthineers, honoraria from LabRoots and ADLM, research funding from Siemens Healthineers, Bioporto, and IDEXX, and free instrumentation for research purposes from Bruker, and serves on the Boards of ADLM and Clinical Chemistry.