Emergency Management of Insulinoma and Other Causes of Hypoglycemia

Karo syrup can be used by owners at home to help control hypoglycemia.
Figure from www.gotoaid.com.

Hypoglycemia is a medical emergency that may result in seizures and permanent brain damage (1-5). Mild cases of hypoglycemia can quickly become serious and life threatening if the low blood glucose concentration continues to fall.

Untreated, severe, prolonged hypoglycemia can lead to stupor, coma, and even death in some animals.

Emergency Management of Hypoglycemia

Oral glucose administration
Owners who witness a hypoglycemic seizure can be instructed to rub a sugar solution (e.g., Karo syrup or honey) on their pet’s gums. Most animals will respond rapidly. However, owners should be warned not to place their hands directly into the mouth of an animal that is having a seizure and not to pour a sugar solution into the mouth of an unconscious pet (2,3,6).

If the animal responds to intravenous or oral glucose administration, it then should be fed a small, high-protein meal and kept as quiet as possible. Owners who notice a pet is becoming weak may prevent a hypoglycemic seizure by feeding.

Intravenous glucose administration
All patients with serious neurologic signs referable to hypoglycemia should be treated immediately by intravenous administration of a 50% dextrose solution (1-5 ml is given slowly over 10 minutes).  If the animal responds clinically, continuous intravenous administration of fluids with a 5% dextrose solution should be considered  (2,3,6). Some clinicians prefer to dilute the initial dose in 5% dextrose or sterile water to create a 20-25% solution prior to injection and thereby reduce the osmolality of the infused solution.

Regardless of the glucose concentration chosen to be administered in an emergency, it is important to keep in mind that it is not necessary to completely normalize the serum glucose concentration, but rather, to eliminate the clinical signs related to hypoglycemia.

Intravenous glucagon infusion
When glucose is administered intravenously to a patient with insulinoma, the tumor may be stimulated to release massive amounts of insulin, leading to severe hypoglycemia. This may result in a viscous cycle of the patient receiving larger volumes and more frequent dosing of intravenous dextrose even as clinical signs become more severe (2,3,6,7).

In dogs with insulinoma, intravenous glucagon should be considered if hypoglycemia and associated clinical signs cannot be not stabilized with infusions of dextrose alone. Glucagon stimulates hepatic gluconeogensis and glycogenolysis, thereby raising the circulating glucose concentrations.

One milligram of lyophilized glucagon USP should be reconstituted according to package directions and mixed with 1 liter of 0.9% saline solution. This resulting 1.0 µg/ml solution is given at 5-10 ng/kg/minute (2,6,7). The rate of infusion is adjusted, as needed, to maintain the serum glucose at a concentration of 50-100 mg/dl.

When the dog is able to eat and maintain its own blood sugar, and/or other surgical or medical therapy is used to treat the insulinoma, the glucagon infusion may be slowly tapered over 1-2 days as the serum glucose and clinical signs are monitored (6,7).

Complications of Prolonged Hypoglycemia and Its Treatment

Acquired seizure disorder
Prolonged hypoglycemia can cause focal laminar and pseudolaminar necrosis of the cerebral cortex, which can result in an acquired seizure disorder (2-6). Anticonvulsants may be required long-term for some animals recovering from hypoglycemic seizures.

If seizures persist despite the correction of hypoglycemia, cerebral hypoxia and edema may be responsible. Glucocorticoids, mannitol, or both, should be administered to help treat cerebral edema. Diazepam and phenobarbital may be required to control the seizures. However, we should also consider the possibility that a condition other than hypoglycemia may be the cause of the seizures.

Secondary hypokalemia
Uptake of glucose by cells is accompanied by the transport of potassium from the circulation to the intracellular space. This can result in severe hypokalemia in some cases (2,6). Therefore, the serum potassium concentration should be monitored in patients receiving dextrose infusions and animals supplemented with potassium in most cases (e.g., 16 mEq KCl per liter of intravenous fluids). This is particularly important for animals that are unable or refuse to eat.

References:

1. Elie MS, Zerbe CA. Insulinoma in dogs, cats, and ferrets. Compend Contin Educ Vet 1995;17:51-59.
2. Feldman EC, Nelson RW. Canine and Feline Endocrinology and Reproduction. 3rd ed. St Louis: Elsevier Saunders; 2004;616–644.
3. Kintzer PP. Insulinoma and other gastrointestinal tract tumours In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;148-155.
4. Goutal CM, Brugmann BL, Ryan KA. Insulinoma in dogs: a review. J Am Anim Hosp Assoc  2012;48:151-163. 
5. Kraje AC. Hypoglycemia and irreversible neurologic complications in a cat with insulinoma. J Am Vet Med Assoc 2003;223:812-814.
6. Meleo KA, Peterson ME. Treatment of insulinoma in the dog, cat, and ferret In: Bonagura JD,Twedt DC, eds. Kirk's Current Veterinary Therapy, Volume XV. Philadelphia: Saunders Elsevier, 2013 (in press)
7. Fischer JR, Smith SA, Harkin KR. Glucagon constant-rate infusion: a novel strategy for the management of hyperinsulinemic-hypoglycemic crisis in the dog. J Am Anim Hosp Assoc 2000;36:27-32.

Insulinoma in Dogs, Cat, and Ferrets: Confirming the Diagnosis

Insulinoma, or functional beta-cell tumor, originates from the islet cells of the endocrine portion of the pancreas (1,2). Insulinoma has been described most commonly in dogs and ferrets, and less commonly in cats. Although insulinoma cells produce a variety of polypeptides, most animals with insulinoma are examined because of clinical signs related to hyperinsulinism and resultant hypoglycemia.

Signalment
Insulinoma has been reported in dogs ranging from 3-15 years old but is most common in dogs older than 8 years old (3-8). Insulinoma is very common in domestic ferrets, with an age range from 2-7 years (9-11). No sex predilection has been reported in dogs, but male ferrets seem to be affected more commonly than females. Insulinoma appears to be a rare condition in cats, with only five cats having been reported; these cats ranged in age from 12-17 years (12-15).

Clinical Signs
Clinical signs in animals with insulinoma are caused by hyperinsulinism, which leads to hypoglycemia. In response to a low blood glucose concentration, catecholamines, glucagon, cortisol, ACTH, and growth hormone are released.

When there is a drop in blood glucose in clinically normal animals, these hormones (i.e., catecholamines, glucagon, cortisol, and growth hormone), in conjunction with a decrease in circulating insulin, help prevent progressive and potentially dangerously low blood glucose concentration. In animals with insulinoma, insulin is secreted even in the face of hypoglycemia and the increase in the counterregulatory hormones listed above. In these patients, the blood glucose is not stabilized, but continues to fall.

Dogs with insulinoma may be examined because of clinical signs related to neuroglycopenic symptoms produced by glucose deprivation of the central nervous system (e.g., hypoglycemia). Less commonly, some animals show adrenergic symptoms caused by catecholamines such as epinephrine (e.g., nervousness, tachycardia) (1,2,16).

The most common complaint for dogs with insulinoma is seizures (1-8). Other signs include collapse, lethargy, weakness, ataxia, mental dullness, muscle fasciculation, trembling, and nervousness (Table 1). Similar signs have been reported in cats with insulinoma (1,12-15). Peripheral neuropathy in association with insulinoma and hypoglycemia has been rarely reported in dogs with insulinoma (17,18).

Table 1: Clinical signs associated with insulinoma in dogs, cats, or ferrets (from reference 2).

Ferrets with insulinoma also commonly show signs of weakness and lethargy (9-11). As in dogs, these symptoms may be episodic. However, seizures are relatively uncommon in this species. Ptyalism is a clinical sign associated with insulinoma in ferrets that has not been described in dogs. The cause of this sign is not known, but ptyalism in ferrets may indicate nausea.

Confirming the Diagnosis— Fulfilling Whipple's Triad
A complete history may lead the clinician to suspect that a patient’s presenting clinical signs are related to hypoglycemia, and thus consider insulinoma as a differential diagnosis. A plasma glucose concentration of 40 mg/dl (2 mmol/L) or less supports the conclusion that the signs are caused by hypoglycemia (16).

If administration of glucose relieves the clinical signs of weakness, disorientation, seizures or trembling, we can conclude that these symptoms are caused by hypoglycemia. This full fills Whipple's triad (i.e, signs of hypoglycemia, biochemical confirmation of low blood glucose at time of clinical signs, and relief of clinical signs after glucose administration) (19,20). This positive response may be seen in animals with hypoglycemia for any reason, however, and is not diagnostic of insulinoma.

Other Causes of Hypoglycemia
In addition to insulinoma, there are many other possible causes of hypoglycemia in animals (1,2,16,21) (Table 2).

Table 2: Causes of hypoglycemia in the mature animal

Many of these differential diagnoses can be ruled out quickly during the initial history and physical examination. After consideration of these diseases is eliminated, insulinoma should be seriously considered in a mature patient with clinical signs of hypoglycemia.

Confirming the Diagnosis—Documenting Hyperinsulinemia
Hyperinsulinism is best diagnosed by the interpretation of serum insulin and glucose concentrations obtained from the patient at the same time. If the clinician suspects hyperinsulinism at the time of initial examination of an animal showing signs of hypoglycemia, serum samples for glucose and insulin measurements are best obtained at that time.

If attempts are made to document hyperinsulinism at a later date, blood samples should be obtained after fasting when the glucose is less than 50 mg/dl (<3.0 mmol/L). It is essential that patients suspected of having hyperinsulinism fast under supervision to allow intervention should signs of hypoglycemia occur.

A high insulin concentration in any animal with concurrent hypoglycemia is consistent with hyperinsulinism (1,2,16,22). If a hypoglycemic patient has an insulin concentration that is within the reference range, the animal again should fast, and the test should be repeated when two consecutive serum glucose readings of 50 mg/dl or less are obtained. If the patient is consistently hypoglycemic, an insulin level within the normal range is considered inappropriate and the patient likely has hyperinsulinism.

Identifying the Pancreatic Nodule
Whenever possible, abdominal ultrasound should be performed in dogs and cats with suspected insulinoma. It can be difficult to detect small pancreatic nodules via ultrasound, but it may be helpful in identifying abdominal metastases (2,22,23). In all species, abdominal ultrasonography may help rule out other neoplasms as a cause of hypoglycemia.

Computed tomography (CT) can also be used to accurately identify pancreatic nodules, and this procedure may be helpful in surgical planning (23).

Bottom Line

Although an accurate diagnosis of insulinoma can generally be made by clinical pathologic testing, histologic examination is required for a definitive diagnosis. Exploratory celiotomy is recommended in all patients with insulinoma if the owner wishes to pursue treatment, but long-term medical management can be helpful in many of these animals.

In my next post, I'll be discussing emergency management of hypoglycemia associated with insulinoma.  This is a critical issue—  if we can't control the immediate clinical signs of hypoglycemia, we will never be able to proceed to definitive or long-term treatment of this serious disorder.

References:

1. Elie MS, Zerbe CA. Insulinoma in dogs, cats, and ferrets. Compend Contin Educ Vet 1995;17:51-59.
2. Kintzer PP. Insulinoma and other gastrointestinal tract tumours In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;148-155.
3. Kruth SA, Feldman EC, Kennedy PC. Insulin-secreting islet cell tumors: establishing a diagnosis and the clinical course for 25 dogs. J Am Vet Med Assoc 1982;181:54-58. 
4. Leifer CE, Peterson ME, Matus RE. Insulin-secreting tumor: diagnosis and medical and surgical management in 55 dogs. J Am Vet Med Assoc 1986;188:60-64. 
5. Schrauwen E. Clinical peripheral polyneuropathy associated with canine insulinoma. Vet Rec 1991;128:211-212.
6. Trifonidou MA, Kirpensteijn J, Robben JH. A retrospective evaluation of 51 dogs with insulinoma. Vet Q 1998;20 Suppl 1:S114-115. 
7. Madarame H, Kayanuma H, Shida T, et al. Retrospective study of canine insulinomas: eight cases (2005-2008). J Vet Med Sci 2009;71:905-911. 
8. Goutal CM, Brugmann BL, Ryan KA. Insulinoma in dogs: a review. J Am Anim Hosp Assoc  2012;48:151-163. 
9. Caplan ER, Peterson ME, Mullen HS, et al. Diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: 57 cases (1986-1994). J Am Vet Med Assoc 1996;209:1741-1745.
10. Ehrhart N, Withrow SJ, Ehrhart EJ, et al. Pancreatic beta cell tumor in ferrets: 20 cases (1986-1994). J Am Vet Med Assoc 1996;209:1737-1740.
11. Weiss CA, Williams BH, Scott MV. Insulinoma in the ferret: clinical findings and treatment comparison of 66 cases.  J Am Anim Hosp Assoc 1998;34:471-475.
12. McMillan FD, Feldman EC. Functional pancreatic islet cell tumor in a cat. J Am Anim Hosp Assoc 1985;21:741-746.
13. Hawks D, Peterson ME, Hawkins KL, et al. Insulin-secreting pancreatic (islet cell) carcinoma in a cat. J Vet Intern Med 1992;6:193-196.
14. Kraje AC. Hypoglycemia and irreversible neurologic complications in a cat with insulinoma. J Am Vet Med Assoc 2003;223:812-814.
15. Greene SN, Bright RM. Insulinoma in a cat. J Small Anim Pract 2008;49:38-40. 
16. Schoeman JP. Investigation of hypoglycaemia In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;259-264.
17. Schrauwen E, Van Ham L, Desmidt M, et al. Peripheral polyneuropathy associated with insulinoma in the dog: Clinical, pathological, and electrodiagnostic features. Prog Vet Neurol 1996;7:16-19.
18. Braund KG, Steiss JE, Amling KA, et al. Insulinoma and subclinical peripheral neuropathy in two dogs. J Vet Intern Med 1987;1:86-90. 
19. Ariamkina OL, Doroshenko GM, Petrenko LV. On diagnostic value of Whipple's triad: a case of insulinoma diagnosis. Klin Med (Mosk) 1997;75:61-63. 
20. Hirshberg B, Livi A, Bartlett DL, et al. Forty-eight-hour fast: the diagnostic test for insulinoma. J Clin Endo Metab 2000;85:3222-3226. 
21. Murphy LA, Coleman AE. Xylitol toxicosis in dogs. Vet Clin North Am Small Anim Pract 2012;42:307-312. 
22. Goutal CM, Brugmann BL, Ryan KA. Insulinoma in dogs: a review. J Am Anim Hosp Assoc 2012;48:151-163. 
23. Robben JH, Pollak YW, Kirpensteijn J, et al. Comparison of ultrasonography, computed tomography, and single-photon emission computed tomography for the detection and localization of canine insulinoma. J Vet Intern Med 2005;19:15-22. 

Top Endocrine Publications of 2012: Insulinoma and Other Causes of Hypoglycemia

Insulinoma in a dog, showing up a solidary pancreatic nodule

In my fourth compilation of the canine and feline endocrine publications of 2012, I’m going back to disorders of the pancreas. But now let’s move on to islet cell tumors (e.g., insulinoma) and other causes of hypoglycemia.

Listed below are 13 research papers written in 2012 that review endocrine hypoglycemia in dogs and cats. These include new aspects in pathogenesis of hypoglycemia associated with insulin-secreting tumors of the pancreas (1,3,5), portosystemic shunts (2), or hypoadrenocorticism (4,8).

Other papers review the hypoglycemia and liver toxicosis associated with xylitol toxicosis in dogs (7,10), as well as the hypoglycemia associated with insulin treatment of diabetes mellitus (6,11-13).

2012 Papers on Canine and Feline Islet Cell Tumors of the Pancreas and Other Causes for Hypoglycemia:

1. Buishand FO, van Erp MG, Groenveld HA, et al. Expression of insulin-like growth factor-1 by canine insulinomas and their metastases. Vet J 2012;191:334-340. 
2. Collings AJ, Gow AG, Marques A, et al. A prospective study of basal insulin concentrations in dogs with congenital portosystemic shunts. J Small Anim Pract 2012;53:228-233. 
3. Goutal CM, Brugmann BL, Ryan KA. Insulinoma in dogs: a review. J Am Anim Hosp Assoc 2012;48:151-163. 
4. Gow AG, Gow DJ, Bell R, et al. Insulin concentrations in dogs with hypoadrenocorticism. Res Vet Sci 2012;93:97-99. 
5. Hambrook LE, Kudnig ST. Tumor thrombus formation in two dogs with insulinomas. J Am Vet Med Assoc 2012;241:1065-1069. 
6. Hugler S. Diabetic alert dogs: A good nose for hypoglycemia. Dtsch Med Wochenschr 2012;137:p25. 
7. Imai A, Nishita T, Ichihara N, et al. Binding affinity of anti-xylitol antibodies to canine hepatic vessels. Vet Immunol Immunopathol 2012;149:108-111. 
8. Kasabalis D, Bodina E, Saridomichelakis MN. Severe hypoglycemia in a cat with primary hypoadrenocorticism. J Feline Med Surg 2012;14:755-758. 
9. Mazaki-Tovi M, Segev G, Yas-Natan E, et al. Serum gastrin concentrations in dogs with liver disorders. Vet Rec 2012;171:19. 
10. Murphy LA, Coleman AE. Xylitol toxicosis in dogs. Vet Clin North Am Small Anim Pract 2012;42:307-312. 
11. Niessen SJ. Glucagon: are we missing a (life-saving) trick? J Vet Emerg Crit Care (San Antonio) 2012;22:523-525. 
12. Niessen SJ, Powney S, Guitian J, et al. Evaluation of a quality-of-life tool for dogs with diabetes mellitus. J Vet Intern Med 2012;26:953-961. 
13. Zeugswetter FK, Schornsteiner E, Haimel G, et al. Metabolic and hormonal responses to subcutaneous glucagon in healthy beagles. J Vet Emerg Crit Care (San Antonio) 2012;22:558-563. (See my blog post entitled "Use of a Glucagon Emergency Kit for Insulin-Induced Hypoglycemia" to see my review of this paper.)

What's the Best Insulin for Diabetic Cats and Dogs?

With the reintroduction of Vetsulin (porcine insulin zinc suspension) to the U.S. market (1-3), I've received a number of questions concerning the use of this insulin in dogs and cats. Here are the major questions I've received:

• Is this the initial insulin of choice for dogs? 
• How about for cats? Should this be an insulin that we turn to for our newly diagnosed feline diabetics?  
• If we have a dog or cat that isn't responding well to the current insulin, should they be switched to Vetsulin?
• How will this insulin compare to NPH, glargine (Lantus), detemir (Levemir), or PZI (ProZinc) in cats or dogs?  How does the cost compare to these other insulin preparations?

Is this the initial insulin of choice for dogs?
In my opinion, the answer to that is yes, this is the initial insulin of choice for most dogs. Because it's composed of both short and long-acting insulin components, Vetsulin helps control postprandial hyperglycemia and has a longer duration of action than NPH in most dogs (4-7). The cost is approximately the same as NPH, but less than the insulin analogues (e.g., detemir and glargine). See more below, where I do my cost-comparisons for the various insulin preparations.

Is this the initial insulin of choice for cats?
A number of studies have proven that Vetsulin will certainly control hyperglycemia in cats, especially if combined with a low carbohydrate diet (<10% of calories) (8-11). However, the duration of action may be too short in some diabetic cats, and most feel that the longer acting insulin preparations (glargine, detemir or ProZinc) work better to maintain better control of hyperglycemia in cats (11-13).

Of course, maintaining glycemic control throughout the day is more important if diabetic remission is the goal (13,15,16)— if not, Vetsulin might be less expensive and certainly would be more than adequate in most cats (8-10).

If we have a dog or cat that isn't responding well to the current insulin, should they be switched to Vetsulin?
For diabetic dogs not regulated on NPH, the answer is a definite "yes." For cats that are on NPH, Vetsulin would certainly be a better choice, but switching to one of the  longer-acting insulin preparations (insulin glargine, detemir, or PZI) would be a better choice.

How will this insulin compare to NPH, detemir, glargine, or PZI in cats or dogs?  
In dogs, I generally use 3 insulin preparations: Vetsulin, NPH, and insulin detemir. Again, the main problem with NPH insulin in dogs is that the duration of action is too short, and many of these dogs will respond better to Vetsulin. Insulin detemir is the most potent insulin we have for use in dogs, with a longer duration of action (14), but it's much more expensive than either NPH or Vetsulin.

In cats, I never use NPH because of it's short duration, but I will sometimes use Vetsulin as an intermediate-acting insulin.  Most authorities would rank insulin glargine as the first choice of insulin in cats, then insulin detemir or PZI (not the compounded product (17), but FDA-approved ProZinc), then Vetsulin, then finally NPH as a very last choice.

How does the cost of Vetsulin compare to these other insulin preparations?
NPH insulin, insulin glargine, and insulin detemir are all available as 10-mL vials at a U-100 insulin concentration. The retail prices of an individual vial of NPH insulin, insulin glargine, and insulin detemir vary, depending on the pharmacy and its location. I checked out the prices today at my local Walmart, CVS, and Rite Aid pharmacies, and here are the range of prices I was quoted:

• NPH —$24.88 (Walmart's ReliOn brand) to $100.39 (CVS) and $107.99 (Rite Aid)!
• Glargine — $152.84 (Walmart) to $169.99 (CVS) and $188.99 (Rite Aid)
• Detemir — $160.32 (Walmart) to $190.99 (CVS)

Vetsulin (porcine insulin zinc suspension) is available as a 10-mL vial of insulin in a U-40 insulin concentration. The wholesale cost of the updated Vetsulin product is inexpensive ($23.97 per vial), similar to the original product. In most veterinary practices, the retail price of a vial of Vetsulin will be approximately $50.

Therefore, the cost of a bottle of Vetsulin is similar or even less than the price of NPH insulin and costs much less (about 25-30%) than that of insulin glargine or detemir.  But remember, the total amount of insulin in a vial of NPH, glargine, and detemir (all U-100 insulins) is 1000 units, where a vial of Vetsulin (a U-40 insulin) contains 400 units, only 40% as much. So in the end, the cost per unit of most of these insulins preparations woud generally turn out to be similar.

Therefore, the cost of all of these insulin preparations would be similar in most diabetic cats and smaller dogs receiving a typical daily dose of insulin (0.5-0.7 U/kg). Because the concentration of Vetsulin is 40 U/mL, owners of larger diabetic dogs, owner will often go through a bottle within a few days. In these dogs, if insulin resistance is present, it's sometimes cheaper to use a U-100 insulin, such as detemir.

However, the bottom line is clear: it doesn't matter how much money we are saving if the insulin isn't working. Instead, for most pet owners, it's more important use an insulin preparation that best controls the diabetic state. And for most diabetic dogs, that insulin would be Vetsulin, followed by insulin detemir.

References:

1. Peterson ME. Vetsulin Insulin Updated and Approved for Release in USA. Insights into Veterinary Endocrinology. April 17, 2013.
2. Vetsulin website. www.vetsulin.com
3. Vetsulin website: Veterinary Product Updates. www.vetsulin.com/vet/Product_Update.aspx
4. Monroe WE, Laxton D, Fallin EA, et al. Efficacy and safety of a purified porcine insulin zinc suspension for managing diabetes mellitus in dogs. J Vet Intern Med 2005;19:675-682. 
5. Fleeman LM, Rand JS, Morton JM. Pharmacokinetics and pharmacodynamics of porcine insulin zinc suspension in eight diabetic dogs. Vet Rec 2009;164:232-237. 
6. Nelson RW. Canine diabetes mellitus In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Seventh Edition ed. St. Louis: Saunders Elsevier, 2010;1449-1474.
7. Davison LJ. Canine diabetes mellitus In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;116-132.
8. Martin GJ, Rand JS. Pharmacology of a 40 IU/ml porcine lente insulin preparation in diabetic cats: findings during the first week and after 5 or 9 weeks of therapy. J Feline Med Surg 2001;3:23-30. 
9. Martin GJ, Rand JS. Control of diabetes mellitus in cats with porcine insulin zinc suspension. Vet Rec 2007;161:88-94. 
10. Michiels L, Reusch CE, Boari A, et al. Treatment of 46 cats with porcine lente insulin—a prospective, multicentre study. J Feline Med Surg 2008;10:439-451. 
11. Rand JS. Feline diabetes mellitus In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;133-147.
12. Marshall RD, Rand JS, Morton JM. Glargine and protamine zinc insulin have a longer duration of action and result in lower mean daily glucose concentrations than lente insulin in healthy cats. J Vet Pharmacol Ther 2008;31:205-212.
13. Marshall RD, Rand JS, Morton JM. Treatment of newly diagnosed diabetic cats with glargine insulin improves glycaemic control and results in higher probability of remission than protamine zinc and lente insulins. J Feline Med Surg 2009;11:683-691. 
14. Sako T, Mori A, Lee P, et al. Time-action profiles of insulin detemir in normal and diabetic dogs. Res Vet Sci 2011;90:396-403.
15. Roomp K, Rand J. Intensive blood glucose control is safe and effective in diabetic cats using home monitoring and treatment with glargine. J Feline Med Surg 2009;11:668-682.
16. Roomp K, Rand J. Evaluation of detemir in diabetic cats managed with a protocol for intensive blood glucose control. J Feline Med Surg 2012;14:566-572.
17. Scott-Moncrieff JC, Moore GE, Coe J, et al. Characteristics of commercially manufactured and compounded protamine zinc insulin. J Am Vet Med Assoc 2012;240:600-605. 
18. Drugstore.com. www.drugstore.com. (accessed 2006 Oct 3).
19. http://www.relion.com/diabetes/insulin

Predicting Which Dogs will Develop Hypocalcemia after Parathyroidectomy for Primary Hyperparathyroidism

Evaluation of preoperative serum concentrations of ionized calcium and parathyroid hormone as predictors of hypocalcemia following parathyroidectomy in dogs with primary hyperparathyroidism: 17 cases (2001-2009)

Melissa Arbaugh, Daniel Smeak, and Eric Monnet

Journal of the American Veterinary Medical Association  2012;24:233-236

In dogs, primary hyperparathyroidism causes hypercalemia through the excessive secretion of parathyroid hormone (PTH), usually by an adenoma affecting one of the parathyroid glands (1-6). Such autonomous hypersecretion of PTH by the parathyroid tumor may lead to disuse atrophy of the remaining parathyroid glands (7).

Removal or ablation of the affected parathyroid tumor(s) is the treatment of choice for dogs with primary hyperparathyroid (1-6,8). Following surgery, these dogs may develop transient hypocalcemia while the suppressed parathyroid glands regain appropriate function. If hypocalcemia occurs, it generally does so within the first 7 days following surgery, and affected dogs often require extensive hospitalization and monitoring of serum ionized calcium concentrations (1-6,8).

It has been suggested that hyperparathyroid dogs with more severe hypercalcemia (total serum calcium concentrations > 14 mg/dL) should be started on supplementation with oral calcium and vitamin D prior to surgery (4,5). These dogs with more severe hypercalcemia may be at a greater risk for developing iatrogenic hypocalcemia after parathyroidectomy, since it is likely that their severe hypercalcemia would result in atrophy of the remaining parathyroid glands. These recommendations, however, are not based on clinical studies. If it turns out that theses dog did not actually require the calcium and vitamin D supplementation to prevent clinical hypocalcemia, then these supplements become unnecessary treatments that may delay recovery of the suppressed parathyroid glands to normal function.

Currently, there are no published predictors to indicate which hyperparathyroid dogs will become hypocalcemic and require calcium supplementation following parathyroidectomy. The purpose of the study reported by Arbaugh, et al (9) was to evaluate predictors of postoperative hypocalcemia in dogs following parathyroidectomy. They hypothesized that both the absolute preoperative calcium concentrations and its rate of decrease would aid in predicting clinical hypocalcemia.

Objective—To determine whether preoperative serum ionized calcium (iCa) or parathyroid hormone (PTH) concentrations help predict postoperative hypocalcemia following parathyroidectomy in dogs with primary hyperparathyroidism.

Design—Retrospective case series.

Animals— 17 dogs with primary hyperparathyroidism treated with parathyroidectomy.

Procedures—Medical records were evaluated from years 2001 to 2009. Data evaluated included age, breed, sex, clinical signs, diagnostic tests performed, preoperative and postoperative iCa concentrations, preoperative PTH concentrations, and whether calcium supplementation was provided following surgery. Two groups were identified on the basis of whether dogs became hypocalcemic (iCa < 1.2 mmol/L) following parathyroidectomy.

Results—12 dogs developed hypocalcemia after surgery. Preoperative (within 24 hours before surgery) iCa concentrations for the hypocalcemic group (mean ± SD, 1.82 ± 0.22 mmol/L) and the nonhypocalcemic group (1.83 ± 0.29 mmol/L) were not significantly different.

Calcium concentrations decreased in a linear fashion during the 24 hours following parathyroidectomy, and the slopes of the decrease over that time were not significantly different between the 2 groups of dogs. Preoperative PTH concentrations were not significantly different between the hypocalcemic and nonhypocalcemic groups.

Conclusions and Clinical Relevance —Preoperative iCa and PTH concentrations were not predictive of postoperative hypocalcemia in dogs undergoing parathyroidectomy for primary hyperparathyroidism. Future studies to evaluate whether calcium supplementation should be provided on an individual basis with perhaps more emphasis on clinical signs than iCa concentrations after surgery may be warranted.

My Bottom Line

Can preoperative calcium or PTH predict postoperative hypocalcemia?
The results of the this study by Arbaugh (9) indicate that preoperative serum concentrations of iCa or PTH concentrations are not reliable predictors of postoperative hypocalcemia in dogs undergoing parathyroidectomy for primary hyperparathyroidism.

These results agree with studies reported in human patients with  primary hyperparathyroidism, in which no difference could be detected in the preoperative serum values for calcium or PTH and the development of postoperative hypocalcemia (10). In addition, in a recent study by Milovancev and Schmiedt (11) of 62 dogs that had parathyroidectomy for primary hyperparathyroidism, these investigators arrived at the same conclusion. No correlation existed between preoperative serum total and ionized calcium concentrations and the development of postoperative hypocalcemia in these 62 dogs (11).

The hypothesis that dogs with higher preoperative serum concentrations of calcium would have more severe postoperative hypocalcemia versus dogs with lower preoperative serum concentrations of calcium would appear on the surface to be a logical one.  However, since this hypothesis is not supported by any these research studies (9-11), it must, therefore, be seriously questioned.

How often does postoperative hypocalcemia develop?
In the present study by Arbaugh (9), only 4 (24%) of the 17 dogs developed postoperative hypocalcemia, but none of the dogs developed clinical signs of hypocalcemia, even those with low iCa concentrations. Similarly, in the recent study reported by Milovancev and Schmiedt (11), approximately one-third of their 62 dogs became hypocalcemic following parathyroidectomy, but only a quarter of those dogs developed associated clinical signs. Of all 62 dogs in that study, less than 10% developed clinical hypocalcemia and required treatment with calcium and vitamin D (11).

When does postoperative hypocalcemia require treatment?
Overall, these two studies indicate an important fact about the need for treatment— that is, although postoperative hypocalcemia is a common complication in dogs after surgical parathyroidectomy, associated life-threatening clinical signs (e.g., tetany, seizures) are very uncommon (9,11).

These findings suggest that dogs treated with parathyroidectomy should be supplemented with calcium and vitamin D only when they have clinical signs of hypocalcemia, rather than started on these medications prophylactically or based solely on the postoperative iCa concentration.

References:

1. Berger B, Feldman EC. Primary hyperparathyroidism in dogs: 21 cases (1976-1986). J Am Vet Med Assoc 1987;191:350-356. 
2. Bonczynski J. Primary hyperparathyroidism in dogs and cats. Clin Tech Small Anim Pract 2007;22:70-74. 
3. Feldman EC, Hoar B, Pollard R, et al. Pretreatment clinical and laboratory findings in dogs with primary hyperparathyroidism: 210 cases (1987-2004). J Am Vet Med Assoc 2005;227:756-761.  
4. Gear RN, Neiger R, Skelly BJ, et al. Primary hyperparathyroidism in 29 dogs: diagnosis, treatment, outcome and associated renal failure. J Small Anim Pract 2005;46:10-16. 
5. Feldman EC, Nelson RW. Hypercalcemia and primary hyperparathyroidism. Canine and Feline Endocrinology and Reproduction. 3rd ed. St. Louis: Saunders Elsevier, 2004;660–715.
6. Skelly BJ. Hyperparathyroidism In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;43-55.
7. Thiele J, Kärner J, Fischer R. Ultrastructural morphometry on human parathyroid tissue. Morphological and functional implications. J Submicrosc Cytol Pathol. 1988;20:491-500. 
8. Rasor L, Pollard R, Feldman EC. Retrospective evaluation of three treatment methods for primary hyperparathyroidism in dogs. J Am Anim Hosp Assoc 2007;43:70-77. 
9. Arbaugh M, Smeak D, Monnet E. Evaluation of preoperative serum concentrations of ionized calcium and parathyroid hormone as predictors of hypocalcemia following parathyroidectomy in dogs with primary hyperparathyroidism: 17 cases (2001-2009). J Am Vet Med Assoc 2012;241:233-236. 
10. Strickland PL, Recabaren J. Are preoperative serum calcium, parathyroid hormone, and adenoma weight predictive of postoperative hypocalcemia? Am Surg 2002;68:1080-1082. 
11. Milovancev M, Schmiedt CW. Preoperative factors associated with postoperative hypocalcemia in dogs with primary hyperparathyroidism that underwent parathyroidectomy: 62 cases (2004-2009). J Am Vet Med Assoc 2013;242:507-515. 

Vetsulin Insulin Updated and Approved For Release in USA

Merck Animal Health just announced that Vetsulin (porcine insulin zinc suspension) will again be available for distribution in the USA this week (1). This is welcome news, especially for dogs with diabetes mellitus that are not well regulated with other available insulin preparations, such as human recombinant NPH. Most authorities consider Vetsulin, known as Caninsulin outside the USA (2), to be the initial insulin of choice for treatment of dogs with diabetes mellitus (3-6). It can also be used to successfully manage cats with diabetes (7,8), but we have many other good options for treatment of feline diabetes.

As you may know, Vetsulin has had its share of problems in the last few years, which lead to the FDA having it withdrawn from the US market. All of these manufacturing issues have been resolved, and the FDA has re-approved the drug for release.

How is the "new" Vetsulin different than the original insulin product?

According to the company, the updated Vetsulin product has the same characteristics as the original insulin preparation, and the action in diabetic patients is expected to be the same as before. There have been no changes in the safety profile of Vetsulin for use in either dogs or cats.

The only difference in the Vetsulin itself is a slight change in the listed proportion of the short-acting amorphous fraction and long-acting crystalline fraction. The short-acting amorphous fraction is now listed as 35% (it was 30%), whereas the long-acting crystalline fraction is now reported as 65% (it was 70%).

Vetsulin will now be available only in 10-ml vials. The 2-5 ml vial formulation of Vetsulin has been discontinued.

Two important differences in the updated Vetsulin Package Insert: (9)

1. Prior to use, vials of Vetsulin should be shaken thoroughly (manually) until a homogeneous, uniformly, milky suspension is obtained. Foam on the surface of the suspension formed during shaking should be allowed to disperse before the product is used.
2. Vetsulin contents should be used within 42 days after the vial is first punctured.  

For the first new vials of Vetsulin that are released, the initial product dating will be only 12 months from the manufactured date. The company expects that Vetsulin will eventually have a 24-month shelf-life.

Additional information can be found on the Veterinary Home Page under the Product Update section (10). Also, see the package insert for full information regarding contraindications, warnings, and precautions (9).

Bottom line:

It's great to have Vetsulin, an FDA-approved insulin for use in both dogs and cats, back on the market. The company has done a great deal of work showing that Vetsulin is stable and effective, and they have made a few minor but good improvements in this insulin preparation.

Vigorous manual shaking of the Vetsulin? How will that impact accurate dosing?
We have all been taught that vigorous shaking will interfere with accurate dosing because of air bubbles that form when the insulin bottle is shaken (5,11). When the dose is drawn up, the insulin suspension may come out into the syringe together with lots of air bubbles.  In addition, we tend to worry that too vigorous shaking may damage the insulin protein structure itself. For these reasons, we generally instruct owners to gently roll the insulin vial than to vigorously shake it!

According to the company, the instructions are now to give the Vetsulin bottle a vigorous shake on initial use; then let it settle for a few minutes to let the bubbles rise before drawing up the dose. Upon subsequent insulin dosing, the bottle should only need a quick shake (less vigorous) to resuspend the insulin prior to drawing up the insulin dose.

Why this difference in vigorous shaking vs. rolling? I don't know, but I suspect it has something to do with the way the study was designed and input from the FDA. In any case, Merck Animal Health actually has done the studies to prove that this vigorous manual shaking does not interfere with the measured insulin concentration in the product (10).

Why was the proportion in the short-and long-acting insulin fractions changed? 
According to the company, the proportions of the short-acting amorphous fraction and long-acting crystalline fraction have not actually changed, as compared to the original Vetsulin product. Instead, the new stated ratio (65% long-acting and 35% short-acting insulin) represents a more accurate reflection of the actual composition of both the original and updated Vetsulin product (10).

Nevertheless, on a clinical basis, such a small change in the short- vs. the long-acting insulin fractions would not be expected to make any difference in the animal's glycemic control, even if the composition of the product did change slightly.

Vetsulin (porcine insulin zinc suspension), with it's 2 peaks of insulin activity

Must Vetsulin really be discarded after only 42 days? 
The Vetsulin product insert (9) clearly states that the product should be used within "42 days of first vial puncture."

Has the efficacy really been determined to decrease after 42 days?  The answer to that is no — it's almost certain that the potency of this preparation will extend many days (or even weeks) longer than this 42 days.  However, because Merck Animal Health only did the FDA studies for a period of 42 days, that's what they have been required to put on the package insert. But remember, in addition to efficacy, we must also worry about bacterial contamination secondary to repeated puncture of the insulin vial (11).

Why such a short expiration date? 
Again, for the first new vials of Vetsulin that are released, the initial product dating will be only 12 months from the manufactured date. This is not because the updated Vetsulin is unstable, but the FDA requires ongoing studies of the insulin's duration. The company expects that Vetsulin will eventually have a 24-month shelf-life (10).

Since we do not have to worry about Vetsulin disappearing again, at least anytime soon, I wouldn't recommend purchasing too many insulin vials now, since the initial "expiration" date will be only a few months.

References:

1. Vetsulin website. www.vetsulin.com
2. Caninsulin website. www.caninsulin.com
3. Monroe WE, Laxton D, Fallin EA, et al. Efficacy and safety of a purified porcine insulin zinc suspension for managing diabetes mellitus in dogs. J Vet Intern Med 2005;19:675-682. 
4. Fleeman LM, Rand JS, Morton JM. Pharmacokinetics and pharmacodynamics of porcine insulin zinc suspension in eight diabetic dogs. Vet Rec 2009;164:232-237. 
5. Nelson RW. Canine diabetes mellitus In: Ettinger SJ,Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Seventh Edition ed. St. Louis: Saunders Elsevier, 2010;1449-1474.
6. Davison LJ. Canine diabetes mellitus In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;116-132.
7. Michiels L, Reusch CE, Boari A, et al. Treatment of 46 cats with porcine lente insulin—a prospective, multicentre study. J Feline Med Surg 2008;10:439-451. 
8. Martin GJ, Rand JS. Control of diabetes mellitus in cats with porcine insulin zinc suspension. Vet Rec 2007;161:88-94. 
9. Vetsulin Package Insert. www.vetsulin.com/PDF/Vetsulin-Package-Insert.pdf
10. Vetsulin website: Veterinary Product Updates. www.vetsulin.com/vet/Product_Update.aspx
11. American Diabetes Association. Insulin administration. Diabetes Care 2001;24:1984-1987. 

Intraoperative Parathyroid Hormone Monitoring in Dogs with Hyperparathyroidism

Intraoperative Parathyroid Hormone Concentration to Confirm Removal of Hypersecretory Parathyroid Tissue and Time to Postoperative Normocalcaemia in Nine Dogs with Primary Hyperparathyroidism

K.J. Graham, M. Wilkinson, J. Culvenor, N.K. Dhand, and R.K. Churcher

Australian Veterinary Journal 2012;90:203-209.

Primary hyperparathyroidism is a relatively uncommon disease in dogs but must always be considered as a differential cause of hypercalcemia, particularly in an older, relatively asymptomatic dog with no evidence for malignancy (1-5). In primary hyperparathyroidism, parathyroid gland function is autonomous and nonresponsive to inhibition.  In approximately 90% of dogs, single parathyroid adenomas are responsible, but two adenomas, hyperplastic nodules or, rarely, carcinomas have been reported (2,6)

In human patients suffering from primary hyperparathyroidism, intraoperative parathyroid hormone (PTH) measurement is a highly sensitive and specific tool for determining successful removal of autonomously functioning parathyroid tissue (7-10). In this procedure, PTH is measured just prior to parathyroidectomy and at least 10 minutes after parathyroid tumor removal.  Since PTH has a plasma half life of less than 5 minutes (11), removal of a parathyroid tumor leads to a rapid fall in circulating PTH concentrations within minutes. The criterion for a positive test result varies between studies, but is generally accepted that > 50% decrease in PTH values indicates successful parathyroid tumor removal (7-10). A recent prospective veterinary study of 12 dogs demonstrated a greater than 50% reduction in parathyroidectomy PTH in all dogs, confirming correct removal of the hypersecretory gland (12).

The aims of this study by Graham et al (13) were to determine if serum PTH concentrations would decrease after successful parathyroidectomy and whether manipulation or dissection of the target gland would cause a transient increase in PTH, given its rapid half-life.

Objective of Study — To determine whether the intraoperative parathyroid hormone concentration (PTH) during parathyroidectomy can be used to indicate a cure in dogs with primary hyperparathyroidism. A secondary objective was to determine the time taken for the postoperative serum calcium concentration to normalize.

Design — Retrospective study (2005–2010) from a private referral hospital in Sydney, New South Wales, Australia.

Procedure — Nine client-owned dogs underwent surgical parathyroidectomy for naturally occurring primary hyperparathyroidism. The first PTH sample was taken immediately after induction of anesthesia and prior to manipulation of parathyroid tissue.  A second sample was collected as the abnormal parathyroid gland was being manipulated or excised in 6 dogs. A final PTH sample was taken 20–30 minutes after completion of parathyroidectomy in all dogs.

The concentration of ionized calcium (iCa) was measured at various time points postoperatively until it normalized, then stabilized or decreased below reference ranges. Statistical analysis compared the mean pre-, intra- and post-parathyroidectomy PTH concentration and the average rate of decline of iCa concentration postoperative.

Assays—Blood for PTH measurement was collected from the jugular vein and placed into plain tubes on ice for 30 minutes, centrifuged at 2500g and the serum transferred to a plain tube for storage at 4°C until assay. The serum specimens were assayed for PTH within 2 hours of collection, and results were available by the afternoon of surgery.

Intact serum PTH was assayed using an in-house, quick chemiluminescent enzyme immunometric assay, which is a modification of the Immulite PTH assay (Immulite Turbo: Siemens Medical Solutions Diagnostics).

Results – Serum PTH concentrations fell significantly when measured before and shortly after parathyroidectomy from mean pre-PTH value of 168.51 pg/mL to mean post-parathyroidectomy value of 29.20 pg/mL (Fig. 1). Intraoperative manipulation of the parathyroid tumor resulted in a significant increase in the mean PTH value to 279.78 pg/mL (Fig. 1). The average rate of decline of iCa concentration to within the reference range (1.12-1.40 mmol/L) occurred after 24 hours postoperatively.

Figure 1: Mean PTH concentration before surgery, at time of manipulation of the parathyroid tumor, and 20 min after parathyroidectomy.

Conclusions – Intraoperative measurements of PTH can be used clinically to determine cure of primary hyperparathyroidism. Parathyroid hormone increases significantly during parathyroid gland manipulation. Plasma iCa concentration returns to within the reference range on average 24 hours after successful parathyroidectomy. Not all dogs require vitamin D or calcium supplementation postoperatively.

My Bottom Line:

In this study (13), the investigators were able to document that use of intraoperative PTH in dogs undergoing parathyroidectomy for primary hyperparathyroidism can be a very powerful technique to determine successful surgical removal of the parathyroid tumor and predict outcome. Following parathyroid tumor removal, serum PTH fell dramatically when remeasured at 20-30 minutes postoperatively (see Figure 1). This agrees with another study performed in dogs in which serum PTH fell by >50% when rechecked 30-45 minutes after excision of the diseased parathyroid gland (12).

The finding that PTH concentrations can rise dramatically after manipulation of the abnormal parathyroid tissue highlights the importance of waiting at least 20 minutes to collect the final PTH sample after adenoma removal. This interval ensures that circulating PTH has had enough time to fall to it nadir level before collection.  If collected too soon, the PTH may still be too high, leading one to believe that the surgery was not successful.

Is intraoperative PTH testing practical?
The cost and availability of PTH assays is a major limiting factor for use of intra-operative PTH testing in dogs. Very few veterinary practices, even large speciality hospitals, will have access for the dedicated lab equipment needed to perform these assays. A nearby human facility was used in this case series reported by Graham (13), whereas the only other veterinary case series (12) performed quick parathyroid testing with a in-house, dedicated machine.

And that brings up the biggest down-side to this method —is it feasible to have the results of PTH testing back during the surgical procedure so that we know that all of the involved parathyroid tissue has been removed hyperfunctioning gland prior to closing?  If the PTH samples must be sent out to an outside lab for analysis, that typically will take a few days to receive the PTH results. Such a delay overrides or negates almost all of the advantages of this diagnostic technique.

Remember, as shown in this present study (13), we can judge the success of the operation on the basis of serial serum ionized calcium concentrations, which normalize by 24 hours after successful surgery. We do not need a serum PTH measurement to tell us that operation for hyperparathyroidism has been successful, at least not in most dogs with this disease.

References:

1. Berger B, Feldman EC. Primary hyperparathyroidism in dogs: 21 cases (1976-1986). J Am Vet Med Assoc 1987;191:350-356. 
2. Feldman EC, Hoar B, Pollard R, et al. Pretreatment clinical and laboratory findings in dogs with primary hyperparathyroidism: 210 cases (1987-2004). J Am Vet Med Assoc 2005;227:756-761. 
3. Gear RN, Neiger R, Skelly BJ, et al. Primary hyperparathyroidism in 29 dogs: diagnosis, treatment, outcome and associated renal failure. J Small Anim Pract 2005;46:10-16.  
4. Skelly BJ. Hyperparathyroidism In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;43-55.
5. Jores K, Kessler M. Primary hyperparathyroidism in the dog. Diagnosis, therapy and postoperative management in 19 dogs. Tierarztliche Praxis Ausgabe K, Kleintiere/Heimtiere 2011;39:389-396.
6. Sawyer ES, Northrup NC, Schmiedt CW, et al. Outcome of 19 dogs with parathyroid carcinoma after surgical excision. Vet Comp Oncol 2012;10:57-64. 
7. Irvin GL, 3rd, Solorzano CC, Carneiro DM. Quick intraoperative parathyroid hormone assay: surgical adjunct to allow limited parathyroidectomy, improve success rate, and predict outcome. World J Surg 2004;28:1287-1292. 
8. Sharma J, Milas M, Berber E, et al. Value of intraoperative parathyroid hormone monitoring. Ann Surg Oncol 2008;15:493-498. 
9. Richards ML, Thompson GB, Farley DR, et al. An optimal algorithm for intraoperative parathyroid hormone monitoring. Arch Surg 2011;146:280-285. 
10. Carneiro-Pla D. Contemporary and practical uses of intraoperative parathyroid hormone monitoring. Endocr Pract 2011;17 Suppl 1:44-53. 
11. Bieglmayer C, Prager G, Niederle B. Kinetic analyses of parathyroid hormone clearance as measured by three rapid immunoassays during parathyroidectomy. Clin Chem 2002;48:1731-1738.
12. Ham K, Greenfield CL, Barger A, et al. Validation of a rapid parathyroid hormone assay and intraoperative measurement of parathyroid hormone in dogs with benign naturally occurring primary hyperparathyroidism. Vet Surg 2009;38:122-132.  
13. Graham KJ, Wilkinson M, Culvenor J, et al. Intraoperative parathyroid hormone concentration to confirm removal of hypersecretory parathyroid tissue and time to postoperative normocalcaemia in nine dogs with primary hyperparathyroidism. Aust Vet J 2012;90:203-209. 

Top Endocrine Publications of 2012: Canine & Feline Parathyroid & Calcium Disorders

In my third compilation of the canine and feline endocrine publications of 2012, I’m moving on to disorders of the parathyroid gland, including the clinical problems of hypercalcemia and hypocalcemia.

Listed below are 22 research papers written in 2012 that deal with a variety of topics and issues related to calcium, parathyroid or vitamin D metabolism.

These range from iatrogenic hypoparathyroidism following parathyroid or thyroid surgery (1,20,21) to dietary hypocalcemia in growing dogs (2,14); from paraneoplastic hypercalcemia (3) to puerperal tetany (eclampsia) and hypocalcemia associated with whelping (6); and from dietary and animal-related factors associated with urinary calcium and calcium oxalate stones (7,8) to circulating PTH concentrations in cats with secondary hyperparathyroidism due to renal disease (9).

Other papers discuss the use of intraoperative PTH measurements during parathyroidectomy to help predict cure in dogs with primary hyperparathyroidism (10) to characterization of a mutation that causes vitamin D-dependent rickets in cats (11); from a study of hypovitaminosis D in dogs with endotoxemia (12) to the effect of prednisolone therapy on calcium and vitamin D metabolism in dogs (16,17); from studies of the outcome of dogs with thyroid or parathyroid carcinoma treated with surgical excision (20,21) to an evaluation of calcium and phosphate homeostasis in hyperthyroid cats with chronic kidney disease.

References:

1. Arbaugh M, Smeak D, Monnet E. Evaluation of preoperative serum concentrations of ionized calcium and parathyroid hormone as predictors of hypocalcemia following parathyroidectomy in dogs with primary hyperparathyroidism: 17 cases (2001-2009). J Am Vet Med Assoc 2012;241:233-236. 
2. Becker N, Kienzle E, Dobenecker B. Calcium deficiency: a problem in growing and adult dogs: two case reports. Tierarztl Prax Ausg K Kleintiere Heimtiere 2012;40:135-139. 
3. Bergman PJ. Paraneoplastic hypercalcemia. Top Companion Anim Med 2012;27:156-158. 
4. Brockley LK, Heading KL, Jardine JE, et al. Polyostotic lymphoma with multiple pathological fractures in a six-month-old cat. J Feline Med Surg 2012;14:285-291. 
5. Corbee RJ, Tryfonidou MA, Meij BP, et al. Vitamin D status before and after hypophysectomy in dogs with pituitary-dependent hypercortisolism. Domest Anim Endocrinol 2012;42:43-49. 
6. Davidson AP. Reproductive causes of hypocalcemia. Top Companion Anim Med 2012;27:165-166. 
7. Dijcker JC, Hagen-Plantinga EA, Everts H, et al. Dietary and animal-related factors associated with the rate of urinary oxalate and calcium excretion in dogs and cats. Vet Rec 2012;171:46. 
8. Dijcker JC, Kummeling A, Hagen-Plantinga EA, et al. Urinary oxalate and calcium excretion by dogs and cats diagnosed with calcium oxalate urolithiasis. Vet Rec 2012;171:646. 
9. Finch NC, Syme HM, Elliott J. Parathyroid hormone concentration in geriatric cats with various degrees of renal function. J Am Vet Med Assoc 2012;241:1326-1335. 
10. Graham KJ, Wilkinson M, Culvenor J, et al. Intraoperative parathyroid hormone concentration to confirm removal of hypersecretory parathyroid tissue and time to postoperative normocalcaemia in nine dogs with primary hyperparathyroidism. Aust Vet J 2012;90:203-209. 
11. Grahn RA, Ellis MR, Grahn JC, et al. A novel CYP27B1 mutation causes a feline vitamin D-dependent rickets type IA. J Feline Med Surg 2012;14:587-590. 
12. Holowaychuk MK, Birkenheuer AJ, Li J, et al. Hypocalcemia and hypovitaminosis D in dogs with induced endotoxemia. J Vet Intern Med 2012;26:244-251. 
13. Hong HH, Chou TA, Yang JC, et al. The potential effects of cholecalciferol on bone regeneration in dogs. Clin Oral Implants Res 2012;23:1187-1192. 
14. Hutchinson D, Freeman LM, McCarthy R, et al. Seizures and severe nutrient deficiencies in a puppy fed a homemade diet. J Am Vet Med Assoc 2012;241:477-483. 
15. Kovalik M, Mellanby RJ, Evans H, et al. Ciclosporin therapy is associated with minimal changes in calcium metabolism in dogs with atopic dermatitis. Vet Dermatol 2012;23:481-491. 
16. Kovalik M, Thoday KL, Berry J, et al. Prednisolone therapy for atopic dermatitis is less effective in dogs with lower pretreatment serum 25-hydroxyvitamin D concentrations. Vet Dermatol 2012;23:125-130, e127-128.
17. Kovalik M, Thoday KL, Evans H, et al. Short-term prednisolone therapy has minimal impact on calcium metabolism in dogs with atopic dermatitis. Vet J 2012;193:439-442. 
18. Nunamaker EA, Sherman JG. Oral administration of lanthanum dioxycarbonate does not alter bone morphology of normal cats. J Vet Pharmacol Ther 2012;35:193-197. 
19. Pineda C, Aguilera-Tejero E, Raya AI, et al. Feline parathyroid hormone: validation of hormonal assays and dynamics of secretion. Domest Anim Endocrinol 2012;42:256-264. 
20. Sawyer ES, Northrup NC, Schmiedt CW, et al. Outcome of 19 dogs with parathyroid carcinoma after surgical excision. Vet Comp Oncol 2012;10:57-64. 
21. Tuohy JL, Worley DR, Withrow SJ. Outcome following simultaneous bilateral thyroid lobectomy for treatment of thyroid gland carcinoma in dogs: 15 cases (1994-2010). J Am Vet Med Assoc 2012;241:95-103. 
22. Williams TL, Elliott J, Syme HM. Calcium and phosphate homeostasis in hyperthyroid cats - associations with development of azotaemia and survival time. J Small Anim Pract 2012;53:561-571. 

Gene Therapy: A Cure for Canine Diabetes

Beagle dog, from Wikipedia commons

Treatment of Diabetes and Long-term Survival Following Insulin and Glucokinase Gene Therapy

David Callejas, Christopher J. Mann, Eduard Ayuso, Ricardo Lage, Iris Grifoll, Carles Roca, Anna Andaluz, Rafael Ruiz-de Gopegui, Joel Montané, Sergio Muñoz, Tura Ferre, Virginia Haurigot, Shangzhen Zhou, Jesús Ruberte, Federico Mingozzi, Katherine A. High, Felix Garcia, and Fatima Bosch

Diabetes (Published online before print) February 1, 2013, doi:10.2337/db12-1113

In both man and dogs, diabetes is a chronic disease for which there is no cure. Almost all diabetic dogs need insulin replacement therapy to survive, but glycemic control is never perfect (1-3). Therefore, long-term complications of diabetes (e.g., cataracts) disease are frequently observed (4-6). In addition, burden of our traditional treatment methods takes a toll on the owners of diabetic pets and results in a proportion of pets being euthanized instead of being treated (7). If pet owners do decide to treat their diabetic pets, they commonly report a significant treatment burden, with a number of negative effects on various aspects of the pet’s and owner’s lifestyle (7,8).

Gene therapy for diabetes
Obviously, a less arduous treatment option for controlling diabetes mellitus in dogs would be welcomed.  Gene therapy has the potential to provide this alternative method of treatment for animals as well as for humans. Simplistically, in gene therapy, particular strings of DNA are "inserted" into cells such that the machinery of the cell takes the DNA, transcribes it into RNA, and then makes the protein that is encoded in the new DNA. This technique allows us to force cells to make proteins that they might not otherwise make, and in the last decade it has started to see success in small trials to treat a number of diseases.

As it turns out, however, designing and targeting a string of DNA at a cell is not easy (8,9). Currently, most investigators use what is called viral vectors to get the DNA inside cells— basically, this involves taking a virus that contains the DNA we want to insert, and infect cells with the virus. The virus enters the cell, where it can integrate its DNA segment into the chromosomes of the host cell (8).

For diabetes, experimental gene therapy has generally involved injection of protein-coding genes into skeletal muscle or the liver. This exploits these tissues' intrinsic ability to read such genes and subsequently constitutively produce the corresponding protein, which is then secreted into the general circulation by virtue of the extensive vascularization of peripheral muscles (10-14).

Study reporting successful gene therapy for canine diabetes
To date, most gene therapy has involved studies in diabetic mice. As reported by Callejas et al. in the  journal Diabetes (15), these researchers from the Center of Animal Biotechnology and Gene Therapy, Universitat Autonoma de Barcelona, Spain, used gene therapy to treat and "cure" 5 Beagle dogs with insulin-deficient diabetes.

This same research group had already tested this type of therapy on mice (10), but the excellent results obtained for the first time with large animals lays the foundation for the clinical translation of this gene therapy approach to veterinary medicine and eventually for diabetic human patients.

The diabetic state in these dogs had been induced by injection of a mixture of streptozotocin and alloxan, both of which are toxic to beta cells (16). After becoming diabetic, the dogs were treated with one of the following:

1. Insulin glargine, administered twice daily
2. Intramuscular injection of the glucokinase gene alone 
3. Intramuscular injection of the insulin gene alone
4. Intramuscular injection of both the insulin and glucokinase genes. 

Not surprisingly, without treatment, the dogs were markedly hyperglycemic. The dogs on twice daily insulin were better controlled but remained hyperglycemic.

To test both the insulin and glucose kinase vectors together, the researchers injected 5 dogs with one or two doses of the viral vectors. These dogs quickly regained normoglycemia, and even the higher doses were tolerated well; none of the dog had any side effects. Unlike the dogs given insulin alone, the dogs given both genes together responded much better to an oral glucose tolerance test, spiking slightly up to between 200 and 300 mg/dL, but stayed well below the levels seen with the diabetic dogs.

The dogs gained weight, had lower serum fructosamine levels, and experienced long-term remission of the diabetes without secondary complications. Therapy that included only the insulin gene or the glucose kinase gene but not both did not result in the same level of success.

Click here to see a video of the treated dogs in diabetic remission.

My Bottom Line:

Since the 1922 breakthrough discovery of Banting and Best (17), who corrected hyperglycemia in dogs using pancreatic extracts, exogenous insulin administration has been the mainstay of diabetes therapy (1-3). Alternative therapies have been studied, but thus far only a handful of approaches, mainly involving allo- or xeno-transplantation of pancreatic islets, have reached clinical application (18).

The authors of this landmark study (15) took a different approach: instead of trying to transplant or manufacture beta cells that respond to glucose and produce insulin, they used dual gene therapy to develop a “glucose sensor” in skeletal muscle that permitted long-term, normoglycemia in this canine model of diabetes.

The gene therapy used in these dogs consisted of a single session of intramuscular injections to introduce gene therapy vectors, with two ultimate objectives: to express the insulin gene, on the one hand, and that of glucokinase, on the other. Glucokinase is an enzyme that regulates the uptake of glucose from the blood (19). When both insulin and glucokinase genes act simultaneously, they function together as a glucose sensor to automatically regulate the uptake of glucose from the blood, thus reducing hyperglycemia (20).

The results of this study look very promising indeed. The dogs in the study, once treated, experienced long-term normoglycemia, both in the fasting or fed state, with no need for exogenous insulin therapy. There were no episodes of hypoglycemia, even after the dogs were exercised. In addition, all dogs regained lost body weight and developed no secondary complications 4 years after treatment.

Hopefully, this study will provide the basis for the initiation of clinical studies in dogs (and cats?) with naturally-occurring diabetes. Such veterinary clinical trials should also help investigators in preparing for the use of this approach in humans patients with diabetes.  

References:

1. Davison LJ, Herrtage ME, Catchpole B. Study of 253 dogs in the United Kingdom with diabetes mellitus. Vet Rec 2005;156:467-471.
2. Nelson RW. Canine diabetes mellitus In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Seventh ed. St. Louis: Saunders Elsevier, 2010;1449-1474.
3. Davison LJ. Canine diabetes mellitus In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;116-132.
4. Munana KR. Long-term complications of diabetes mellitus, Part I: Retinopathy, nephropathy, neuropathy. Vet Clin North Am Small Anim Pract 1995;25:715-730. 
5. Basher AW, Roberts SM. Ocular manifestations of diabetes mellitus: diabetic cataracts in dogs. Vet Clin North Am Small Anim Pract 1995;25:661-676. 
6. Beam S, Correa MT, Davidson MG. A retrospective-cohort study on the development of cataracts in dogs with diabetes mellitus: 200 cases. Vet Ophthalmol 1999;2:169-172. 
7. Niessen SJ, Powney S, Guitian J, et al. Evaluation of a quality-of-life tool for dogs with diabetes mellitus. J Vet Intern Med 2012;26:953-961. 
8. Bertolaso M, Olsson J, Picardi A, et al. Gene therapy and enhancement for diabetes (and other diseases): the multiplicity of considerations. Diabetes Metab Res Rev 2010;26:520-524. 
9. Alenzi FQ, Lotfy M, Tamimi WG, et al. Review: Stem cells and gene therapy. Lab Hematol 2010;16:53-73. 
10. Mas A, Montane J, Anguela XM, et al. Reversal of type 1 diabetes by engineering a glucose sensor in skeletal muscle. Diabetes 2006;55:1546-1553.  
11. Wong MS, Hawthorne WJ, Manolios N. Gene therapy in diabetes. Self Nonself 2010;1:165-175. 
12. Bagley J, Paez-Cortez J, Tian C, et al. Gene therapy in type 1 diabetes. Crit Rev Immunol 2008;28:301-324. 
13. Niessen SJ, Fernandez-Fuente M, Mahmoud A, et al. Novel diabetes mellitus treatment: mature canine insulin production by canine striated muscle through gene therapy. Domest Anim Endocrinol 2012;43:16-25. 
14. Ren B, O'Brien BA, Byrne MR, et al. Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy. J Gene Med 2013;15:28-41. 
15. Callejas D, Mann CJ, Ayuso E, et al. Treatment of diabetes and long-term survival following insulin and glucokinase gene therapy. Diabetes 2013 (in press). 
16. Anderson HR, Stitt AW, Gardiner TA, et al. Induction of alloxan/streptozotocin diabetes in dogs: a revised experimental technique. Lab Anim 1993;27:281-285. 
17. Banting FG, Best CH, Collip JB, et al. Pancreatic extracts in the treatment of diabetes mellitus. Can Med Assoc J 1922;12:141-146. 
18. Robertson RP. Islet transplantation a decade later and strategies for filling a half-full glass. Diabetes 2010;59:1285-1291.  
19. Printz RL, Magnuson MA, Granner DK. Mammalian glucokinase. Annu Rev Nutr 1993;13:463-496. 
20. Otaegui PJ, Ferre T, Pujol A, et al. Expression of glucokinase in skeletal muscle: a new approach to counteract diabetic hyperglycemia. Hum Gene Ther 2000;11:1543-1552. 

Managing Dogs with Insulin-Sensitive, Brittle Diabetes

My problem patient is a 13-year-old, female-spayed Yorkie with brittle diabetes mellitus. In contrast to many diabetic dogs that I see, this dog is very insulin sensitive, with frequent bouts of hypoglycemia. Fortunately, these hypoglycemic episodes do not appear to be causing clinical problems (i.e., no weakness or seizures have been observed), and she seems to be doing great at home. We have the owner feeding her several times a day to prevent hypoglycemia.

To complicate diabetic control in this dog, the owner's elderly father cares for the dog during the day. On the weekends, the owner works quite late so she "pre-fills" the insulin syringe in the morning so her father can give the insulin shot later that day. I've told the owner that the NPH insulin particles will likely be out of suspension by the evening (unless her father remembers to roll the syringe to remix the insulin suspension), but the owner says she doesn't have much choice.  I'm not sure how this is affecting the diabetic control, but it cannot be helping. We try to do her glucose curves toward the end of the week after the owner (rather than the father) has been injecting the insulin herself.

Below are two recent blood glucose curves, the first done about a month ago and the second one just done this week:

Glucose Curve 1

• 0800 hr—372 mg/dl (Fed; Gave 1 unit of NPH, SC)
• 1000 hr—109 mg/dl
• 1130 hr—Fed small meal
• 1200 hr—98 mg/dl
• 1400 hr—75 mg/dl
• 1530 hr—Fed small meal
• 1600 hr—67 mg/dl
• 1800 hr—214 mg/dl

I thought this curve indicated pretty good glycemic control. However, I found her latest glucose curve to be especially concerning.

Glucose Curve 2

• 0800 hr—310 mg/dl (Fed; Gave 1 unit of NPH, SC)
• 1000 hr—65 mg/dl
• 1015 hr—Fed small meal
• 1100 hr—101 mg/dl
• 1300 hr—24 mg/dl (Sample sent to lab confirmed hypoglycemia: 45 mg/dl)
• 1310 hr—Fed small meal (ate well)
• 1400 hr—98 mg/dl
• 1600 hr—240 mg/dl
• 1800 hr—270 mg/dl

Even with the very low blood glucose value at 1300 hr, this dog was acting fine and was not exhibiting any signs of hypoglycemia.

So I'm at a loss. I told the owner to decrease to 0.5 unit of the NPH twice daily and to feed more at each meal. However, I'm worried that it's going to be very difficult to give such a tiny dose of insulin. Can the NPH be diluted?

I'm also wondering if part of the problem here is a too short of a duration for NPH or a Somogyi effect (hypoglycemia-induced hyperglycemia). Should we try a different insulin?

My Response:

Obviously, this dog has a higher insulin sensitivity than is the typical dog with diabetes mellitus. Such an insulin-sensitive diabetic will require smaller amounts of insulin to lower blood glucose levels than the average canine diabetic with "normal" sensitivity or insulin resistance.

The glucose-lowering effect of insulin varies from diabetic dog to dog, depending on a number of factors including age, degree of insulin deficiency, and concurrent disease (1).  Generally speaking, having a higher sensitivity to insulin makes it easier to regulate the diabetic patient.  However, there are times when this increased sensitivity can be problematic. As in this dog, having high insulin sensitivity will increase the risk of hypoglycemia and can even make it difficult to accurately draw up a small enough dose.

Minimizing hypoglycemia in dogs with increased insulin sensitivity
So what can we do to minimize iatrogenic hypoglycemia in the insulin-sensitive diabetic?  Factors to consider include the following (1-4):

• Insulin dose
• Type of insulin (short, intermediate, or long-acting)
• Timing of food ingestion to insulin injection
• Exercise
• Decreased clearance of insulin

Lowering the insulin dose— You certainly could dilute the NPH insulin and lower the insulin dose to 0.5 U twice a day. Both Eli Lilly and Novo Nordisk make diluents for their brand of NPH insulin, but these diluents can be difficult to obtain (5).  Insulin can also be diluted with sterile water or saline solution, but these insulin solutions are not as stable.

A better option is to use a U-100 insulin syringe with 0.5 unit markings. Again, these syringes may be difficult to find, but are generally available at WalMart  (ask for ReliOn U-100 3/10 cc insulin syringes marked in 1/2 unit dosing).

Using a less potent type of insulin — In general, short-acting insulins (e.g., regular insulin, NPH, lente) have a more potent hypoglycemic effect than do the long-acting insulin preparations (protamine zinc insulin, glargine).  The reason for this is mainly because short-acting insulins are absorbed faster and result in higher levels of circulating insulin concentrations (1,6-8). Long-acting preparations, such as glargine (Lantus) or protamine zinc insulin (PZI, ProZinc) are absorbed much slower and result in much lower peak circulating insulin concentrations (1,6-10). The big exception to this rule is the long-acting insulin preparation detemir (Levemir), which turns out to be a very potent insulin in dogs (11).

Adjusting the timing of food ingestion and insulin injection—In general, I like to have owners inject insulin about 30 minutes before the dog eats in order to prevent severe post-prandial hyperglycemia and help regulate the dog's diabetic state (12).  In most dogs, I strongly discourage the feeding of extra meals or snack throughout the day, since it commonly leads to poor glycemic control when using twice daily insulin injections.

In dogs with severe insulin sensitivity, however, I would try the opposite approach. In these dogs, I feed the dog about 30 to 60 minutes before injecting the insulin to encourage a rise in blood sugar to help prevent the hypoglycemia that commonly occurs in these dogs shortly after the insulin injection.

Accounting for the effect of exercise—Although it does not appear to be a contributory factor in this dog, physical activity is well known to have insulin sensitizing effects, and this can also present a higher risk of hypoglycemia for patients on insulin (13). The insulin sensitizing effects of exercise can sometimes last for hours so it is important to be aware of the increased risk of hypoglycemia in these dogs.

Exclude disorders that result in a decreased clearance of insulin—The liver is responsible for about half of the total insulin degradation with the kidney responsible for the rest (14,15). Therefore, the development of severe liver disease or chronic kidney disease can contribute to the increased insulin sensitivity seen in a dog with diabetes. Again, this does not appear to be a contributory factor in your dog.

My Bottom Line

You certainly could lower the NPH insulin dose, but I don't think that's the best solution for this dog.  I would recommend switching to a longer-acting insulin preparation, such as ProZinc or Lantus.  Although not generally considered as first-choice insulin preparations, both of these long-acting insulin preparations have been reported to be effective in dogs (9,10). Because they are slowly absorbed, both of these insulins have a less potent hypoglycemic effect as compared with NPH insulin.

However, we can't forget about the "premixing" of the insulin dose that the owner does on the weekends, can we! When we throw that into the equation, my choice becomes more obvious. Let's change to Lantus, which is a solution so the insulin won't precipitate out of suspension like the premixed NPH insulin would likely do.

References:

1. Nelson RW. Canine diabetes mellitus. In: Ettinger SJ, Feldman EC (eds). Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Seventh Edition. Saunders Elsevier, St Louis. 2010;1782-1796.
2. Cryer PE, Davis SN, Shamoon H. Hypoglycemia in diabetes. Diabetes Care 2003;26:1902-1912. 
3. Cryer PE. Hypoglycemia risk reduction in type 1 diabetes. Exp Clin Endocrinol Diabetes 2001;109 Suppl 2:S412-423. 
4. Boyle PJ, Zrebiec J. Management of diabetes-related hypoglycemia. South Med J 2007;100:183-194. 
5. Insulin administration. Diabetes Care 2001;24:1984-1987. 
6. Goeders LA, Esposito LA, Peterson ME. Absorption kinetics of regular and isophane (NPH) insulin in the normal dog. Domest Anim Endocrinol 1987;4:43-50. 
7. Wallace MS, Peterson ME, Nichols CE. Absorption kinetics of regular, isophane, and protamine zinc insulin in normal cats. Domest Anim Endocrinol 1990;7:509-515. 
8. Clark M, Thomaseth K, Heit M, et al. Pharmacokinetics and pharmacodynamics of protamine zinc recombinant human insulin in healthy dogs. J Vet Pharmacol Ther 2012;35:342-350. 
9. Maggiore AD, Nelson RW, Dennis J, et al. Efficacy of protamine zinc recombinant human insulin for controlling hyperglycemia in dogs with diabetes mellitus. J Vet Intern Med 2012;26:109-115. 
10. Fracassi F, Boretti FS, Sieber-Ruckstuhl NS, et al. Use of insulin glargine in dogs with diabetes mellitus. Vet Rec 2012;170:52. 
11. Sako T, Mori A, Lee P, et al. Time-action profiles of insulin detemir in normal and diabetic dogs. Res Vet Sci 2011;90:396-403.
12. Cobry E, McFann K, Messer L, et al. Timing of meal insulin boluses to achieve optimal postprandial glycemic control in patients with type 1 diabetes. Diabetes Technol Ther 2010;12:173-177. 
13. Zisser H, Gong P, Kelley CM, et al. Exercise and diabetes. Int J Clin Pract Suppl 2011:71-75. 
14. Duckworth WC, Hamel FG, Peavy DE. Hepatic metabolism of insulin. Am J Med 1988;85:71-76. 
15. Rabkin R, Ryan MP, Duckworth WC. The renal metabolism of insulin. Diabetologia 1984;27:351-357. 
16. Gilor C, Graves TK. Synthetic insulin analogs and their use in dogs and cats. Vet Clin North America Small Anim Prac 2010;40:297-307.