ß-Hydroxybutyrate (BHB) is the main ketone body produced during ketosis and diabetic ketosis. This article demonstrates how quantitatively measuring plasma/serum BHB levels can give a direct indication of blood ketone levels and provide a more accurate method of diagnosing and managing ketosis than via traditional nitroprusside-based urine dipstick testing. Rapid identification of ketosis through BHB testing can improve clinical management and patient care in diabetes.

by Dr Cormac Kilty and Al Blanco

What is Ketosis?
Ketosis occurs when the body begins to break down its stored fats in response to a low supply of energy (glucose) to produce ketone bodies. These water soluble by-products of fatty acid metabolism are then used by the body as alternative energy sources to reduce tissue demand for glucose.

Ketone bodies are always present in the blood and are normally broken down into carbon dioxide and water. However, ketone build-up in the blood (ketonemia) can result from both physiological and pathological causes. Physiological ketosis, leading to a mild to moderate build-up, can result from prolonged exercise, fasting or a high-fat diet. If the cause is pathological, then ultimately the excessive build-up of ketones causes an acid/base imbalance known as ketoacidosis.  Pathological causes of ketosis include: diabetes mellitus, alcoholism, glycogen storage disease, alkalosis, ingestion of isopropyl alcohol, and salicylate poisoning. If not diagnosed and treated, ketoacidosis is potentially fatal.

The ketone bodies produced during ketosis within the liver are ß-Hydroxybutyrate (BHB), acetoacetate (AcAc) and acetone, where BHB is the predominant ketone body (78%) which is metabolized from AcAc [Figure 1]. The ketone body ratio, which is the ratio of BHB to AcAc, is approximately 1:1 in healthy people, but this can rise to nearly 6:1 after prolonged fasting and even 10:1 in cases of acute pathological ketosis [1].

Diabetic Ketoacidosis
Pathological ketosis most commonly arises due to diabetes mellitus (DM), a metabolic disease resulting in chronically high blood sugar. This occurs due to glucose under-utilisation and over-production in response to either: 1) an inability to produce and secrete insulin (Type 1 diabetes), or 2) insulin resistance (Type 2 diabetes) [2].

Diabetic ketoacidosis (DKA) is a life threatening complication of untreated or poorly managed diabetes which is most typically seen in the setting of Type 1 diabetes; in these cases the lack of insulin prevents the body from utilizing glucose for energy. This is because insulin acts on cell receptors to assist with glucose absorption, so in its absence cells are unable to take in, and subsequently metabolize, glucose. When the body senses glucose is not readily available, DKA occurs as fat is broken down instead. Furthermore, blood glucose levels rise (usually higher than 300 mg/dL) due to the over production of glucose by the liver to try to compensate for the problem. However, this additional glucose also cannot be metabolized without insulin [Figure 2] resulting in hyperglycemia. Although more common in patients with Type 1 diabetes, patients with Type 2 diabetes are also at risk of developing DKA during catabolic stress in the setting of trauma, surgery or infection [5]. There is also a subset of patients with Type 2 diabetes who are prone to ketosis.  They present with transient and severe beta cell dysfunction and the clinical course is variable. 

Rapid diagnosis of DKA is essential because a delay in starting insulin treatment is associated with an increase in morbidity and mortality [4]. Before insulin treatment was available, DKA was once the leading cause of death among Type 1 diabetics. Even now there is still a high mortality rate of 5 to 10% in developed countries, and it is the leading cause of death in pediatrics and young adults [5].

Testing for DKA
Like glucose, ketones can be tested or monitored in either urine or blood. Historically, DKA has been identified using a colorimetric semi-quantitative method for detection of ketones in urine. Nitroprusside turns purple in the presence of acetoacetate (Figure 3).  Although it is simple and rapid the dipstick nitroprusside test has several limitations, primarily that it only measures acetoacetate. False positives can result from interference by drugs such as L-Dopa, Captopril and other ACE inhibitors. False negatives can also occur because nitroprusside does not test for the presence of BHB which is the predominant ketone in DKA (>0.27 mmol/L is abnormal). Consequently, tests that only recognise the presence of AcAc will underestimate the total ketone body concentration (6). Furthermore, monitoring AcAc levels by using nitroprusside testing during DKA treatment can be misleading because a patient in DKA converts BHB to AcAc and acetone with insulin treatment. Therefore, nitroprusside tests will have a stronger reaction than prior to treatment, even though ketoacidosis is actually improving. This is because the fall in AcAc lags behind the improvement in ketoacidosis. By monitoring BHB levels instead, clinicians are able to assess the patient’s direct response to DKA treatment and ascertain immediately when ketoacidosis is resolved.

There are several different methods of testing for BHB in blood, plasma or serum, these include gas chromatography and capillary electrophoresis.  Such methodologies are specific, but they are more complex procedures that are not amenable to all hospital laboratory or clinic testing. In addition, the turnaround time can be longer than the one to two minutes of the nitroprusside method.

Rapid β-Hydroxybutyrate measurement
An enzymatic assay is also available for direct quantitative measurement of BHB in blood. This is rapid, has minimal cross reactivity with interfering substances and can be performed on both automated laboratory instrumentation (for plasma/serum), or using whole blood samples on devices at the point of care.  An example of this assay is presented by the β-Hydroxybutyrate LiquiColor® Reagent System (Stanbio, Boerne, TX, USA). Figure 4 details the enzymatic reaction which gives a purple colour proportional to the concentration of BHB; where normal levels are 0 – 0.3 mM/L, ketosis is >0.3 mM/L and possible ketoacidosis is >5 mM/L.

Recent prospective studies have shown that blood BHB enzymatic testing has a far superior specificity in comparison to the nitroprusside urine test [7].  One such study prospectively screened for DKA in emergency department (ED) patients who had a blood sugar of >250 mg/dL, regardless of the reason for the ED visit. Both a urine dipstick and a point of care capillary BHB test were performed, with both tests displaying an acceptable sensitivity of at least 98%. However, the BHB was markedly more specific at 78.6%, in comparison to the urine dipstick (35.1% specificity) [7].  The American Diabetes Association discourages the use of urine nitroprusside testing and instead recommends quantitative serum BHB testing for diagnosing and monitoring ketoacidosis [8]. Furthermore, the Association recommends that blood ketone determinations that rely on the nitroprusside reaction should only be used as an adjunct to diagnose DKA and should not be used to monitor DKA treatment due to the lag in decrease in AcAc after resolution of ketoacidosis. In contrast, specific measurement of BHB in blood can be used for diagnosis and monitoring of DKA [9, 10].

Clinical advantages of BHB
As BHB testing is rapid and more specific than urine nitroprusside testing for ketones; it can be used to identify ketosis in multiple settings. BHB in serum and plasma can be used to clinically diagnose and monitor the disease status and severity of diabetes mellitus, alcoholism, as well as starvation-induced ketosis. It may also have potential application for diagnosing and monitoring glycogen storage disease, high fat/low carbohydrate diets, ingestion of isopropyl alcohol and salicylate poisoning. BHB testing is rapid and more specific than urine nitroprusside testing for ketones since it tests for the main ketone produced during ketosis (78%).

During ketosis, BHB levels increase more than the levels of acetone and acetoacetate, clearly indicating the patient’s trend in metabolic status. Consequently, quantitative, objective BHB results provide a better tool for determining and monitoring ketosis than qualitative nitroprusside testing that detects only 22% of ketones present during ketosis.

BHB testing gives the earliest detection of clinically significant ketosis, enabling clinicians to diagnose DKA with confidence based on quantifiable results. Rapid identification of ketosis through BHB testing can improve clinical management and patient care [4]. As such, early detection could enable shorter triage times and faster treatment of patients which could in turn lead to improved clinical outcomes and Emergency Department efficiency, and decreased turnaround times [11]. Furthermore, unnecessary patient admissions could also be avoided through faster and more accurate patient assessments for ketosis and ketoacidosis, particularly within the Emergency Department, which may also result in ever important cost savings.

Acknowledgement
The authors thank Dr. James H. Nichols, Ph.D., DABCC, FACB, Professor of Pathology, Tufts University School of Medicine and Medical Director for Clinical Chemistry at Baystate Health in Springfield, MA. This manuscript is based on a presentation given by Dr. Nichols at the July, 2012 American Association for Clinical Chemistry meeting in Los Angeles.

References
1. Laffel L. Diabetes/Metabolism Research and Reviews 1999; 15:412-426.
2. Shoback, edited by David G. Gardner, Dolores (2011). Greenspan’s basic & clinical endocrinology (9th ed. ed.). New York: McGraw-Hill Medical. pp. Chapter 17
3. Kitabchi AE, et al. Diabetes Care 2009; 32(7):1335-1343.
4. Singh RK, et al. Diabet Med 1997;14:482-486.
5. Felner E, et al. Pediatrics 2001;108:735-740.
6. Sacks DB, et al.  Diabetes Care 2004:34:e61-e99.
7. Arora S, et al. Diabetes Care. 2011; 34(4):852-4.
8. American Diabetes Association. Diabetes Care 2010; 33 (Suppl 1); S62-69.
9. Sacks DB, et al.  Diabetes Care 2011; 34:1419-1423
10. Savage MW, et al. Diabetic Medicine 2011; 28(5):508-515.
11. Foreback C, Former Director of Clinical Chemistry, Henry Ford Hospital, Detroit, MI, White Paper, Clinical effectiveness of Beta-Hydroxybutryate assays in a clinical decision unit, (1998).

The authors
Dr Cormac Kilty
EKF Diagnostics Holdings plc, UK
Tel. +44 (0)2920 710 570
E-mail: cormackilty@ekfdiagnostics.com

Al Blanco
Stanbio Laboratory (An EKF Diagnostics company), USA
Tel. +1 (0)830 249 0772

The interest in newborn screening for lysosomal storage disorders (LSDs) has increased significantly due to newly developed enzyme replacement therapies, the need for early diagnosis, and advances in technical developments. However, testing for lysosomal storage disorders in newborn screening (NBS) raises many challenges for primary health care and their providers. The high frequency of late-onset mutations makes lysosomal storage disorders a broad health problem beyond childhood, as well as a challenge for diagnosis and therapy.

by Professor David C. Kasper

Clinical Background
Lysosomal storage disorders (LSDs) may be an attractive candidate for newborn screening (NBS). These disorders result in the accumulation of macromolecular substrates that would normally be degraded by enzymes involved in lysosomal metabolism [1]. Although individual LSDs are rare, their combined incidence has been estimated at 1 per 7,700 live births for Caucasians [2]. LSDs have a progressive course, and can present at any age affecting any number of tissues and organ systems [3]. In most cases, treatment is directed toward symptomatic care of secondary complications. The development of novel diagnostic techniques was strengthened by the availability of treatment strategies including enzyme replacement, stem cell transplantation and substrate reduction although limitations of these therapies still exist [4]. Nonetheless, early diagnosis and treatment is essential for optimal treatment thus leading to the support of implementing LSDs to the NBS panel. However, the current experience of nationwide screening for LSDs is still limited.

Laboratory diagnostics
The increased technological capacity implies that expanded NBS programmes can now identify a broader range of conditions where early detection and pre-symptomatic treatment result in clinical benefit. However, the technology for a simultaneous screening of several enzyme activities related to LSDs from more or less one single blood sample was initially complicated, time-consuming and laborious but finally new protocols and technologies are now available that allow a simplified screening procedure. For future implementation of high-throughput LSD assays in routine clinical diagnostics, sample handling and mass spectrometric analysis has to be simplified; specifically, sample pre-treatment, speed of analysis and finally detection must become more integrated [5]. In this context it is also mandatory to achieve high laboratory standards in terms of technical proficiency and reproducibility of results. Hereby, quality control materials provided by the Newborn Screening Quality Assurance Program at the Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) are available [6].

Protocols for analysing lysosomal enzyme activities evolve continuously. In addition to fluorescent methods, using, for example, 4-methylumbelliferone, efforts have been made to use tandem mass spectrometry (MS/MS) particularly for high-throughput analysis in routine newborn screening laboratories. MS/MS procedures were refined and optimised, but the complexity of sample preparation prior to mass spectrometry still remains. Drawbacks of these protocols were the need of liquid-liquid extraction (LLE), solid phase extraction (SPE), and the handling with hazardous organic compounds such as ethyl acetate. Novel aspects such as online multi-dimensional chromatography prior to flow injection analysis facilitate ease-of-use sample introduction and increased speed of analysis. Our research group previously reported the use of TurboFlow (Turbulent Flow Chromatography) for online sample clean-up to remove matrix interferences such as salts, proteins and detergents for the analysis of lysosomal enzyme activities in dried blood spot samples [7]. Subsequently, purified analytes of interest that were removed from potential matrix interferences were transferred from a TurboFlow column to an analytical column for ultra high performance liquid chromatography (UHPLC) separation prior to MS/MS analysis in order to separate enzymatic products from residual substrate. This simplified protocol has recently been evaluated in a comprehensive pilot screening of more than 8,500 newborns to demonstrate the technical feasibility and robustness [8]. Moreover, the incubation time was reduced tremendously from 12–16h to 3h [9]. However, novel buffer systems for the combined incubation of more than 6 or 9 enzymes simultaneously are on the horizon including substrates for mucopolysaccharidosis type II, IVA and VI [10]. These new buffer systems might allow the incubation of several enzymes in one reaction vial, and help to reduce costs for personnel, consumables and reagents. We conclude that multiplex MS/MS screening assays are reliable for nationwide LSD NBS, and for selective metabolite screening in high-risk population.

In our experience, comparing biochemical with genetic data of affected patients, we did not observe any correlation between mutation and lack of enzyme activity measured biochemically by MS/MS, nor could type of mutation be estimated by the level of decreased enzyme activity. However, it is mandatory to confirm biochemically suspected cases by genetic mutation analysis.

The nationwide LSD screening experience
The nationwide screening for LSDs is the beginning of a new category of disorders that will confront us with challenging topics regarding NBS. Currently, routine newborn screening for LSDs has been introduced for Pompe disease in Taiwan and for Krabbe disease in the State of New York, respectively. The Austrian Newborn Screening Center and others, for example in Washington State and Italy, have successfully started pilot studies using multiplexed MS/MS screening assays.

We report the results of a comprehensive pilot screening of ~35,000 newborns for four LSDs using a multiplex MS/MS based assay including genetic mutation analysis [11]. Our results revealed a surprisingly high number of enzyme deficiencies among a predominantly Caucasian population in a Central European country. The results finally confirmed 15 newborns with at least one mutation including diminished lysosomal enzyme activity, demonstrating the high overall incidence of 1 : 2315 among the Austrian population. Frequency, positive predictive value and technical practicability make nationwide NBS for LSDs technical feasible. In our screening, the positive cases contribute predominantly to Fabry disease with an incidence of late-onset Fabry disease of 1 : 4100 among the Austrian population. Fabry disease is found among all ethnic, racial, and demographic groups and is not restricted to a specific ethnic background. Our results are concurrent with those from Spada et al. who reported a high incidence of 1 : 3100 for late-onset and 1 : 37 000 for the classic phenotype [12]. Furthermore, several studies have shown that patients with renal insufficiency, cerebral infarctions, or left ventricular hypertrophy of unknown aetiology might suffer from Fabry disease [13]. We conclude that a putative NBS may be beneficial to identify severe clinical cases and but has the drawbacks of detecting mild forms, late onsets and asymptomatic cases.

Future perspectives
The high incidence of the late-onset phenotypes in Fabry, Gaucher and Pompe disease raises the question when genetic screening for this disease should be undertaken, in the neonatal period or at early maturity. Clearly, early detection, genetic counselling, and therapeutic intervention are beneficial for the classic phenotype but the time of screening for the late-onset variants of Fabry and other treatable diseases may raise concerns. A recent study revealed that long-term treatment led to substantial and sustained clinical benefits; however advanced cardiac and renal disease cannot be reversed later on making early diagnosis crucial. NBS is less controversial for infantile Pompe. In Taiwan, first prospective Pompe screening including the initiation of treatment before onset of obvious symptoms and significant irreversible muscle damage clearly demonstrated the benefit for infants. The central nervous system cannot be treated by enzyme replacement therapies for neuronopathic LSDs like for Gaucher II and Niemann-Pick A, and thus highlights the importance of consented genotyping and phenotype prediction after biochemical first-line screening. Apart the potential clinical benefit for patients, NBS for LSDs can provide reproductive risk information for parents and future adults. This situation is common for screening of metabolic disorders as they are inherited predominately in a recessive manner.
In conclusion, our study shows that Pompe, Gaucher and Fabry are frequent disorders with great public health implications. Even though the American College of Medical Genetics (ACMG) ranked LSDs with low priority in 2006, two LSDs including Pompe and Krabbe were finally nominated for consideration by the federal advisory committee. Currently, three states initiated NBS for LSDs, three other states have passed legislation [14]. LSDs belong to a new category of disorders for which population-based screening assays exist, and new high-throughput screening assays and novel treatment strategies are on the horizon for many others. Challenges of the future may include the implementation of the LSDs in routine NBS, dealing with the identification of late-onset phenotypes, and optimal therapy schemes potentially including cost-intensive enzyme replacement therapies.

References
1. Wenger DA, Coppola S, and Liu SL. Insights into the diagnosis and treatment of lysosomal storage diseases. Arch Neurol 2003; 60(3): 322–328.
2. Ranierri E, et al. Pilot neonatal screening program for lysosomal storage disorders, using lamp-1. Southeast Asian J Trop Med Public Health 1999; 30(Suppl 2): 111–113.
3. Beck M. Variable clinical presentation in lysosomal storage disorders. J Inherit Metab Dis 2001; 24(Suppl 2): 47–51; discussion 45–46.
4. Beck M. Therapy for lysosomal storage disorders. IUBMB Life 2010; 62(1): 33–40.
5. Annesley T, et al. Mass spectrometry in the clinical laboratory: how have we done, and where do we need to be? Clin Chem 2009; 55(6): 1236–1239.
6. De Jesus VR, et al. Development and evaluation of quality control dried blood spot materials in newborn screening for lysosomal storage disorders. Clin Chem 2009; 55(1): 158–64.
7. Kasper DC, et al. The application of multiplexed, multi-dimensional ultra-high-performance liquid chromatography/tandem mass spectrometry to the high-throughput screening of lysosomal storage disorders in newborn dried bloodspots. Rapid Commun Mass Spectrom 2010; 24(7): 986–994.
8. Metz TF, et al. Simplified newborn screening protocol for lysosomal storage disorders. Clin Chem 2011; 57(9): 1286–1294.
9. Mechtler TP, et al. Short-incubation mass spectrometry assay for lysosomal storage disorders in newborn and high-risk population screening. Journal of Chromatography B 2012; in press.
10. Gelb MH, and Scott CR. Screening for three lysosomal storage diseases in a NBS laboratory and the potential to expand to a nine-plex assay. APHL Newborn Screening and Genetics Testing Symposium San Diego, CA, USA; 7–10 November, 2011.
11. Mechtler TP, et al. Neonatal screening for lysosomal storage disorders: feasibility and incidence from a nationwide study in Austria. Lancet 2012; 379(9813): 335–341.
12. Spada M, et al. High incidence of later-onset fabry disease revealed by newborn screening. Am J Hum Genet 2006; 79(1): 31–40.
13. Monserrat L, et al. Prevalence of fabry disease in a cohort of 508 unrelated patients with hypertrophic cardiomyopathy. J Am Coll Cardiol 2007; 50(25): 2399–2403.
14. Zhou H, Fernhoff P, and Vogt RF. Newborn bloodspot screening for lysosomal storage disorders. Journal of Pediatrics 2011; 159: 7–13.

The author
David Kasper, PhD
Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria
e-mail: david.kasper@meduniwien.ac.at

The introduction of infliximab and adalimumab, monoclonal antibodies against TNF-α, to induce and maintain clinical remission in patients with moderate to severe inflammatory bowel disease has generated new perspectives managing these disorders [1]. However, about a third of all CED patients in clinical studies treated with TNF-α antibodies exhibited no primary response (primary treatment failures) and up to 40% of patients, after having exhibited primary therapy response, show a decrease in efficacy with increasing duration of therapy (secondary treatment failures*) and do need multiple dose adjustments to re-induce or maintain clinical response.

by Dr J. Stein

Clinically important factors predicting treatment response include brief disease duration, a predominantly inflammatory disease course and disease involving the colon, non-smoker status and moderate to severe disease activity (overview in Yanai and Hanauer [2]).  At first, the development of antibodies against infliximab in the sense of anti-drug antibodies (ADA) [3] occurring especially in patients undergoing episodic administration of IFX (36–61% of cases) [4] was proposed as the primary cause of this phenomenon. It is now considered increasingly questionable that ADA alone are responsible for therapy failure in patients treated with IFX. In fact, multiple studies have reported IFX trough levels that were either undetectable or very low despite the absence of ADA [5-7], which points to other factors that may influence the pharmacokinetics of these agents. A retrospective analysis of the ACT1 and ACT2 studies found an inverse correlation with serum albumin concentrations [8]. Albumin concentrations < 3 g/dl correlated with a significantly poorer initial response (primary non-responders). Several strategies can be undertaken in cases of loss of response: dose escalation (increasing the dose or shortening the interval), switching to another anti-TNF-α drug, or changing to other immunosuppressive drugs. The decision as to which is the best option for the management of these patients remains largely empirical. Data from studies suggests that measurement of anti-TNF-α trough levels and ADAs could be useful in therapeutic drug monitoring in IBD patients, as part of an individualized therapy. Figures 1a and 1b summarize an algorithm for the management of TNF-α antibody therapy based on currently available data. Methods used to detect anti-TNF-α drug concentrations and ADA concentrations are mainly based on enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) or less frequently EMSA (electrophoretic mobility shift assay). Compared to the more complex RIA- or EMSA- based detection methods, the most commonly used and, as a rule, easily performed enzyme-coupled immunoabsorptive assays (EIA) exhibit some limitations which are important for the timing of measurement: Since anti-TNF-α drugs are able to bind to antibodies to form immune complexes, they cannot be detected by ELISA and their presence can only be ascertained by detectable ADAs regardless of the serum levels of the anti-TNF-a drug. However, when ADAs are negative, it is important to know the levels of the anti-TNF-α drugs: if anti-TNF-α serum levels are undetectable, the result is a true negative, but if anti-TNF-α levels are detectable and ADA levels are negative, the result is considered inconclusive because it might be either a true negative result or a false negative result if the antibodies have bound to the anti-TNF-α drug. Therefore, anti-TNF-α drug concentrations should be determined when the drug levels are expected to be lowest, i.e. just before the next administration of the drug (trough level) and at the same time antibody titres are measured to enable further interpretation. When an EIA is used, the optimum trough level stands at > 4–5 μg/ml [5,8], compared with cut-off values of > 1 μg/ml with RIA [9,10].

_{* Defined as recurrence following initially effective remission maintenance with TNF-α antibodies.}

References
1. Chaparro M, et al. Aliment Pharmacol Ther 2012; 35: 971–986.
2. Yanai H, et al. Am J Gastroenterol. 2011; 106: 685–98.
3. Baert F, et al. N Engl J Med. 2003; 348(7): 601–608.
4. Cassinotti A, et al. Pract Gastroenterol. 2010; 34:11–20.
5. Maser EA, et al. Clin Gastroenterol Hepatol. 2006; 4:1248–1254.
6. St Clair EW, et al. Arthritis Rheum. 2002; 46: 1451–9.
7. Fasanmade AA, et al. Int J Clin Pharmacol Ther. 2010; 48: 297–308.
8. Seow CH, et al. Gut. 2010; 59: 49–54.
9. Steenholdt C, et al. Scand J Gastroenterol. 2011; 46: 310–318.
10. Bendtzen K, et al. Scand J Gastroenterol. 2009; 44: 774–781.

The author
J. Stein, MD, PhD
Crohn Colitis Centre
Frankfurt, Germany